US20070218489A1

US20070218489A1 - Compositions for use in identification of bacteria

Info

Publication number: US20070218489A1
Application number: US11/685,603
Authority: US
Inventors: Rangarajan Sampath; Thomas Hall; David Ecker; Lawrence Blyn
Original assignee: Ibis Biosciences Inc
Current assignee: Ibis Biosciences Inc
Priority date: 2003-09-11
Filing date: 2007-03-13
Publication date: 2007-09-20
Also published as: US20120122101A1; US8242254B2; US8288523B2; US20070224614A1; US20070248969A1; US20120122103A1; US20120122097A1; US20070243544A1; US20070238116A1; US20120122096A1; US20120122099A1; US7956175B2; US20120122100A1; US8394945B2; US8013142B2; US20120122098A1; US20120122102A1

Abstract

The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.

Description

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No. 11/409,535, filed Apr. 21, 2006, which is a continuation-in-part of U.S. application Ser. No. 11/060,135, filed Feb. 17, 2005 which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/545,425 filed Feb. 18, 2004; U.S. Provisional Application Ser. No. 60/559,754, filed Apr. 5, 2004; U.S. Provisional Application Ser. No. 60/632,862, filed Dec. 3, 2004; U.S. Provisional Application Ser. No. 60/639,068, filed Dec. 22, 2004; and U.S. Provisional Application Ser. No. 60/648,188, filed Jan. 28, 2005. U.S. application Ser. No. 11/409,535 is a also continuation-in-part of U.S. application Ser. No. 10/728,486, filed Dec. 5, 2003 which claims the benefit of priority to U.S. Provisional Application Ser. No. 60/501,926, filed Sep. 11, 2003. U.S. application Ser. No. 11/409,535 also claims the benefit of priority to: U.S. Provisional Application Ser. No. 60/674,118, filed Apr. 21, 2005; U.S. Provisional Application Ser. No. 60/705,631, filed Aug. 3, 2005; U.S. Provisional Application Ser. No. 60/732,539, filed Nov. 1, 2005; and U.S. Provisional Application Ser. No. 60/773,124, filed Feb. 13, 2006. Each of the above-referenced U.S. Applications is incorporated herein by reference in its entirety. Methods disclosed in U.S. application Ser. Nos. 09/891,793, 10/156,608, 10/405,756, 10/418,514, 10/660,122, 10,660,996, 10/660,997, 10/660,998, 10/728,486, 11/060,135, and 11/073,362, are commonly owned and incorporated herein by reference in their entirety for any purpose.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States Government support under CDC contract RO1 CI000099-01. The United States Government has certain rights in the invention.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled DIBIS0083USC12SEQ.txt, created on Mar. 13, 2007 which is 252 Kb in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

A problem in determining the cause of a natural infectious outbreak or a bioterrorist attack is the sheer variety of organisms that can cause human disease. There are over 1400 organisms infectious to humans; many of these have the potential to emerge suddenly in a natural epidemic or to be used in a malicious attack by bioterrorists (Taylor et al. Philos. Trans. R. Soc. London B. Biol. Sci., 2001, 356, 983-989). This number does not include numerous strain variants, bioengineered versions, or pathogens that infect plants or animals.
Much of the new technology being developed for detection of biological weapons incorporates a polymerase chain reaction (PCR) step based upon the use of highly specific primers and probes designed to selectively detect certain pathogenic organisms. Although this approach is appropriate for the most obvious bioterrorist organisms, like smallpox and anthrax, experience has shown that it is very difficult to predict which of hundreds of possible pathogenic organisms might be employed in a terrorist attack. Likewise, naturally emerging human disease that has caused devastating consequence in public health has come from unexpected families of bacteria, viruses, fungi, or protozoa. Plants and animals also have their natural burden of infectious disease agents and there are equally important biosafety and security concerns for agriculture.
A major conundrum in public health protection, biodefense, and agricultural safety and security is that these disciplines need to be able to rapidly identify and characterize infectious agents, while there is no existing technology with the breadth of function to meet this need. Currently used methods for identification of bacteria rely upon culturing the bacterium to effect isolation from other organisms and to obtain sufficient quantities of nucleic acid followed by sequencing of the nucleic acid, both processes which are time and labor intensive.
Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. DNA chips with specific probes can only determine the presence or absence of specifically anticipated organisms. Because there are hundreds of thousands of species of benign bacteria, some very similar in sequence to threat organisms, even arrays with 10,000 probes lack the breadth needed to identify a particular organism.
The present invention provides oligonucleotide primers and compositions and kits containing the oligonucleotide primers, which define bacterial bioagent identifying amplicons and, upon amplification, produce corresponding amplification products whose molecular masses provide the means to identify bacteria, for example, at and below the species taxonomic level.

SUMMARY OF THE INVENTION

The present invention provides compositions, kits and methods for rapid identification and quantification of bacteria by molecular mass and base composition analysis.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 288.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1269.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 288 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1269.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 698.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1420.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 698 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1420.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 217.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1167
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 217 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1167.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 399.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1041.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 399 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1041.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 430.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1321.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 430 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1321.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 174.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 853.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 174 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 853.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 172.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1360.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 172 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1360.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 456 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1261 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 288:1269, 698:1420, 217:1167, 399:1041, 430:1321, 174:853, and 172:1360.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 315.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1379.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 315 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1379.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 346.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 955.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 346 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 955.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 504.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1409.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 504 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1409.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 323.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1068.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 323 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1068.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 479.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 938.
Another embodiment is an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 479 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 938.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 681 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1022 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 315:1379, 346:955, 504:1409, 323:1068, 479:938.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 583.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 923.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 583 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 923.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 454.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1418.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 454 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1418.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 250.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 902.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 250 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 902.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 384.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 878.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 384 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 878.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 694.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1215.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 694 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1215.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 194.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1173.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 194 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1173.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 375.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 890.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 375 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 890.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 656.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1224.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 656 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1224.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 618.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1157.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 618 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1157.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 302.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 852.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 302 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 852.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 199.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 889.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 199 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 889.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 596.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1169.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 596 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1169.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 150.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1242.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 150 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1242.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1069.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1069.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1168.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 166 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1168.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 583 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 923 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 454:1418, 250:902, 384:878, 694:1215, 194:1173, 375:890, 656:1224, 618:1157, 302:852, 199:889, 596:1169, 150:1242, 166:1069 and 166:1168.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 437.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1137.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 437 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1137.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 530.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 891.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 530 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 891.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 474.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 869.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 474 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 869.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 268.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1284.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 268 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1284.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 418.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1301.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 418 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1301.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 318.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1300.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 318 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1300.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 440.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1076.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 440 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1076.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 219.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1013.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 219 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1013.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 437 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1137 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 530:891, 474:869, 268:1284, 418:1301, 318:1300, 440:1076 and 219:1013.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 325.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1163.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 325 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1163.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 278.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1039.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 278 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1039.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 465.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1037.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 465 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1037.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 148.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1172.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 148 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1172.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 190.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1254.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 190 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1254.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 266.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1094.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 266 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1094.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 508.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1297.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 508 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1297.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 259.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1060.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 259 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1060.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 325 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1163 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 278:1039: 465:1037, 148:1172, 190:1254, 266:1094, 508:1297 and 259:1060.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 376.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1265.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 376 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1265.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 267.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1341.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 267 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1341.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 705.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1056.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 705 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1056.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 710.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1259.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 710 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1259.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 374.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1111.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 374 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1111.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 545.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 978.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 545 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 978.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 249.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1095.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 249 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1095.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 195.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1376.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 195 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1376.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 311.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1014.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 311 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1014.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 365.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1052.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 365 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1052.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 527.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1071.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 527 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1071.
One embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 490.
Another embodiment is an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1182.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 490 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1182.
Another embodiment is a kit comprising an oligonucleotide primer pair including an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 376 and an oligonucleotide primer 14 to 35 nucleobases in length having at least 70% sequence identity with SEQ ID NO: 1265 and further comprising one or more primer pairs wherein each member of said one or more primer pairs is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by SEQ ID NOs: 267:1341, 705:1056, 710:1259, 374:1111, 545:978, 249:1095, 195:1376, 311:1014, 365:1052, 527:1071 and 490:1182.
In some embodiments, either or both of the primers of a primer pair composition contain at least one modified nucleobase such as 5-propynyluracil or 5-propynylcytosine for example.
In some embodiments, either or both of the primers of the primer pair comprises at least one universal nucleobase such as inosine for example.
In some embodiments, either or both of the primers of the primer pair comprises at least one non-templated T residue on the 5′-end.
In some embodiments, either or both of the primers of the primer pair comprises at least one non-template tag.
In some embodiments, either or both of the primers of the primer pair comprises at least one molecular mass modifying tag.
In some embodiments, the present invention provides primers and compositions comprising pairs of primers, and kits containing the same, and methods for use in identification of bacteria. The primers are designed to produce amplification products of DNA encoding genes that have conserved and variable regions across different subgroups and genotypes of bacteria.
Some embodiments are kits that contain one or more of the primer pair compositions. In some embodiments, each member of the one or more primer pairs of the kit is of a length of 14 to 35 nucleobases and has 70% to 100% sequence identity with the corresponding member from any of the primer pairs listed in Table 2.
Some embodiments of the kits contain at least one calibration polynucleotide for use in quantitiation of bacteria in a given sample, and also for use as a positive control for amplification.
Some embodiments of the kits contain at least one anion exchange functional group linked to a magnetic bead.
In some embodiments, the present invention also provides methods for identification of bacteria. Nucleic acid from the bacterium is amplified using the primers described above to obtain an amplification product. The molecular mass of the amplification product is measured. Optionally, the base composition of the amplification product is determined from the molecular mass. The molecular mass or base composition is compared with a plurality of molecular masses or base compositions of known analogous bacterial identifying amplicons, wherein a match between the molecular mass or base composition and a member of the plurality of molecular masses or base compositions identifies the bacterium. In some embodiments, the molecular mass is measured by mass spectrometry in a modality such as electrospray ionization (ESI) time of flight (TOF) mass spectrometry or ESI Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, for example. Other mass spectrometry techniques can also be used to measure the molecular mass of bacterial bioagent identifying amplicons.
In some embodiments, the present invention is also directed to a method for determining the presence or absence of a bacterium in a sample. Nucleic acid from the sample is amplified using the composition described above to obtain an amplification product. The molecular mass of the amplification product is determined. Optionally, the base composition of the amplification product is determined from the molecular mass. The molecular mass or base composition of the amplification product is compared with the known molecular masses or base compositions of one or more known analogous bacterial bioagent identifying amplicons, wherein a match between the molecular mass or base composition of the amplification product and the molecular mass or base composition of one or more known bacterial bioagent identifying amplicons indicates the presence of the bacterium in the sample. In some embodiments, the molecular mass is measured by mass spectrometry.
In some embodiments, the present invention also provides methods for determination of the quantity of an unknown bacterium in a sample. The sample is contacted with the composition described above and a known quantity of a calibration polynucleotide comprising a calibration sequence. Nucleic acid from the unknown bacterium in the sample is concurrently amplified with the composition described above and nucleic acid from the calibration polynucleotide in the sample is concurrently amplified with the composition described above to obtain a first amplification product comprising a bacterial bioagent identifying amplicon and a second amplification product comprising a calibration amplicon. The molecular masses and abundances for the bacterial bioagent identifying amplicon and the calibration amplicon are determined. The bacterial bioagent identifying amplicon is distinguished from the calibration amplicon based on molecular mass and comparison of bacterial bioagent identifying amplicon abundance and calibration amplicon abundance indicates the quantity of bacterium in the sample. In some embodiments, the base composition of the bacterial bioagent identifying amplicon is determined.
In some embodiments, the present invention provides methods for detecting or quantifying bacteria by combining a nucleic acid amplification process with a mass determination process. In some embodiments, such methods identify or otherwise analyze the bacterium by comparing mass information from an amplification product with a calibration or control product. Such methods can be carried out in a highly multiplexed and/or parallel manner allowing for the analysis of as many as 300 samples per 24 hours on a single mass measurement platform. The accuracy of the mass determination methods in some embodiments of the present invention permits allows for the ability to discriminate between different bacteria such as, for example, various genotypes and drug resistant strains of Staphylococcus aureus.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary of the invention, as well as the following detailed description of the invention, is better understood when read in conjunction with the accompanying drawings which are included by way of example and not by way of limitation.
FIG. 1: process diagram illustrating a representative primer pair selection process.
FIG. 2: process diagram illustrating an embodiment of the calibration method.
FIG. 3: common pathogenic bacteria and primer pair coverage. The primer pair number in the upper right hand corner of each polygon indicates that the primer pair can produce a bioagent identifying amplicon for all species within that polygon.
FIG. 4: a representative 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples (labeled NHRC samples) closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.
FIG. 5: a representative mass spectrum of amplification products indicating the presence of bioagent identifying amplicons of Streptococcus pyogenes, Neisseria meningitidis, and Haemophilus influenzae obtained from amplification of nucleic acid from a clinical sample with primer pair number 349 which targets 23S rRNA. Experimentally determined molecular masses and base compositions for the sense strand of each amplification product are shown.
FIG. 6: a representative mass spectrum of amplification products representing a bioagent identifying amplicon of Streptococcus pyogenes, and a calibration amplicon obtained from amplification of nucleic acid from a clinical sample with primer pair number 356 which targets rplB. The experimentally determined molecular mass and base composition for the sense strand of the Streptococcus pyogenes amplification product is shown.
FIG. 7: a representative mass spectrum of an amplified nucleic acid mixture which contained the Ames strain of Bacillus anthracis, a known quantity of combination calibration polynucleotide (SEQ ID NO: 1464), and primer pair number 350 which targets the capC gene on the virulence plasmid pX02 of Bacillus anthracis. Calibration amplicons produced in the amplification reaction are visible in the mass spectrum as indicated and abundance data (peak height) are used to calculate the quantity of the Ames strain of Bacillus anthracis.

DEFINITIONS

As used herein, the term “abundance” refers to an amount. The amount may be described in terms of concentration which are common in molecular biology such as “copy number,” “pfu or plate-forming unit” which are well known to those with ordinary skill. Concentration may be relative to a known standard or may be absolute.
As used herein, the term “amplifiable nucleic acid” is used in reference to nucleic acids that may be amplified by any amplification method. It is contemplated that “amplifiable nucleic acid” also comprises “sample template.”
As used herein the term “amplification” refers to a special case of nucleic acid replication involving template specificity. It is to be contrasted with non-specific template replication (i.e., replication that is template-dependent but not dependent on a specific template). Template specificity is here distinguished from fidelity of replication (i.e., synthesis of the proper polynucleotide sequence) and nucleotide (ribo- or deoxyribo-) specificity. Template specificity is frequently described in terms of “target” specificity. Target sequences are “targets” in the sense that they are sought to be sorted out from other nucleic acid. Amplification techniques have been designed primarily for this sorting out. Template specificity is achieved in most amplification techniques by the choice of enzyme. Amplification enzymes are enzymes that, under conditions they are used, will process only specific sequences of nucleic acid in a heterogeneous mixture of nucleic acid. For example, in the case of Qβ replicase, MDV-1 RNA is the specific template for the replicase (D. L. Kacian et al., Proc. Natl. Acad. Sci. USA 69:3038 [1972]). Other nucleic acid will not be replicated by this amplification enzyme. Similarly, in the case of T7 RNA polymerase, this amplification enzyme has a stringent specificity for its own promoters (Chamberlin et al., Nature 228:227 [1970]). In the case of T4 DNA ligase, the enzyme will not ligate the two oligonucleotides or polynucleotides, where there is a mismatch between the oligonucleotide or polynucleotide substrate and the template at the ligation junction (D. Y. Wu and R. B. Wallace, Genomics 4:560 [1989]). Finally, Taq and Pfu polymerases, by virtue of their ability to function at high temperature, are found to display high specificity for the sequences bounded and thus defined by the primers; the high temperature results in thermodynamic conditions that favor primer hybridization with the target sequences and not hybridization with non-target sequences (H. A. Erlich (ed.), PCR Technology, Stockton Press [1989]).
As used herein, the term “amplification reagents” refers to those reagents (deoxyribonucleotide triphosphates, buffer, etc.), needed for amplification, excluding primers, nucleic acid template, and the amplification enzyme. Typically, amplification reagents along with other reaction components are placed and contained in a reaction vessel (test tube, microwell, etc.).
As used herein, the term “analogous” when used in context of comparison of bioagent identifying amplicons indicates that the bioagent identifying amplicons being compared are produced with the same pair of primers. For example, bioagent identifying amplicon “A” and bioagent identifying amplicon “B”, produced with the same pair of primers are analogous with respect to each other. Bioagent identifying amplicon “C”, produced with a different pair of primers is not analogous to either bioagent identifying amplicon “A” or bioagent identifying amplicon “B”.
As used herein, the term “anion exchange functional group” refers to a positively charged functional group capable of binding an anion through an electrostatic interaction. The most well known anion exchange functional groups are the amines, including primary, secondary, tertiary and quaternary amines.
The term “bacteria” or “bacterium” refers to any member of the groups of eubacteria and archaebacteria.
As used herein, a “base composition” is the exact number of each nucleobase (for example, A, T, C and G) in a segment of nucleic acid. For example, amplification of nucleic acid of Staphylococcus aureus strain carrying the lukS-PV gene with primer pair number 2095 (SEQ ID NOs: 456:1261) produces an amplification product 117 nucleobases in length from nucleic acid of the lukS-PV gene that has a base composition of A35 G17 C19 T46 (by convention—with reference to the sense strand of the amplification product). Because the molecular masses of each of the four natural nucleotides and chemical modifications thereof are known (if applicable), a measured molecular mass can be deconvoluted to a list of possible base compositions. Identification of a base composition of a sense strand which is complementary to the corresponding antisense strand in terms of base composition provides a confirmation of the true base composition of an unknown amplification product. For example, the base composition of the antisense strand of the 139 nucleobase amplification product described above is A46 G19 C17 T35.
As used herein, a “base composition probability cloud” is a representation of the diversity in base composition resulting from a variation in sequence that occurs among different isolates of a given species. The “base composition probability cloud” represents the base composition constraints for each species and is typically visualized using a pseudo four-dimensional plot.
In the context of this invention, a “bioagent” is any organism, cell, or virus, living or dead, or a nucleic acid derived from such an organism, cell or virus. Examples of bioagents include, but are not limited, to cells, (including but not limited to human clinical samples, bacterial cells and other pathogens), viruses, fungi, protists, parasites, and pathogenicity markers (including but not limited to: pathogenicity islands, antibiotic resistance genes, virulence factors, toxin genes and other bioregulating compounds). Samples may be alive or dead or in a vegetative state (for example, vegetative bacteria or spores) and may be encapsulated or bioengineered. In the context of this invention, a “pathogen” is a bioagent which causes a disease or disorder.
As used herein, a “bioagent division” is defined as group of bioagents above the species level and includes but is not limited to, orders, families, classes, clades, genera or other such groupings of bioagents above the species level.
As used herein, the term “bioagent identifying amplicon” refers to a polynucleotide that is amplified from a bioagent in an amplification reaction and which 1) provides sufficient variability to distinguish among bioagents from whose nucleic acid the bioagent identifying amplicon is produced and 2) whose molecular mass is amenable to a rapid and convenient molecular mass determination modality such as mass spectrometry, for example.
As used herein, the term “biological product” refers to any product originating from an organism. Biological products are often products of processes of biotechnology. Examples of biological products include, but are not limited to: cultured cell lines, cellular components, antibodies, proteins and other cell-derived biomolecules, growth media, growth harvest fluids, natural products and bio-pharmaceutical products.
The terms “biowarfare agent” and “bioweapon” are synonymous and refer to a bacterium, virus, fungus or protozoan that could be deployed as a weapon to cause bodily harm to individuals. Military or terrorist groups may be implicated in deployment of biowarfare agents.
In context of this invention, the term “broad range survey primer pair” refers to a primer pair designed to produce bioagent identifying amplicons across different broad groupings of bioagents. For example, the ribosomal RNA-targeted primer pairs are broad range survey primer pairs which have the capability of producing bacterial bioagent identifying amplicons for essentially all known bacteria. With respect to broad range primer pairs employed for identification of bacteria, a broad range survey primer pair for bacteria such as 16S rRNA primer pair number 346 (SEQ ID NOs: 202:1110) for example, will produce an bacterial bioagent identifying amplicon for essentially all known bacteria.
The term “calibration amplicon” refers to a nucleic acid segment representing an amplification product obtained by amplification of a calibration sequence with a pair of primers designed to produce a bioagent identifying amplicon.
The term “calibration sequence” refers to a polynucleotide sequence to which a given pair of primers hybridizes for the purpose of producing an internal (i.e: included in the reaction) calibration standard amplification product for use in determining the quantity of a bioagent in a sample. The calibration sequence may be expressly added to an amplification reaction, or may already be present in the sample prior to analysis.
The term “clade primer pair” refers to a primer pair designed to produce bioagent identifying amplicons for species belonging to a clade group. A clade primer pair may also be considered as a “speciating” primer pair which is useful for distinguishing among closely related species.
The term “codon” refers to a set of three adjoined nucleotides (triplet) that codes for an amino acid or a termination signal.
In context of this invention, the term “codon base composition analysis,” refers to determination of the base composition of an individual codon by obtaining a bioagent identifying amplicon that includes the codon. The bioagent identifying amplicon will at least include regions of the target nucleic acid sequence to which the primers hybridize for generation of the bioagent identifying amplicon as well as the codon being analyzed, located between the two primer hybridization regions.
As used herein, the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides such as an oligonucleotide or a target nucleic acid) related by the base-pairing rules. For example, for the sequence “5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.” Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids. Either term may also be used in reference to individual nucleotides, especially within the context of polynucleotides. For example, a particular nucleotide within an oligonucleotide may be noted for its complementarity, or lack thereof, to a nucleotide within another nucleic acid strand, in contrast or comparison to the complementarity between the rest of the oligonucleotide and the nucleic acid strand.
The term “complement of a nucleic acid sequence” as used herein refers to an oligonucleotide which, when aligned with the nucleic acid sequence such that the 5′ end of one sequence is paired with the 3′ end of the other, is in “antiparallel association.” Certain bases not commonly found in natural nucleic acids may be included in the nucleic acids of the present invention and include, for example, inosine and 7-deazaguanine. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can determine duplex stability empirically considering a number of variables including, for example, the length of the oligonucleotide, base composition and sequence of the oligonucleotide, ionic strength and incidence of mismatched base pairs. Where a first oligonucleotide is complementary to a region of a target nucleic acid and a second oligonucleotide has complementary to the same region (or a portion of this region) a “region of overlap” exists along the target nucleic acid. The degree of overlap will vary depending upon the extent of the complementarity.
In context of this invention, the term “division-wide primer pair” refers to a primer pair designed to produce bioagent identifying amplicons within sections of a broader spectrum of bioagents For example, primer pair number 352 (SEQ ID NOs: 687:1411), a division-wide primer pair, is designed to produce bacterial bioagent identifying amplicons for members of the Bacillus group of bacteria which comprises, for example, members of the genera Streptococci, Enterococci, and Staphylococci. Other division-wide primer pairs may be used to produce bacterial bioagent identifying amplicons for other groups of bacterial bioagents.
As used herein, the term “concurrently amplifying” used with respect to more than one amplification reaction refers to the act of simultaneously amplifying more than one nucleic acid in a single reaction mixture.
As used herein, the term “drill-down primer pair” refers to a primer pair designed to produce bioagent identifying amplicons for identification of sub-species characteristics or confirmation of a species assignment. For example, primer pair number 2146 (SEQ ID NOs: 437:1137), a drill-down Staphylococcus aureus genotyping primer pair, is designed to produce Staphylococcus aureus genotyping amplicons. Other drill-down primer pairs may be used to produce bioagent identifying amplicons for Staphylococcus aureus and other bacterial species.
The term “duplex” refers to the state of nucleic acids in which the base portions of the nucleotides on one strand are bound through hydrogen bonding the their complementary bases arrayed on a second strand. The condition of being in a duplex form reflects on the state of the bases of a nucleic acid. By virtue of base pairing, the strands of nucleic acid also generally assume the tertiary structure of a double helix, having a major and a minor groove. The assumption of the helical form is implicit in the act of becoming duplexed.
As used herein, the term “etiology” refers to the causes or origins, of diseases or abnormal physiological conditions.
The term “gene” refers to a DNA sequence that comprises control and coding sequences necessary for the production of an RNA having a non-coding function (e.g., a ribosomal or transfer RNA), a polypeptide or a precursor. The RNA or polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence so long as the desired activity or function is retained.
The terms “homology,” “homologous” and “sequence identity” refer to a degree of identity. There may be partial homology or complete homology. A partially homologous sequence is one that is less than 100% identical to another sequence. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is otherwise identical to another 20 nucleobase primer but having two non-identical residues has 18 of 20 identical residues (18/20=0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of a primer 20 nucleobases in length would have 15/20=0.75 or 75% sequence identity with the 20 nucleobase primer. In context of the present invention, sequence identity is meant to be properly determined when the query sequence and the subject sequence are both described and aligned in the 5′ to 3′ direction. Sequence alignment algorithms such as BLAST, will return results in two different alignment orientations. In the Plus/Plus orientation, both the query sequence and the subject sequence are aligned in the 5′ to 3′ direction. On the other hand, in the Plus/Minus orientation, the query sequence is in the 5′ to 3′ direction while the subject sequence is in the 3′ to 5′ direction. It should be understood that with respect to the primers of the present invention, sequence identity is properly determined when the alignment is designated as Plus/Plus. Sequence identity may also encompass alternate or modified nucleobases that perform in a functionally similar manner to the regular nucleobases adenine, thymine, guanine and cytosine with respect to hybridization and primer extension in amplification reactions. In a non-limiting example, if the 5-propynyl pyrimidines propyne C and/or propyne T replace one or more C or T residues in one primer which is otherwise identical to another primer in sequence and length, the two primers will have 100% sequence identity with each other. In another non-limiting example, Inosine (I) may be used as a replacement for G or T and effectively hybridize to C, A or U (uracil). Thus, if inosine replaces one or more C, A or U residues in one primer which is otherwise identical to another primer in sequence and length, the two primers will have 100% sequence identity with each other. Other such modified or universal bases may exist which would perform in a functionally similar manner for hybridization and amplification reactions and will be understood to fall within this definition of sequence identity.
As used herein, “housekeeping gene” refers to a gene encoding a protein or RNA involved in basic functions required for survival and reproduction of a bioagent. Housekeeping genes include, but are not limited to genes encoding RNA or proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like.
As used herein, the term “hybridization” is used in reference to the pairing of complementary nucleic acids. Hybridization and the strength of hybridization (i.e., the strength of the association between the nucleic acids) is influenced by such factors as the degree of complementary between the nucleic acids, stringency of the conditions involved, and the T_mof the formed hybrid. “Hybridization” methods involve the annealing of one nucleic acid to another, complementary nucleic acid, i.e., a nucleic acid having a complementary nucleotide sequence. The ability of two polymers of nucleic acid containing complementary sequences to find each other and anneal through base pairing interaction is a well-recognized phenomenon. The initial observations of the “hybridization” process by Marmur and Lane, Proc. Natl. Acad. Sci. USA 46:453 (1960) and Doty et al., Proc. Natl. Acad. Sci. USA 46:461 (1960) have been followed by the refinement of this process into an essential tool of modern biology.
The term “in silico” refers to processes taking place via computer calculations. For example, electronic PCR (ePCR) is a process analogous to ordinary PCR except that it is carried out using nucleic acid sequences and primer pair sequences stored on a computer formatted medium.
As used herein, “intelligent primers” are primers that are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and, upon amplification, yield amplification products which ideally provide enough variability to distinguish individual bioagents, and which are amenable to molecular mass analysis. By the term “highly conserved,” it is meant that the sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity among all, or at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of species or strains.
The “ligase chain reaction” (LCR; sometimes referred to as “Ligase Amplification Reaction” (LAR) described by Barany, Proc. Natl. Acad. Sci., 88:189 (1991); Barany, PCR Methods and Applic., 1:5 (1991); and Wu and Wallace, Genomics 4:560 (1989) has developed into a well-recognized alternative method for amplifying nucleic acids. In LCR, four oligonucleotides, two adjacent oligonucleotides which uniquely hybridize to one strand of target DNA, and a complementary set of adjacent oligonucleotides, that hybridize to the opposite strand are mixed and DNA ligase is added to the mixture. Provided that there is complete complementarity at the junction, ligase will covalently link each set of hybridized molecules. Importantly, in LCR, two probes are ligated together only when they base-pair with sequences in the target sample, without gaps or mismatches. Repeated cycles of denaturation, hybridization and ligation amplify a short segment of DNA. LCR has also been used in combination with PCR to achieve enhanced detection of single-base changes. However, because the four oligonucleotides used in this assay can pair to form two short ligatable fragments, there is the potential for the generation of target-independent background signal. The use of LCR for mutant screening is limited to the examination of specific nucleic acid positions.
The term “locked nucleic acid” or “LNA” refers to a nucleic acid analogue containing one or more 2′-O, 4′-C-methylene-o-D-ribofuranosyl nucleotide monomers in an RNA mimicking sugar conformation. LNA oligonucleotides display unprecedented hybridization affinity toward complementary single-stranded RNA and complementary single- or double-stranded DNA. LNA oligonucleotides induce A-type (RNA-like) duplex conformations. The primers of the present invention may contain LNA modifications.
As used herein, the term “mass-modifying tag” refers to any modification to a given nucleotide which results in an increase in mass relative to the analogous non-mass modified nucleotide. Mass-modifying tags can include heavy isotopes of one or more elements included in the nucleotide such as carbon-13 for example. Other possible modifications include addition of substituents such as iodine or bromine at the 5 position of the nucleobase for example.
The term “mass spectrometry” refers to measurement of the mass of atoms or molecules. The molecules are first converted to ions, which are separated using electric or magnetic fields according to the ratio of their mass to electric charge. The measured masses are used to identity the molecules.
The term “microorganism” as used herein means an organism too small to be observed with the unaided eye and includes, but is not limited to bacteria, virus, protozoans, fungi; and ciliates.
The term “multi-drug resistant” or multiple-drug resistant” refers to a microorganism which is resistant to more than one of the antibiotics or antimicrobial agents used in the treatment of said microorganism.
The term “multiplex PCR” refers to a PCR reaction where more than one primer set is included in the reaction pool allowing 2 or more different DNA targets to be amplified by PCR in a single reaction tube.
The term “non-template tag” refers to a stretch of at least three guanine or cytosine nucleobases of a primer used to produce a bioagent identifying amplicon which are not complementary to the template. A non-template tag is incorporated into a primer for the purpose of increasing the primer-duplex stability of later cycles of amplification by incorporation of extra G-C pairs which each have one additional hydrogen bond relative to an A-T pair.
The term “nucleic acid sequence” as used herein refers to the linear composition of the nucleic acid residues A, T, C or G or any modifications thereof, within an oligonucleotide, nucleotide or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single or double stranded, and represent the sense or antisense strand
As used herein, the term “nucleobase” is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).
The term “nucleotide analog” as used herein refers to modified or non-naturally occurring nucleotides such as 5-propynyl pyrimidines (i.e., 5-propynyl-dTTP and 5-propynyl-dTCP), 7-deaza purines (i.e., 7-deaza-dATP and 7-deaza-dGTP). Nucleotide analogs include base analogs and comprise modified forms of deoxyribonucleotides as well as ribonucleotides.
The term “oligonucleotide” as used herein is defined as a molecule comprising two or more deoxyribonucleotides or ribonucleotides, preferably at least 5 nucleotides, more preferably at least about 13 to 35 nucleotides. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotide may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. Because mononucleotides are reacted to make oligonucleotides in a manner such that the 5′ phosphate of one mononucleotide pentose ring is attached to the 3′ oxygen of its neighbor in one direction via a phosphodiester linkage, an end of an oligonucleotide is referred to as the “5′-end” if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentose ring and as the “3′-end” if its 3′ oxygen is not linked to a 5′ phosphate of a subsequent mononucleotide pentose ring. As used herein, a nucleic acid sequence, even if internal to a larger oligonucleotide, also may be said to have 5′ and 3′ ends. A first region along a nucleic acid strand is said to be upstream of another region if the 3′ end of the first region is before the 5′ end of the second region when moving along a strand of nucleic acid in a 5′ to 3′ direction. All oligonucleotide primers disclosed herein are understood to be presented in the 5′ to 3′ direction when reading left to right. When two different, non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3′ end of one oligonucleotide points towards the 5′ end of the other, the former may be called the “upstream” oligonucleotide and the latter the “downstream” oligonucleotide. Similarly, when two overlapping oligonucleotides are hybridized to the same linear complementary nucleic acid sequence, with the first oligonucleotide positioned such that its 5′ end is upstream of the 5′ end of the second oligonucleotide, and the 3′ end of the first oligonucleotide is upstream of the 3′ end of the second oligonucleotide, the first oligonucleotide may be called the “upstream” oligonucleotide and the second oligonucleotide may be called the “downstream” oligonucleotide.
In the context of this invention, a “pathogen” is a bioagent which causes a disease or disorder.
As used herein, the terms “PCR product,” “PCR fragment,” and “amplification product” refer to the resultant mixture of compounds after two or more cycles of the PCR steps of denaturation, annealing and extension are complete. These terms encompass the case where there has been amplification of one or more segments of one or more target sequences.
The term “peptide nucleic acid” (“PNA”) as used herein refers to a molecule comprising bases or base analogs such as would be found in natural nucleic acid, but attached to a peptide backbone rather than the sugar-phosphate backbone typical of nucleic acids. The attachment of the bases to the peptide is such as to allow the bases to base pair with complementary bases of nucleic acid in a manner similar to that of an oligonucleotide. These small molecules, also designated anti gene agents, stop transcript elongation by binding to their complementary strand of nucleic acid (Nielsen, et al. Anticancer Drug Des. 8:53 63). The primers of the present invention may comprise PNAs.
The term “polymerase” refers to an enzyme having the ability to synthesize a complementary strand of nucleic acid from a starting template nucleic acid strand and free dNTPs.
As used herein, the term “polymerase chain reaction” (“PCR”) refers to the method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, hereby incorporated by reference, that describe a method for increasing the concentration of a segment of a target sequence in a mixture of genomic DNA without cloning or purification. This process for amplifying the target sequence consists of introducing a large excess of two oligonucleotide primers to the DNA mixture containing the desired target sequence, followed by a precise sequence of thermal cycling in the presence of a DNA polymerase. The two primers are complementary to their respective strands of the double stranded target sequence. To effect amplification, the mixture is denatured and the primers then annealed to their complementary sequences within the target molecule. Following annealing, the primers are extended with a polymerase so as to form a new pair of complementary strands. The steps of denaturation, primer annealing, and polymerase extension can be repeated many times (i.e., denaturation, annealing and extension constitute one “cycle”; there can be numerous “cycles”) to obtain a high concentration of an amplified segment of the desired target sequence. The length of the amplified segment of the desired target sequence is determined by the relative positions of the primers with respect to each other, and therefore, this length is a controllable parameter. By virtue of the repeating aspect of the process, the method is referred to as the “polymerase chain reaction” (hereinafter “PCR”). Because the desired amplified segments of the target sequence become the predominant sequences (in terms of concentration) in the mixture, they are said to be “PCR amplified.” With PCR, it is possible to amplify a single copy of a specific target sequence in genomic DNA to a level detectable by several different methodologies (e.g., hybridization with a labeled probe; incorporation of biotinylated primers followed by avidin-enzyme conjugate detection; incorporation of 32P-labeled deoxynucleotide triphosphates, such as dCTP or dATP, into the amplified segment). In addition to genomic DNA, any oligonucleotide or polynucleotide sequence can be amplified with the appropriate set of primer molecules. In particular, the amplified segments created by the PCR process itself are, themselves, efficient templates for subsequent PCR amplifications.
The term “polymerization means” or “polymerization agent” refers to any agent capable of facilitating the addition of nucleoside triphosphates to an oligonucleotide. Preferred polymerization means comprise DNA and RNA polymerases.
As used herein, the terms “pair of primers,” or “primer pair” are synonymous. A primer pair is used for amplification of a nucleic acid sequence. A pair of primers comprises a forward primer and a reverse primer. The forward primer hybridizes to a sense strand of a target gene sequence to be amplified and primes synthesis of an antisense strand (complementary to the sense strand) using the target sequence as a template. A reverse primer hybridizes to the antisense strand of a target gene sequence to be amplified and primes synthesis of a sense strand (complementary to the antisense strand) using the target sequence as a template.
The primers are designed to bind to highly conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which ideally provide enough variability to distinguish each individual bioagent, and which are amenable to molecular mass analysis. In some embodiments, the highly conserved sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity, or between about 99-100% identity. The molecular mass of a given amplification product provides a means of identifying the bioagent from which it was obtained, due to the variability of the variable region. Thus design of the primers requires selection of a variable region with appropriate variability to resolve the identity of a given bioagent. Bioagent identifying amplicons are ideally specific to the identity of the bioagent.
Properties of the primers may include any number of properties related to structure including, but not limited to: nucleobase length which may be contiguous (linked together) or non-contiguous (for example, two or more contiguous segments which are joined by a linker or loop moiety), modified or universal nucleobases (used for specific purposes such as for example, increasing hybridization affinity, preventing non-templated adenylation and modifying molecular mass) percent complementarity to a given target sequences.
Properties of the primers also include functional features including, but not limited to, orientation of hybridization (forward or reverse) relative to a nucleic acid template. The coding or sense strand is the strand to which the forward priming primer hybridizes (forward priming orientation) while the reverse priming primer hybridizes to the non-coding or antisense strand (reverse priming orientation). The functional properties of a given primer pair also include the generic template nucleic acid to which the primer pair hybridizes. For example, identification of bioagents can be accomplished at different levels using primers suited to resolution of each individual level of identification. Broad range survey primers are designed with the objective of identifying a bioagent as a member of a particular division (e.g., an order, family, genus or other such grouping of bioagents above the species level of bioagents). In some embodiments, broad range survey intelligent primers are capable of identification of bioagents at the species or sub-species level. Other primers may have the functionality of producing bioagent identifying amplicons for members of a given taxonomic genus, clade, species, sub-species or genotype (including genetic variants which may include presence of virulence genes or antibiotic resistance genes or mutations). Additional functional properties of primer pairs include the functionality of performing amplification either singly (single primer pair per amplification reaction vessel) or in a multiplex fashion (multiple primer pairs and multiple amplification reactions within a single reaction vessel).
As used herein, the terms “purified” or “substantially purified” refer to molecules, either nucleic or amino acid sequences, that are removed from their natural environment, isolated or separated, and are at least 60% free, preferably 75% free, and most preferably 90% free from other components with which they are naturally associated. An “isolated polynucleotide” or “isolated oligonucleotide” is therefore a substantially purified polynucleotide.
The term “reverse transcriptase” refers to an enzyme having the ability to transcribe DNA from an RNA template. This enzymatic activity is known as reverse transcriptase activity. Reverse transcriptase activity is desirable in order to obtain DNA from RNA viruses which can then be amplified and analyzed by the methods of the present invention.
The term “ribosomal RNA” or “rRNA” refers to the primary ribonucleic acid constituent of ribosomes. Ribosomes are the protein-manufacturing organelles of cells and exist in the cytoplasm. Ribosomal RNAs are transcribed from the DNA genes encoding them.
The term “sample” in the present specification and claims is used in its broadest sense. On the one hand it is meant to include a specimen or culture (e.g., microbiological cultures). On the other hand, it is meant to include both biological and environmental samples. A sample may include a specimen of synthetic origin. Biological samples may be animal, including human, fluid, solid (e.g., stool) or tissue, as well as liquid and solid food and feed products and ingredients such as dairy items, vegetables, meat and meat by-products, and waste. Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc. Environmental samples include environmental material such as surface matter, soil, water, air and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, utensils, disposable and non-disposable items. These examples are not to be construed as limiting the sample types applicable to the present invention. The term “source of target nucleic acid” refers to any sample that contains nucleic acids (RNA or DNA). Particularly preferred sources of target nucleic acids are biological samples including, but not limited to blood, saliva, cerebral spinal fluid, pleural fluid, milk, lymph, sputum and semen.
As used herein, the term “sample template” refers to nucleic acid originating from a sample that is analyzed for the presence of “target” (defined below). In contrast, “background template” is used in reference to nucleic acid other than sample template that may or may not be present in a sample. Background template is often a contaminant. It may be the result of carryover, or it may be due to the presence of nucleic acid contaminants sought to be purified away from the sample. For example, nucleic acids from organisms other than those to be detected may be present as background in a test sample.
A “segment” is defined herein as a region of nucleic acid within a target sequence.
The “self-sustained sequence replication reaction” (3SR) (Guatelli et al., Proc. Natl. Acad. Sci., 87:1874-1878 [1990], with an erratum at Proc. Natl. Acad. Sci., 87:7797 [1990]) is a transcription-based in vitro amplification system (Kwok et al., Proc. Natl. Acad. Sci., 86:1173-1177 [1989]) that can exponentially amplify RNA sequences at a uniform temperature. The amplified RNA can then be utilized for mutation detection (Fahy et al., PCR Meth. Appl., 1:25-33 [1991]). In this method, an oligonucleotide primer is used to add a phage RNA polymerase promoter to the 5′ end of the sequence of interest. In a cocktail of enzymes and substrates that includes a second primer, reverse transcriptase, RNase H, RNA polymerase and ribo- and deoxyribonucleoside triphosphates, the target sequence undergoes repeated rounds of transcription, cDNA synthesis and second-strand synthesis to amplify the area of interest. The use of 3SR to detect mutations is kinetically limited to screening small segments of DNA (e.g., 200-300 base pairs).
As used herein, the term ““sequence alignment”” refers to a listing of multiple DNA or amino acid sequences and aligns them to highlight their similarities. The listings can be made using bioinformatics computer programs.
In context of this invention, the term “speciating primer pair” refers to a primer pair designed to produce a bioagent identifying amplicon with the diagnostic capability of identifying species members of a group of genera or a particular genus of bioagents. Primer pair number 2249 (SEQ ID NOs: 430:1321), for example, is a speciating primer pair used to distinguish Staphylococcus aureus from other species of the genus Staphylococcus.
As used herein, a “sub-species characteristic” is a genetic characteristic that provides the means to distinguish two members of the same bioagent species. For example, one viral strain could be distinguished from another viral strain of the same species by possessing a genetic change (e.g., for example, a nucleotide deletion, addition or substitution) in one of the viral genes, such as the RNA-dependent RNA polymerase. Sub-species characteristics such as virulence genes and drug—are responsible for the phenotypic differences among the different strains of bacteria.
As used herein, the term “target” is used in a broad sense to indicate the gene or genomic region being amplified by the primers. Because the present invention provides a plurality of amplification products from any given primer pair (depending on the bioagent being analyzed), multiple amplification products from different specific nucleic acid sequences may be obtained. Thus, the term “target” is not used to refer to a single specific nucleic acid sequence. The “target” is sought to be sorted out from other nucleic acid sequences and contains a sequence that has at least partial complementarity with an oligonucleotide primer. The target nucleic acid may comprise single- or double-stranded DNA or RNA. A “segment” is defined as a region of nucleic acid within the target sequence.
The term “template” refers to a strand of nucleic acid on which a complementary copy is built from nucleoside triphosphates through the activity of a template-dependent nucleic acid polymerase. Within a duplex the template strand is, by convention, depicted and described as the “bottom” strand. Similarly, the non-template strand is often depicted and described as the “top” strand.
As used herein, the term “T_m” is used in reference to the “melting temperature.” The melting temperature is the temperature at which a population of double-stranded nucleic acid molecules becomes half dissociated into single strands. Several equations for calculating the T_mof nucleic acids are well known in the art. As indicated by standard references, a simple estimate of the T_mvalue may be calculated by the equation: T_m=81.5+0.41(% G+C), when a nucleic acid is in aqueous solution at 1 M NaCl (see e.g., Anderson and Young, Quantitative Filter Hybridization, in Nucleic Acid Hybridization (1985). Other references (e.g., Allawi, H. T. & SantaLucia, J., Jr. Thermodynamics and NMR of internal G.T mismatches in DNA. Biochemistry 36, 10581-94 (1997) include more sophisticated computations which take structural and environmental, as well as sequence characteristics into account for the calculation of T_m.
The term “triangulation genotyping analysis” refers to a method of genotyping a bioagent by measurement of molecular masses or base compositions of amplification products, corresponding to bioagent identifying amplicons, obtained by amplification of regions of more than one gene. In this sense, the term “triangulation” refers to a method of establishing the accuracy of information by comparing three or more types of independent points of view bearing on the same findings. Triangulation genotyping analysis carried out with a plurality of triangulation genotyping analysis primers yields a plurality of base compositions that then provide a pattern or “barcode” from which a species type can be assigned. The species type may represent a previously known sub-species or strain, or may be a previously unknown strain having a specific and previously unobserved base composition barcode indicating the existence of a previously unknown genotype.
As used herein, the term “triangulation genotyping analysis primer pair” is a primer pair designed to produce bioagent identifying amplicons for determining species types in a triangulation genotyping analysis.
The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as “triangulation identification.” Triangulation identification is pursued by analyzing a plurality of bioagent identifying amplicons produced with different primer pairs. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
In the context of this invention, the term “unknown bioagent” may mean either: (i) a bioagent whose existence is known (such as the well known bacterial species Staphylococcus aureus for example) but which is not known to be in a sample to be analyzed, or (ii) a bioagent whose existence is not known (for example, the SARS coronavirus was unknown prior to April 2003). For example, if the method for identification of coronaviruses disclosed in commonly owned U.S. patent Ser. No. 10/829,826 (incorporated herein by reference in its entirety) was to be employed prior to April 2003 to identify the SARS coronavirus in a clinical sample, both meanings of “unknown” bioagent are applicable since the SARS coronavirus was unknown to science prior to April, 2003 and since it was not known what bioagent (in this case a coronavirus) was present in the sample. On the other hand, if the method of U.S. patent Ser. No. 10/829,826 was to be employed subsequent to April 2003 to identify the SARS coronavirus in a clinical sample, only the first meaning (i) of “unknown” bioagent would apply since the SARS coronavirus became known to science subsequent to April 2003 and since it was not known what bioagent was present in the sample.
The term “variable sequence” as used herein refers to differences in nucleic acid sequence between two nucleic acids. For example, the genes of two different bacterial species may vary in sequence by the presence of single base substitutions and/or deletions or insertions of one or more nucleotides. These two forms of the structural gene are said to vary in sequence from one another. In the context of the present invention, “viral nucleic acid” includes, but is not limited to, DNA, RNA, or DNA that has been obtained from viral RNA, such as, for example, by performing a reverse transcription reaction. Viral RNA can either be single-stranded (of positive or negative polarity) or double-stranded.
The term “virus” refers to obligate, ultramicroscopic, parasites that are incapable of autonomous replication (i.e., replication requires the use of the host cell's machinery). Viruses can survive outside of a host cell but cannot replicate.
The term “wild-type” refers to a gene or a gene product that has the characteristics of that gene or gene product when isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designated the “normal” or “wild-type” form of the gene. In contrast, the term “modified”, “mutant” or “polymorphic” refers to a gene or gene product that displays modifications in sequence and or functional properties (i.e., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally-occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
As used herein, a “wobble base” is a variation in a codon found at the third nucleotide position of a DNA triplet. Variations in conserved regions of sequence are often found at the third nucleotide position due to redundancy in the amino acid code.

DETAILED DESCRIPTION OF EMBODIMENTS

A. Bioagent Identifying Amplicons
The present invention provides methods for detection and identification of unknown bioagents using bioagent identifying amplicons. Primers are selected to hybridize to conserved sequence regions of nucleic acids derived from a bioagent, and which bracket variable sequence regions to yield a bioagent identifying amplicon, which can be amplified and which is amenable to molecular mass determination. The molecular mass then provides a means to uniquely identify the bioagent without a requirement for prior knowledge of the possible identity of the bioagent. The molecular mass or corresponding base composition signature of the amplification product is then matched against a database of molecular masses or base composition signatures. A match is obtained when an experimentally-determined molecular mass or base composition of an analyzed amplification product is compared with known molecular masses or base compositions of known bioagent identifying amplicons and the experimentally determined molecular mass or base composition is the same as the molecular mass or base composition of one of the known bioagent identifying amplicons. Alternatively, the experimentally-determined molecular mass or base composition may be within experimental error of the molecular mass or base composition of a known bioagent identifying amplicon and still be classified as a match. In some cases, the match may also be classified using a probability of match model such as the models described in U.S. Ser. No. 11/073,362, which is commonly owned and incorporated herein by reference in entirety. Furthermore, the method can be applied to rapid parallel multiplex analyses, the results of which can be employed in a triangulation identification strategy. The present method provides rapid throughput and does not require nucleic acid sequencing of the amplified target sequence for bioagent detection and identification.
Despite enormous biological diversity, all forms of life on earth share sets of essential, common features in their genomes. Since genetic data provide the underlying basis for identification of bioagents by the methods of the present invention, it is necessary to select segments of nucleic acids which ideally provide enough variability to distinguish each individual bioagent and whose molecular mass is amenable to molecular mass determination.
Unlike bacterial genomes, which exhibit conservation of numerous genes (i.e. housekeeping genes) across all organisms, viruses do not share a gene that is essential and conserved among all virus families. Therefore, viral identification is achieved within smaller groups of related viruses, such as members of a particular virus family or genus. For example, RNA-dependent RNA polymerase is present in all single-stranded RNA viruses and can be used for broad priming as well as resolution within the virus family.
In some embodiments of the present invention, at least one bacterial nucleic acid segment is amplified in the process of identifying the bacterial bioagent. Thus, the nucleic acid segments that can be amplified by the primers disclosed herein and that provide enough variability to distinguish each individual bioagent and whose molecular masses are amenable to molecular mass determination are herein described as bioagent identifying amplicons.
In some embodiments of the present invention, bioagent identifying amplicons comprise from about 45 to about 150 nucleobases (i.e. from about 45 to about 200 linked nucleosides), although both longer and short regions may be used. One of ordinary skill in the art will appreciate that the invention embodies compounds of 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, and 150 nucleobases in length, or any range therewithin.
It is the combination of the portions of the bioagent nucleic acid segment to which the primers hybridize (hybridization sites) and the variable region between the primer hybridization sites that comprises the bioagent identifying amplicon. Thus, it can be said that a given bioagent identifying amplicon is “defined by” a given pair of primers.
In some embodiments, bioagent identifying amplicons amenable to molecular mass determination which are produced by the primers described herein are either of a length, size or mass compatible with the particular mode of molecular mass determination or compatible with a means of providing a predictable fragmentation pattern in order to obtain predictable fragments of a length compatible with the particular mode of molecular mass determination. Such means of providing a predictable fragmentation pattern of an amplification product include, but are not limited to, cleavage with chemical reagents, restriction enzymes or cleavage primers, for example. Thus, in some embodiments, bioagent identifying amplicons are larger than 150 nucleobases and are amenable to molecular mass determination following restriction digestion. Methods of using restriction enzymes and cleavage primers are well known to those with ordinary skill in the art.
In some embodiments, amplification products corresponding to bioagent identifying amplicons are obtained using the polymerase chain reaction (PCR) that is a routine method to those with ordinary skill in the molecular biology arts. Other amplification methods may be used such as ligase chain reaction (LCR), low-stringency single primer PCR, and multiple strand displacement amplification (MDA). These methods are also known to those with ordinary skill.
B. Primers and Primer Pairs
In some embodiments, the primers are designed to bind to conserved sequence regions of a bioagent identifying amplicon that flank an intervening variable region and yield amplification products which provide variability sufficient to distinguish each individual bioagent, and which are amenable to molecular mass analysis. In some embodiments, the highly conserved sequence regions exhibit between about 80-100%, or between about 90-100%, or between about 95-100% identity, or between about 99-100% identity. The molecular mass of a given amplification product provides a means of identifying the bioagent from which it was obtained, due to the variability of the variable region. Thus, design of the primers involves selection of a variable region with sufficient variability to resolve the identity of a given bioagent. In some embodiments, bioagent identifying amplicons are specific to the identity of the bioagent.
In some embodiments, identification of bioagents is accomplished at different levels using primers suited to resolution of each individual level of identification. Broad range survey primers are designed with the objective of identifying a bioagent as a member of a particular division (e.g., an order, family, genus or other such grouping of bioagents above the species level of bioagents). In some embodiments, broad range survey intelligent primers are capable of identification of bioagents at the species or sub-species level. Examples of broad range survey primers include, but are not limited to: primer pair numbers: 346 (SEQ ID NOs: 202:1110), 347 (SEQ ID NOs: 560:1278), 348 SEQ ID NOs: 706:895), and 361 (SEQ ID NOs: 697:1398) which target DNA encoding 16S rRNA, and primer pair numbers 349 (SEQ ID NOs: 401:1156) and 360 (SEQ ID NOs: 409:1434) which target DNA encoding 23S rRNA.
In some embodiments, drill-down primers are designed with the objective of identifying a bioagent at the sub-species level (including strains, subtypes, variants and isolates) based on sub-species characteristics which may, for example, include single nucleotide polymorphisms (SNPs), variable number tandem repeats (VNTRs), deletions, drug resistance mutations or any other modification of a nucleic acid sequence of a bioagent relative to other members of a species having different sub-species characteristics. Drill-down intelligent primers are not always required for identification at the sub-species level because broad range survey intelligent primers may, in some cases provide sufficient identification resolution to accomplishing this identification objective. Examples of drill-down primers include, but are not limited to: confirmation primer pairs such as primer pair numbers 351 (SEQ ID NOs: 355:1423) and 353 (SEQ ID NOs: 220:1394), which target the pX01 virulence plasmid of Bacillus anthracis. Other examples of drill-down primer pairs are found in sets of triangulation genotyping primer pairs such as, for example, the primer pair number 2146 (SEQ ID NOs: 437:1137) which targets the arcC gene (encoding carmabate kinase) and is included in an 8 primer pair panel or kit for use in genotyping Staphylococcus aureus, or in other panels or kits of primer pairs used for determining drug-resistant bacterial strains, such as, for example, primer pair number 2095 (SEQ ID NOs: 456:1261) which targets the pv-luk gene (encoding Panton-Valentine leukocidin) and is included in an 8 primer pair panel or kit for use in identification of drug resistant strains of Staphylococcus aureus.
A representative process flow diagram used for primer selection and validation process is outlined in FIG. 1. For each group of organisms, candidate target sequences are identified (200) from which nucleotide alignments are created (210) and analyzed (220). Primers are then designed by selecting appropriate priming regions (230) to facilitate the selection of candidate primer pairs (240). The primer pairs are then subjected to in silico analysis by electronic PCR (ePCR) (300) wherein bioagent identifying amplicons are obtained from sequence databases such as GenBank or other sequence collections (310) and checked for specificity in silico (320). Bioagent identifying amplicons obtained from GenBank sequences (310) can also be analyzed by a probability model which predicts the capability of a given amplicon to identify unknown bioagents such that the base compositions of amplicons with favorable probability scores are then stored in a base composition database (325). Alternatively, base compositions of the bioagent identifying amplicons obtained from the primers and GenBank sequences can be directly entered into the base composition database (330). Candidate primer pairs (240) are validated by testing their ability to hybridize to target nucleic acid by an in vitro amplification by a method such as PCR analysis (400) of nucleic acid from a collection of organisms (410). Amplification products thus obtained are analyzed by gel electrophoresis or by mass spectrometry to confirm the sensitivity, specificity and reproducibility of the primers used to obtain the amplification products (420).
Many of the important pathogens, including the organisms of greatest concern as biowarfare agents, have been completely sequenced. This effort has greatly facilitated the design of primers for the detection of unknown bioagents. The combination of broad-range priming with division-wide and drill-down priming has been used very successfully in several applications of the technology, including environmental surveillance for biowarfare threat agents and clinical sample analysis for medically important pathogens.
Synthesis of primers is well known and routine in the art. The primers may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed.
In some embodiments primers are employed as compositions for use in methods for identification of bacterial bioagents as follows: a primer pair composition is contacted with nucleic acid (such as, for example, bacterial DNA or DNA reverse transcribed from the rRNA) of an unknown bacterial bioagent. The nucleic acid is then amplified by a nucleic acid amplification technique, such as PCR for example, to obtain an amplification product that represents a bioagent identifying amplicon. The molecular mass of each strand of the double-stranded amplification product is determined by a molecular mass measurement technique such as mass spectrometry for example, wherein the two strands of the double-stranded amplification product are separated during the ionization process. In some embodiments, the mass spectrometry is electrospray Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) or electrospray time of flight mass spectrometry (ESI-TOF-MS). A list of possible base compositions can be generated for the molecular mass value obtained for each strand and the choice of the correct base composition from the list is facilitated by matching the base composition of one strand with a complementary base composition of the other strand. The molecular mass or base composition thus determined is then compared with a database of molecular masses or base compositions of analogous bioagent identifying amplicons for known viral bioagents. A match between the molecular mass or base composition of the amplification product and the molecular mass or base composition of an analogous bioagent identifying amplicon for a known viral bioagent indicates the identity of the unknown bioagent. In some embodiments, the primer pair used is one of the primer pairs of Table 2. In some embodiments, the method is repeated using one or more different primer pairs to resolve possible ambiguities in the identification process or to improve the confidence level for the identification assignment.
In some embodiments, a bioagent identifying amplicon may be produced using only a single primer (either the forward or reverse primer of any given primer pair), provided an appropriate amplification method is chosen, such as, for example, low stringency single primer PCR (LSSP-PCR). Adaptation of this amplification method in order to produce bioagent identifying amplicons can be accomplished by one with ordinary skill in the art without undue experimentation.
In some embodiments, the oligonucleotide primers are broad range survey primers which hybridize to conserved regions of nucleic acid encoding the hexon gene of all (or between 80% and 100%, between 85% and 100%, between 90% and 100% or between 95% and 100%) known bacteria and produce bacterial bioagent identifying amplicons.
In some cases, the molecular mass or base composition of a bacterial bioagent identifying amplicon defined by a broad range survey primer pair does not provide enough resolution to unambiguously identify a bacterial bioagent at or below the species level. These cases benefit from further analysis of one or more bacterial bioagent identifying amplicons generated from at least one additional broad range survey primer pair or from at least one additional division-wide primer pair. The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as triangulation identification.
In other embodiments, the oligonucleotide primers are division-wide primers which hybridize to nucleic acid encoding genes of species within a genus of bacteria. In other embodiments, the oligonucleotide primers are drill-down primers which enable the identification of sub-species characteristics. Drill down primers provide the functionality of producing bioagent identifying amplicons for drill-down analyses such as strain typing when contacted with nucleic acid under amplification conditions. Identification of such sub-species characteristics is often critical for determining proper clinical treatment of viral infections. In some embodiments, sub-species characteristics are identified using only broad range survey primers and division-wide and drill-down primers are not used.
In some embodiments, the primers used for amplification hybridize to and amplify genomic DNA, and DNA of bacterial plasmids.
In some embodiments, various computer software programs may be used to aid in design of primers for amplification reactions such as Primer Premier 5 (Premier Biosoft, Palo Alto, Calif.) or OLIGO Primer Analysis Software (Molecular Biology Insights, Cascade, Colo.). These programs allow the user to input desired hybridization conditions such as melting temperature of a primer-template duplex for example. In some embodiments, an in silico PCR search algorithm, such as (ePCR) is used to analyze primer specificity across a plurality of template sequences which can be readily obtained from public sequence databases such as GenBank for example. An existing RNA structure search algorithm (Macke et al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs. In some embodiments, the hybridization conditions applied to the algorithm can limit the results of primer specificity obtained from the algorithm. In some embodiments, the melting temperature threshold for the primer template duplex is specified to be 35° C. or a higher temperature. In some embodiments the number of acceptable mismatches is specified to be seven mismatches or less. In some embodiments, the buffer components and concentrations and primer concentrations may be specified and incorporated into the algorithm, for example, an appropriate primer concentration is about 250 nM and appropriate buffer components are 50 mM sodium or potassium and 1.5 mM Mg²⁺.
One with ordinary skill in the art of design of amplification primers will recognize that a given primer need not hybridize with 100% complementarity in order to effectively prime the synthesis of a complementary nucleic acid strand in an amplification reaction. Moreover, a primer may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event. (e.g., for example, a loop structure or a hairpin structure). The primers of the present invention may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence identity with any of the primers listed in Table 2. Thus, in some embodiments of the present invention, an extent of variation of 70% to 100%, or any range therewithin, of the sequence identity is possible relative to the specific primer sequences disclosed herein. Determination of sequence identity is described in the following example: a primer 20 nucleobases in length which is identical to another 20 nucleobase primer having two non-identical residues has 18 of 20 identical residues (18/20=0.9 or 90% sequence identity). In another example, a primer 15 nucleobases in length having all residues identical to a 15 nucleobase segment of primer 20 nucleobases in length would have 15/20=0.75 or 75% sequence identity with the 20 nucleobase primer.
Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for UNIX, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). In some embodiments, complementarity of primers with respect to the conserved priming regions of viral nucleic acid is between about 70% and about 75% 80%. In other embodiments, homology, sequence identity or complementarity, is between about 75% and about 80%. In yet other embodiments, homology, sequence identity or complementarity, is at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or is 100%.
In some embodiments, the primers described herein comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or at least 99%, or 100% (or any range therewithin) sequence identity with the primer sequences specifically disclosed herein.
One with ordinary skill is able to calculate percent sequence identity or percent sequence homology and able to determine, without undue experimentation, the effects of variation of primer sequence identity on the function of the primer in its role in priming synthesis of a complementary strand of nucleic acid for production of an amplification product of a corresponding bioagent identifying amplicon.
In one embodiment, the primers are at least 13 nucleobases in length. In another embodiment, the primers are less than 36 nucleobases in length.
In some embodiments of the present invention, the oligonucleotide primers are 13 to 35 nucleobases in length (13 to 35 linked nucleotide residues). These embodiments comprise oligonucleotide primers 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleobases in length, or any range therewithin. The present invention contemplates using both longer and shorter primers. Furthermore, the primers may also be linked to one or more other desired moieties, including, but not limited to, affinity groups, ligands, regions of nucleic acid that are not complementary to the nucleic acid to be amplified, labels, etc. Primers may also form hairpin structures. For example, hairpin primers may be used to amplify short target nucleic acid molecules. The presence of the hairpin may stabilize the amplification complex (see e.g., TAQMAN MicroRNA Assays, Applied Biosystems, Foster City, Calif.).
In some embodiments, any oligonucleotide primer pair may have one or both primers with less then 70% sequence homology with a corresponding member of any of the primer pairs of Table 2 if the primer pair has the capability of producing an amplification product corresponding to a bioagent identifying amplicon. In other embodiments, any oligonucleotide primer pair may have one or both primers with a length greater than 35 nucleobases if the primer pair has the capability of producing an amplification product corresponding to a bioagent identifying amplicon.
In some embodiments, the function of a given primer may be substituted by a combination of two or more primers segments that hybridize adjacent to each other or that are linked by a nucleic acid loop structure or linker which allows a polymerase to extend the two or more primers in an amplification reaction.
In some embodiments, the primer pairs used for obtaining bioagent identifying amplicons are the primer pairs of Table 2. In other embodiments, other combinations of primer pairs are possible by combining certain members of the forward primers with certain members of the reverse primers. An example can be seen in Table 2 for two primer pair combinations of forward primer 16S_EC_—789_—810_F (SEQ ID NO:206), with the reverse primers 16S_EC_—880_—894_R (SEQ ID NO: 796), or 16S_EC_—882_—899_R or (SEQ ID NO: 818). Arriving at a favorable alternate combination of primers in a primer pair depends upon the properties of the primer pair, most notably the size of the bioagent identifying amplicon that would be produced by the primer pair, which preferably is between about 45 to about 150 nucleobases in length. Alternatively, a bioagent identifying amplicon longer than 150 nucleobases in length could be cleaved into smaller segments by cleavage reagents such as chemical reagents, or restriction enzymes, for example.
In some embodiments, the primers are configured to amplify nucleic acid of a bioagent to produce amplification products that can be measured by mass spectrometry and from whose molecular masses candidate base compositions can be readily calculated.
In some embodiments, any given primer comprises a modification comprising the addition of a non-templated T residue to the 5′ end of the primer (i.e., the added T residue does not necessarily hybridize to the nucleic acid being amplified). The addition of a non-templated T residue has an effect of minimizing the addition of non-templated adenosine residues as a result of the non-specific enzyme activity of Taq polymerase (Magnuson et al., Biotechniques, 1996, 21, 700-709), an occurrence which may lead to ambiguous results arising from molecular mass analysis.
In some embodiments of the present invention, primers may contain one or more universal bases. Because any variation (due to codon wobble in the 3^rdposition) in the conserved regions among species is likely to occur in the third position of a DNA (or RNA) triplet, oligonucleotide primers can be designed such that the nucleotide corresponding to this position is a base which can bind to more than one nucleotide, referred to herein as a “universal nucleobase.” For example, under this “wobble” pairing, inosine (I) binds to U, C or A; guanine (G) binds to U or C, and uridine (U) binds to U or C. Other examples of universal nucleobases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides, 1995, 14, 1001-1003), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides, 1995, 14, 1053-1056) or the purine analog 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res., 1996, 24, 3302-3306).
In some embodiments, to compensate for the somewhat weaker binding by the wobble base, the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs that bind with greater affinity than the unmodified nucleotide. Examples of these analogs include, but are not limited to, 2,6-diaminopurine which binds to thymine, 5-propynyluracil (also known as propynylated thymine) which binds to adenine and 5-propynylcytosine and phenoxazines, including G-clamp, which binds to G. Propynylated pyrimidines are described in U.S. Pat. Nos. 5,645,985, 5,830,653 and 5,484,908, each of which is commonly owned and incorporated herein by reference in its entirety. Propynylated primers are described in U.S Pre-Grant Publication No. 2003-0170682, which is also commonly owned and incorporated herein by reference in its entirety. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, each of which is incorporated herein by reference in its entirety. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, each of which is incorporated herein by reference in its entirety.
In some embodiments, primer hybridization is enhanced using primers containing 5-propynyl deoxy-cytidine and deoxy-thymidine nucleotides. These modified primers offer increased affinity and base pairing selectivity.
In some embodiments, non-template primer tags are used to increase the melting temperature (T_m) of a primer-template duplex in order to improve amplification efficiency. A non-template tag is at least three consecutive A or T nucleotide residues on a primer which are not complementary to the template. In any given non-template tag, A can be replaced by C or G and T can also be replaced by C or G. Although Watson-Crick hybridization is not expected to occur for a non-template tag relative to the template, the extra hydrogen bond in a G-C pair relative to an A-T pair confers increased stability of the primer-template duplex and improves amplification efficiency for subsequent cycles of amplification when the primers hybridize to strands synthesized in previous cycles.
In other embodiments, propynylated tags may be used in a manner similar to that of the non-template tag, wherein two or more 5-propynylcytidine or 5-propynyluridine residues replace template matching residues on a primer. In other embodiments, a primer contains a modified internucleoside linkage such as a phosphorothioate linkage, for example.
In some embodiments, the primers contain mass-modifying tags. Reducing the total number of possible base compositions of a nucleic acid of specific molecular weight provides a means of avoiding a persistent source of ambiguity in determination of base composition of amplification products. Addition of mass-modifying tags to certain nucleobases of a given primer will result in simplification of de novo determination of base composition of a given bioagent identifying amplicon from its molecular mass.
In some embodiments of the present invention, the mass modified nucleobase comprises one or more of the following: for example, 7-deaza-2′-deoxyadenosine-5-triphosphate, 5-iodo-2′-deoxyuridine-5′-triphosphate, 5-bromo-2′-deoxyuridine-5′-triphosphate, 5-bromo-2′-deoxycytidine-5′-triphosphate, 5-iodo-2′-deoxycytidine-5′-triphosphate, 5-hydroxy-2′-deoxyuridine-5′-triphosphate, 4-thiothymidine-5′-triphosphate, 5-aza-2′-deoxyuridine-5′-triphosphate, 5-fluoro-2′-deoxyuridine-5′-triphosphate, O6-methyl-2′-deoxyguanosine-5′-triphosphate, N2-methyl-2′-deoxyguanosine-5′-triphosphate, 8-oxo-2′-deoxyguanosine-5′-triphosphate or thiothymidine-5′-triphosphate. In some embodiments, the mass-modified nucleobase comprises ¹⁵N or ¹³C or both ¹⁵N and ¹³C.
In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with a plurality of primer pairs. The advantages of multiplexing are that fewer reaction containers (for example, wells of a 96- or 384-well plate) are needed for each molecular mass measurement, providing time, resource and cost savings because additional bioagent identification data can be obtained within a single analysis. Multiplex amplification methods are well known to those with ordinary skill and can be developed without undue experimentation. However, in some embodiments, one useful and non-obvious step in selecting a plurality candidate bioagent identifying amplicons for multiplex amplification is to ensure that each strand of each amplification product will be sufficiently different in molecular mass that mass spectral signals will not overlap and lead to ambiguous analysis results. In some embodiments, a 10 Da difference in mass of two strands of one or more amplification products is sufficient to avoid overlap of mass spectral peaks.
In some embodiments, as an alternative to multiplex amplification, single amplification reactions can be pooled before analysis by mass spectrometry. In these embodiments, as for multiplex amplification embodiments, it is useful to select a plurality of candidate bioagent identifying amplicons to ensure that each strand of each amplification product will be sufficiently different in molecular mass that mass spectral signals will not overlap and lead to ambiguous analysis results.
C Determination of Molecular Mass of Bioagent Identifying Amplicons
In some embodiments, the molecular mass of a given bioagent identifying amplicon is determined by mass spectrometry. Mass spectrometry has several advantages, not the least of which is high bandwidth characterized by the ability to separate (and isolate) many molecular peaks across a broad range of mass to charge ratio (m/z). Thus mass spectrometry is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product is identified by its molecular mass. The current state of the art in mass spectrometry is such that less than femtomole quantities of material can be readily analyzed to afford information about the molecular contents of the sample. An accurate assessment of the molecular mass of the material can be quickly obtained, irrespective of whether the molecular weight of the sample is several hundred, or in excess of one hundred thousand atomic mass units (amu) or Daltons.
In some embodiments, intact molecular ions are generated from amplification products using one of a variety of ionization techniques to convert the sample to gas phase. These ionization methods include, but are not limited to, electrospray ionization (ES), matrix-assisted laser desorption ionization (MALDI) and fast atom bombardment (FAB). Upon ionization, several peaks are observed from one sample due to the formation of ions with different charges. Averaging the multiple readings of molecular mass obtained from a single mass spectrum affords an estimate of molecular mass of the bioagent identifying amplicon. Electrospray ionization mass spectrometry (ESI-MS) is particularly useful for very high molecular weight polymers such as proteins and nucleic acids having molecular weights greater than 10 kDa, since it yields a distribution of multiply-charged molecules of the sample without causing a significant amount of fragmentation.
The mass detectors used in the methods of the present invention include, but are not limited to, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), time of flight (TOF), ion trap, quadrupole, magnetic sector, Q-TOF, and triple quadrupole.
D. Base Compositions of Bioagent Identifying Amplicons

Although the molecular mass of amplification products obtained using intelligent primers provides a means for identification of bioagents, conversion of molecular mass data to a base composition signature is useful for certain analyses. As used herein, “base composition” is the exact number of each nucleobase (A, T, C and G) determined from the molecular mass of a bioagent identifying amplicon. In some embodiments, a base composition provides an index of a specific organism. Base compositions can be calculated from known sequences of known bioagent identifying amplicons and can be experimentally determined by measuring the molecular mass of a given bioagent identifying amplicon, followed by determination of all possible base compositions which are consistent with the measured molecular mass within acceptable experimental error. The following example illustrates determination of base composition from an experimentally obtained molecular mass of a 46-mer amplification product originating at position 1337 of the 16S rRNA of Bacillus anthracis. The forward and reverse strands of the amplification product have measured molecular masses of 14208 and 14079 Da, respectively. The possible base compositions derived from the molecular masses of the forward and reverse strands for the B. anthracis products are listed in Table 1.

TABLE 1


Possible Base Compositions for B. anthracis 46mer Amplification Product

Calc. Mass	Mass Error	Base	Calc. Mass	Mass Error	Base
Forward	Forward	Composition of	Reverse	Reverse	Composition of
Strand	Strand	Forward Strand	Strand	Strand	Reverse Strand

14208.2935	0.079520	A1 G17 C10 T18	14079.2624	0.080600	A0 G14 C13 T19
14208.3160	0.056980	A1 G20 C15 T10	14079.2849	0.058060	A0 G17 C18 T11
14208.3386	0.034440	A1 G23 C20 T2	14079.3075	0.035520	A0 G20 C23 T3
14208.3074	0.065560	A6 G11 C3 T26	14079.2538	0.089180	A5 G5 C1 T35
14208.3300	0.043020	A6 G14 C8 T18	14079.2764	0.066640	A5 G8 C6 T27
14208.3525	0.020480	A6 G17 C13 T10	14079.2989	0.044100	A5 G11 C11 T19
14208.3751	0.002060	A6 G20 C18 T2	14079.3214	0.021560	A5 G14 C16 T11
14208.3439	0.029060	A11 G8 C1 T26	14079.3440	0.000980	A5 G17 C21 T3
14208.3665	0.006520	A11 G11 C6 T18	14079.3129	0.030140	A10 G5 C4 T27
14208.3890	0.016020	A11 G14 C11 T10	14079.3354	0.007600	A10 G8 C9 T19
14208.4116	0.038560	A11 G17 C16 T2	14079.3579	0.014940	A10 G11 C14 T11
14208.4030	0.029980	A16 G8 C4 T18	14079.3805	0.037480	A10 G14 C19 T3
14208.4255	0.052520	A16 G11 C9 T10	14079.3494	0.006360	A15 G2 C2 T27
14208.4481	0.075060	A16 G14 C14 T2	14079.3719	0.028900	A15 G5 C7 T19
14208.4395	0.066480	A21 G5 C2 T18	14079.3944	0.051440	A15 G8 C12 T11
14208.4620	0.089020	A21 G8 C7 T10	14079.4170	0.073980	A15 G11 C17 T3
—	—	—	14079.4084	0.065400	A20 G2 C5 T19
—	—	—	14079.4309	0.087940	A20 G5 C10 T13

Among the 16 possible base compositions for the forward strand and the 18 possible base compositions for the reverse strand that were calculated, only one pair (shown in bold) are complementary base compositions, which indicates the true base composition of the amplification product. It should be recognized that this logic is applicable for determination of base compositions of any bioagent identifying amplicon, regardless of the class of bioagent from which the corresponding amplification product was obtained.
In some embodiments, assignment of previously unobserved base compositions (also known as “true unknown base compositions”) to a given phylogeny can be accomplished via the use of pattern classifier model algorithms. Base compositions, like sequences, vary slightly from strain to strain within species, for example. In some embodiments, the pattern classifier model is the mutational probability model. On other embodiments, the pattern classifier is the polytope model. The mutational probability model and polytope model are both commonly owned and described in U.S. patent application Ser. No. 11/073,362 which is incorporated herein by reference in entirety.
In one embodiment, it is possible to manage this diversity by building “base composition probability clouds” around the composition constraints for each species. This permits identification of organisms in a fashion similar to sequence analysis. A “pseudo four-dimensional plot” can be used to visualize the concept of base composition probability clouds. Optimal primer design requires optimal choice of bioagent identifying amplicons and maximizes the separation between the base composition signatures of individual bioagents. Areas where clouds overlap indicate regions that may result in a misclassification, a problem which is overcome by a triangulation identification process using bioagent identifying amplicons not affected by overlap of base composition probability clouds.
In some embodiments, base composition probability clouds provide the means for screening potential primer pairs in order to avoid potential misclassifications of base compositions. In other embodiments, base composition probability clouds provide the means for predicting the identity of a bioagent whose assigned base composition was not previously observed and/or indexed in a bioagent identifying amplicon base composition database due to evolutionary transitions in its nucleic acid sequence. Thus, in contrast to probe-based techniques, mass spectrometry determination of base composition does not require prior knowledge of the composition or sequence in order to make the measurement.
The present invention provides bioagent classifying information similar to DNA sequencing and phylogenetic analysis at a level sufficient to identify a given bioagent. Furthermore, the process of determination of a previously unknown base composition for a given bioagent (for example, in a case where sequence information is unavailable) has downstream utility by providing additional bioagent indexing information with which to populate base composition databases. The process of future bioagent identification is thus greatly improved as more BCS indexes become available in base composition databases.
E. Triangulation Identification
In some cases, a molecular mass of a single bioagent identifying amplicon alone does not provide enough resolution to unambiguously identify a given bioagent. The employment of more than one bioagent identifying amplicon for identification of a bioagent is herein referred to as “triangulation identification.” Triangulation identification is pursued by determining the molecular masses of a plurality of bioagent identifying amplicons selected within a plurality of housekeeping genes. This process is used to reduce false negative and false positive signals, and enable reconstruction of the origin of hybrid or otherwise engineered bioagents. For example, identification of the three part toxin genes typical of B. anthracis (Bowen et al., J. Appl. Microbiol., 1999, 87, 270-278) in the absence of the expected signatures from the B. anthracis genome would suggest a genetic engineering event.
In some embodiments, the triangulation identification process can be pursued by characterization of bioagent identifying amplicons in a massively parallel fashion using the polymerase chain reaction (PCR), such as multiplex PCR where multiple primers are employed in the same amplification reaction mixture, or PCR in multi-well plate format wherein a different and unique pair of primers is used in multiple wells containing otherwise identical reaction mixtures. Such multiplex and multi-well PCR methods are well known to those with ordinary skill in the arts of rapid throughput amplification of nucleic acids. In other related embodiments, one PCR reaction per well or container may be carried out, followed by an amplicon pooling step wherein the amplification products of different wells are combined in a single well or container which is then subjected to molecular mass analysis. The combination of pooled amplicons can be chosen such that the expected ranges of molecular masses of individual amplicons are not overlapping and thus will not complicate identification of signals.
F. Codon Base Composition Analysis
In some embodiments of the present invention, one or more nucleotide substitutions within a codon of a gene of an infectious organism confer drug resistance upon an organism which can be determined by codon base composition analysis. The organism can be a bacterium, virus, fungus or protozoan.
In some embodiments, the amplification product containing the codon being analyzed is of a length of about 35 to about 200 nucleobases. The primers employed in obtaining the amplification product can hybridize to upstream and downstream sequences directly adjacent to the codon, or can hybridize to upstream and downstream sequences one or more sequence positions away from the codon. The primers may have between about 70% to 100% sequence complementarity with the sequence of the gene containing the codon being analyzed.
In some embodiments, the codon base composition analysis is undertaken
In some embodiments, the codon analysis is undertaken for the purpose of investigating genetic disease in an individual. In other embodiments, the codon analysis is undertaken for the purpose of investigating a drug resistance mutation or any other deleterious mutation in an infectious organism such as a bacterium, virus, fungus or protozoan. In some embodiments, the bioagent is a bacterium identified in a biological product.
In some embodiments, the molecular mass of an amplification product containing the codon being analyzed is measured by mass spectrometry. The mass spectrometry can be either electrospray (ESI) mass spectrometry or matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Time-of-flight (TOF) is an example of one mode of mass spectrometry compatible with the analyses of the present invention.
The methods of the present invention can also be employed to determine the relative abundance of drug resistant strains of the organism being analyzed. Relative abundances can be calculated from amplitudes of mass spectral signals with relation to internal calibrants. In some embodiments, known quantities of internal amplification calibrants can be included in the amplification reactions and abundances of analyte amplification product estimated in relation to the known quantities of the calibrants.
In some embodiments, upon identification of one or more drug-resistant strains of an infectious organism infecting an individual, one or more alternative treatments can be devised to treat the individual.
G. Determination of the Quantity of a Bioagent
In some embodiments, the identity and quantity of an unknown bioagent can be determined using the process illustrated in FIG. 2. Primers (500) and a known quantity of a calibration polynucleotide (505) are added to a sample containing nucleic acid of an unknown bioagent. The total nucleic acid in the sample is then subjected to an amplification reaction (510) to obtain amplification products. The molecular masses of amplification products are determined (515) from which are obtained molecular mass and abundance data. The molecular mass of the bioagent identifying amplicon (520) provides the means for its identification (525) and the molecular mass of the calibration amplicon obtained from the calibration polynucleotide (530) provides the means for its identification (535). The abundance data of the bioagent identifying amplicon is recorded (540) and the abundance data for the calibration data is recorded (545), both of which are used in a calculation (550) which determines the quantity of unknown bioagent in the sample.
A sample comprising an unknown bioagent is contacted with a pair of primers that provide the means for amplification of nucleic acid from the bioagent, and a known quantity of a polynucleotide that comprises a calibration sequence. The nucleic acids of the bioagent and of the calibration sequence are amplified and the rate of amplification is reasonably assumed to be similar for the nucleic acid of the bioagent and of the calibration sequence. The amplification reaction then produces two amplification products: a bioagent identifying amplicon and a calibration amplicon. The bioagent identifying amplicon and the calibration amplicon should be distinguishable by molecular mass while being amplified at essentially the same rate. Effecting differential molecular masses can be accomplished by choosing as a calibration sequence, a representative bioagent identifying amplicon (from a specific species of bioagent) and performing, for example, a 2-8 nucleobase deletion or insertion within the variable region between the two priming sites. The amplified sample containing the bioagent identifying amplicon and the calibration amplicon is then subjected to molecular mass analysis by mass spectrometry, for example. The resulting molecular mass analysis of the nucleic acid of the bioagent and of the calibration sequence provides molecular mass data and abundance data for the nucleic acid of the bioagent and of the calibration sequence. The molecular mass data obtained for the nucleic acid of the bioagent enables identification of the unknown bioagent and the abundance data enables calculation of the quantity of the bioagent, based on the knowledge of the quantity of calibration polynucleotide contacted with the sample.
In some embodiments, construction of a standard curve where the amount of calibration polynucleotide spiked into the sample is varied provides additional resolution and improved confidence for the determination of the quantity of bioagent in the sample. The use of standard curves for analytical determination of molecular quantities is well known to one with ordinary skill and can be performed without undue experimentation.
In some embodiments, multiplex amplification is performed where multiple bioagent identifying amplicons are amplified with multiple primer pairs which also amplify the corresponding standard calibration sequences. In this or other embodiments, the standard calibration sequences are optionally included within a single vector which functions as the calibration polynucleotide. Multiplex amplification methods are well known to those with ordinary skill and can be performed without undue experimentation.
In some embodiments, the calibrant polynucleotide is used as an internal positive control to confirm that amplification conditions and subsequent analysis steps are successful in producing a measurable amplicon. Even in the absence of copies of the genome of a bioagent, the calibration polynucleotide should give rise to a calibration amplicon. Failure to produce a measurable calibration amplicon indicates a failure of amplification or subsequent analysis step such as amplicon purification or molecular mass determination. Reaching a conclusion that such failures have occurred is in itself, a useful event.
In some embodiments, the calibration sequence is comprised of DNA. In some embodiments, the calibration sequence is comprised of RNA.
In some embodiments, the calibration sequence is inserted into a vector that itself functions as the calibration polynucleotide. In some embodiments, more than one calibration sequence is inserted into the vector that functions as the calibration polynucleotide. Such a calibration polynucleotide is herein termed a “combination calibration polynucleotide.” The process of inserting polynucleotides into vectors is routine to those skilled in the art and can be accomplished without undue experimentation. Thus, it should be recognized that the calibration method should not be limited to the embodiments described herein. The calibration method can be applied for determination of the quantity of any bioagent identifying amplicon when an appropriate standard calibrant polynucleotide sequence is designed and used. The process of choosing an appropriate vector for insertion of a calibrant is also a routine operation that can be accomplished by one with ordinary skill without undue experimentation.
H. Identification of Bacteria
In other embodiments of the present invention, the primer pairs produce bioagent identifying amplicons within stable and highly conserved regions of bacteria. The advantage to characterization of an amplicon defined by priming regions that fall within a highly conserved region is that there is a low probability that the region will evolve past the point of primer recognition, in which case, the primer hybridization of the amplification step would fail. Such a primer set is thus useful as a broad range survey-type primer. In another embodiment of the present invention, the intelligent primers produce bioagent identifying amplicons including a region which evolves more quickly than the stable region described above. The advantage of characterization bioagent identifying amplicon corresponding to an evolving genomic region is that it is useful for distinguishing emerging strain variants or the presence of virulence genes, drug resistance genes, or codon mutations that induce drug resistance.
The present invention also has significant advantages as a platform for identification of diseases caused by emerging bacterial strains such as, for example, drug-resistant strains of Staphylococcus aureus. The present invention eliminates the need for prior knowledge of bioagent sequence to generate hybridization probes. This is possible because the methods are not confounded by naturally occurring evolutionary variations occurring in the sequence acting as the template for production of the bioagent identifying amplicon. Measurement of molecular mass and determination of base composition is accomplished in an unbiased manner without sequence prejudice.
Another embodiment of the present invention also provides a means of tracking the spread of a bacterium, such as a particular drug-resistant strain when a plurality of samples obtained from different locations are analyzed by the methods described above in an epidemiological setting. In one embodiment, a plurality of samples from a plurality of different locations is analyzed with primer pairs which produce bioagent identifying amplicons, a subset of which contains a specific drug-resistant bacterial strain. The corresponding locations of the members of the drug-resistant strain subset indicate the spread of the specific drug-resistant strain to the corresponding locations.
I. Kits
The present invention also provides kits for carrying out the methods described herein. In some embodiments, the kit may comprise a sufficient quantity of one or more primer pairs to perform an amplification reaction on a target polynucleotide from a bioagent to form a bioagent identifying amplicon. In some embodiments, the kit may comprise from one to fifty primer pairs, from one to twenty primer pairs, from one to ten primer pairs, or from two to five primer pairs. In some embodiments, the kit may comprise one or more primer pairs recited in Table 2.
In some embodiments, the kit comprises one or more broad range survey primer(s), division wide primer(s), or drill-down primer(s), or any combination thereof. If a given problem involves identification of a specific bioagent, the solution to the problem may require the selection of a particular combination of primers to provide the solution to the problem. A kit may be designed so as to comprise particular primer pairs for identification of a particular bioagent. A drill-down kit may be used, for example, to distinguish different genotypes or strains, drug-resistant, or otherwise. In some embodiments, the primer pair components of any of these kits may be additionally combined to comprise additional combinations of broad range survey primers and division-wide primers so as to be able to identify a bacterium.
In some embodiments, the kit contains standardized calibration polynucleotides for use as internal amplification calibrants. Internal calibrants are described in commonly owned U.S. Patent Application Ser. No. 60/545,425 which is incorporated herein by reference in its entirety.
In some embodiments, the kit comprises a sufficient quantity of reverse transcriptase (if RNA is to be analyzed for example), a DNA polymerase, suitable nucleoside triphosphates (including alternative dNTPs such as inosine or modified dNTPs such as the 5-propynyl pyrimidines or any dNTP containing molecular mass-modifying tags such as those described above), a DNA ligase, and/or reaction buffer, or any combination thereof, for the amplification processes described above. A kit may further include instructions pertinent for the particular embodiment of the kit, such instructions describing the primer pairs and amplification conditions for operation of the method. A kit may also comprise amplification reaction containers such as microcentrifuge tubes and the like. A kit may also comprise reagents or other materials for isolating bioagent nucleic acid or bioagent identifying amplicons from amplification, including, for example, detergents, solvents, or ion exchange resins which may be linked to magnetic beads. A kit may also comprise a table of measured or calculated molecular masses and/or base compositions of bioagents using the primer pairs of the kit.
Some embodiments are kits that contain one or more survey bacterial primer pairs represented by primer pair compositions wherein each member of each pair of primers has 70% to 100% sequence identity with the corresponding member from the group of primer pairs represented by any of the primer pairs of Table 5. The survey primer pairs may include broad range primer pairs which hybridize to ribosomal RNA, and may also include division-wide primer pairs which hybridize to housekeeping genes such as rplB, tufB, rpoB, rpoC, valS, and infB, for example.
In some embodiments, a kit may contain one or more survey bacterial primer pairs and one or more triangulation genotyping analysis primer pairs such as the primer pairs of Tables 8, 12, 14, 19, 21, 23, or 24. In some embodiments, the kit may represent a less expansive genotyping analysis but include triangulation genotyping analysis primer pairs for more than one genus or species of bacteria. For example, a kit for surveying nosocomial infections at a health care facility may include, for example, one or more broad range survey primer pairs, one or more division wide primer pairs, one or more Acinetobacter baumannii triangulation genotyping analysis primer pairs and one or more Staphylococcus aureus triangulation genotyping analysis primer pairs. One with ordinary skill will be capable of analyzing in silico amplification data to determine which primer pairs will be able to provide optimal identification resolution for the bacterial bioagents of interest.
In some embodiments, a kit may be assembled for identification of strains of bacteria involved in contamination of food. An example of such a kit embodiment is a kit comprising one or more bacterial survey primer pairs of Table 5 with one or more triangulation genotyping analysis primer pairs of Table 12 which provide strain resolving capabilities for identification of specific strains of Campylobacter jejuni.
Some embodiments of the kits are 96-well or 384-well plates with a plurality of wells containing any or all of the following components: dNTPs, buffer salts, Mg²⁺, betaine, and primer pairs. In some embodiments, a polymerase is also included in the plurality of wells of the 96-well or 384-well plates.
Some embodiments of the kit contain instructions for PCR and mass spectrometry analysis of amplification products obtained using the primer pairs of the kits.
Some embodiments of the kit include a barcode which uniquely identifies the kit and the components contained therein according to production lots and may also include any other information relative to the components such as concentrations, storage temperatures, etc. The barcode may also include analysis information to be read by optical barcode readers and sent to a computer controlling amplification, purification and mass spectrometric measurements. In some embodiments, the barcode provides access to a subset of base compositions in a base composition database which is in digital communication with base composition analysis software such that a base composition measured with primer pairs from a given kit can be compared with known base compositions of bioagent identifying amplicons defined by the primer pairs of that kit.
In some embodiments, the kit contains a database of base compositions of bioagent identifying amplicons defined by the primer pairs of the kit. The database is stored on a convenient computer readable medium such as a compact disk or USB drive, for example.
In some embodiments, the kit includes a computer program stored on a computer formatted medium (such as a compact disk or portable USB disk drive, for example) comprising instructions which direct a processor to analyze data obtained from the use of the primer pairs of the present invention. The instructions of the software transform data related to amplification products into a molecular mass or base composition which is a useful concrete and tangible result used in identification and/or classification of bioagents. In some embodiments, the kits of the present invention contain all of the reagents sufficient to carry out one or more of the methods described herein.
While the present invention has been described with specificity in accordance with certain of its embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. In order that the invention disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the invention in any manner.

EXAMPLES

Example 1

Design and Validation of Primers that Define Bioagent Identifying Amplicons for Identification of Bacteria

For design of primers that define bacterial bioagent identifying amplicons, a series of bacterial genome segment sequences were obtained, aligned and scanned for regions where pairs of PCR primers would amplify products of about 45 to about 150 nucleotides in length and distinguish subgroups and/or individual strains from each other by their molecular masses or base compositions. A typical process shown in FIG. 1 is employed for this type of analysis.
A database of expected base compositions for each primer region was generated using an in silico PCR search algorithm, such as (ePCR). An existing RNA structure search algorithm (Macke et al., Nucl. Acids Res., 2001, 29, 4724-4735, which is incorporated herein by reference in its entirety) has been modified to include PCR parameters such as hybridization conditions, mismatches, and thermodynamic calculations (SantaLucia, Proc. Natl. Acad. Sci. U.S.A., 1998, 95, 1460-1465, which is incorporated herein by reference in its entirety). This also provides information on primer specificity of the selected primer pairs.

Table 2 represents a collection of primers (sorted by primer pair number) designed to identify bacteria using the methods described herein. The primer pair number is an in-house database index number. Primer sites were identified on segments of genes, such as, for example, the 16S rRNA gene. The forward or reverse primer name shown in Table 2 indicates the gene region of the bacterial genome to which the primer hybridizes relative to a reference sequence. In Table 2, for example, the forward primer name 16S_EC_—1077_—1106_F indicates that the forward primer (_F) hybridizes to residues 1077-1106 of the reference sequence represented by a sequence extraction of coordinates 4033120 . . . 4034661 from GenBank gi number 16127994 (as indicated in Table 3). As an additional example: the forward primer name BONTA_X52066_—450_—473 indicates that the primer hybridizes to residues 450-437 of the gene encoding Clostridium botulinum neurotoxin type A (BoNT/A) represented by GenBank Accession No. X52066 (primer pair name codes appearing in Table 2 are defined in Table 3. One with ordinary skill knows how to obtain individual gene sequences or portions thereof from genomic sequences present in GenBank. In Table 2, Tp=5-propynyluracil; Cp=5-propynylcytosine; *=phosphorothioate linkage; I=inosine. T. GenBank Accession Numbers for reference sequences of bacteria are shown in Table 3 (below). In some cases, the reference sequences are extractions from bacterial genomic sequences or complements thereof.

TABLE 2


Primer Pairs for Identification of Bacteria

Primer	Forward		Forward	Reverse		Reverse
Pair	Primer	Forward	SEQ ID	Primer	Reverse	SEQ ID
Number	Name	Sequence	NO:	Name	Sequence	NO:

1	16S_EC_1077_1106_F	GTGAGATGTTGGGTTAA	134	16S_EC_1175_1195_R	GACGTCATCCCCACCTT	809
		GTCCCGTAACGAG			CCTC

2	16S_EC_1082_1106_F	ATGTTGGGTTAAGTCCC	38	16S_EC_1175_1197_R	TTGACGTCATCCCCACC	1398
		GCAACGAG			TTCCTC

3	16S_EC_1090_1111_F	TTAAGTCCCGCAACGAT	651	16S_EC_1175_1196_R	TGACGTCATCCCCACCT	1159
		CGCAA			TCCTC

4	16S_EC_1222_1241_F	GCTACACACGTGCTACA	114	16S_EC_1303_1323_R	CGAGTTGCAGACTGCGA	787
		ATG			TCCG

5	16S_EC_1332_1353_F	AAGTCGGAATCGCTAGT	10	16S_EC_1389_1407_R	GACGGGCGGTGTGTACA	806
		AATCG			AG

6	16S_EC_30_54_F	TGAACGCTGGTGGCATG	429	16S_EC_105_126_R	TACGCATTACTCACCCG	897
		CTTAACAC			TCCGC

7	16S_EC_38_64_F	GTGGCATGCCTAATACA	136	16S_EC_101_120_R	TTACTCACCCGTCCGCC	1365
		TGCAAGTCG			GCT

8	16S_EC_49_68_F	TAACACATGCAAGTCGA	152	16S_EC_104_120_R	TTACTCACCCGTCCGCC	1364
		ACG

9	16S_EC_683_700_F	GTGTAGCGGTGAAATGC	137	16S_EC_774_795_R	GTATCTAATCCTGTTTG	839
		G			CTCCC

10	16S_EC_713_732_F	AGAACACCGATGGCGAA	21	16S_EC_789_809_R	CGTGGACTACCAGGGTA	798
		GGC			TCTA

11	16S_EC_785_806_F	GGATTAGAGACCCTGGT	118	16S_EC_880_897_R	GGCCGTACTCCCCAGGC	830
		AGTCC			G

12	16S_EC_785_810_F	GGATTAGATACCCTGGT	119	16S_EC_880_897_2_R	GGCCGTACTCCCCAGGC	830
		AGTCCACGC			G

13	16S_EC_789_810_F	TAGATACCCTGGTAGTC	206	16S_EC_880_894_R	CGTACTCCCCAGGCG	796
		CACGC

14	16S_EC_960_981_F	TTCGATGCAACGCGAAG	672	16S_EC_1054_1073_R	ACGAGCTGACGACAGCC	735
		AACCT			ATG

15	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1061_1078_R	ACGACACGAGCTGACGA	734
					C

16	23S_EC_1826_1843_F	CTGACACCTGCCCGGTG	80	23S_EC_1906_1924_R	GACCGTTATAGTTACGG	805
		C			CC

17	23S_EC_2645_2669_F	TCTGTCCCTAGTACGAG	408	23S_EC_2744_2761_R	TGCTTAGATGCTTTCAG	1252
		AGGACCGG			C

18	23S_EC_2645_2669_	CTGTCCCTAGTACGAGA	83	23S_EC_2751_2767_R	GTTTCATGCTTAGATGC	846
	2_F	GGACCGG			TTTCAGC

19	23S_EC_493_518_F	GGGGAGTGAAAGAGATC	125	23S_EC_551_571_R	ACAAAAGGTACGCCGTC	717
		CTGAAACCG			ACCC

20	23S_EC_493_518_2_F	GGGGAGTGAAAGAGATC	125	23S_EC_551_571_2_R	ACAAAAGGCACGCCATC	716
		CTGAAACCG			ACCC

21	23S_EC_971_992_F	CGAGAGGGAAACAACCC	66	23S_EC_1059_1077_R	TGGCTGCTTCTAAGCCA	1282
		AGACC			AC

22	CAPC_BA_104_131_F	GTTATTTAGCACTCGTT	139	CAPC_BA_180_205_R	TGAATCTTGAAACACCA	1150
		TTTAATCAGCC			TACGTAACG

23	CAPC_BA_114_133_F	ACTCGTTTTTAATCAGC	20	CAPC_BA_185_205_R	TGAATCTTGAAACACCA	1149
		CCG			TACG

24	CAPC_BA_274_303_F	GATTATTGTTATCCTGT	109	CAPC_BA_349_376_R	GTAACCCTTGTCTTTGA	837
		TATGCCATTTGAG			ATTGTATTTGC

25	CAPC_BA_276_296_F	TTATTGTTATCCTGTTA	663	CAPC_BA_358_377_R	GGTAACCCTTGTCTTTG	834
		TGCC			AAT

26	CAPC_BA_281_301_F	GTTATCCTGTTATGCCA	138	CAPC_BA_361_378_R	TGGTAACCCTTGTCTTT	1298
		TTTG			G

27	CAPC_BA_315_334_F	CCGTGGTATTGGAGTTA	59	CAPC_BA_361_378_R	TGGTAACCCTTGTCTTT	1298
		TTG			G

28	CYA_BA_1055_1072_F	GAAAGAGTTCGGATTGG	92	CYA_BA_1112_1130_R	TGTTGACCATGCTTCTT	1352
		G			AG

29	CYA_BA_1349_1370_F	ACAACGAAGTACAATAC	12	CYA_BA_1447_1426_R	CTTCTACATTTTTAGCC	800
		AAGAC			ATCAC

30	CYA_BA_1353_1379_F	CGAAGTACAATACAAGA	64	CYA_BA_1448_1467_R	TGTTAACGGCTTCAAGA	1342
		CAAAAGAAGG			CCC

31	CYA_BA_1359_1379_F	ACAATACAAGACAAAAG	13	CYA_BA_1447_1461_R	CGGCTTCAAGACCCC	794
		AAGG

32	CYA_BA_914_937_F	CAGGTTTAGTACCAGAA	53	CYA_BA_999_1026_R	ACCACTTTTAATAAGGT	728
		CATGCAG			TTGTAGCTAAC

33	CYA_BA_916_935_F	GGTTTAGTACCAGAACA	131	CYA_BA_1003_1025_R	CCACTTTTAATAAGGTT	768
		TGC			TGTAGC

34	INFB_EC_1365_1393_	TGCTCGTGGTGCACAAG	524	INFB_EC_1439_1467_R	TGCTGCTTTCGCATGGT	1248
	F	TAACGGATATTA			TAATTGCTTCAA

35	LEF_BA_1033_1052_F	TCAAGAAGAAAAAGAGC	254	LEF_BA_1119_1135_R	GAATATCAATTTGTAGC	803

36	LEF_BA_1036_1066_F	CAAGAAGAAAAAGAGCT	44	LEF_BA_1119_1149_R	AGATAAAGAATCACGAA	745
		TCTAAAAAGAATAC			TATCAATTTGTAGC

37	LEF_BA_756_781_F	AGCTTTTGCATATTATA	26	LEF_BA_843_872_R	TCTTCCAAGGATAGATT	1135
		TCGAGCCAC			TATTTCTTGTTCG

38	LEF_BA_758_778_F	CTTTTGCATATTATATC	90	LEF_BA_843_865_R	AGGATAGATTTATTTCT	748
		GAGC			TGTTCG

39	LEF_BA_795_813_F	TTTACAGCTTTATGCAC	700	LEF_BA_883_900_R	TCTTGACAGCATCCGTT	1140
		CG			G

40	LEF_BA_883_899_F	CAACGGATGCTGGCAAG	43	LEF_BA_939_958_R	CAGATAAAGAATCGCTC	762
					CAG

41	PAG_BA_122_142_F	CAGAATCAAGTTCCCAG	49	PAG_BA_190_209_R	CCTGTAGTAGAAGAGGT	781
		GGG			AAC

42	PAG_BA_123_145_F	AGAATCAAGTTCCCAGG	22	PAG_BA_187_210_R	CCCTGTAGTAGAAGAGG	774
		GGTTAC			TAACCAC

43	PAG_BA_269_287_F	AATCTGCTATTTGGTCA	11	PAG_BA_326_344_R	TGATTATCAGCGGAAGT	1186
		GG			AG

44	PAG_BA_655_675_F	GAAGGATATACGGTTGA	93	PAG_BA_755_772_R	CCGTGCTCCATTTTTCA	778
		TGTC			G

45	PAG_BA_753_772_F	TCCTGAAAAATGGAGCA	341	PAG_BA_849_868_R	TCGGATAAGCTGCCACA	1089
		CGG			AGG

46	PAG_BA_763_781_F	TGGAGCACGGCTTCTGA	552	PAG_BA_849_868_R	TCGGATAAGCTGCCACA	1089
		TC			AGG

47	RPOC_EC_1018_1045_	CAAAACTTATTAGGTAA	39	RPOC_EC_1095_1124_R	TCAAGCGCCATTTCTTT	959
	F	GCGTGTTGACT			TGGTAAACCACAT

48	RPOC_EC_1018_1045_	CAAAACTTATTAGGTAA	39	RPOC_EC_1095_1124_2_R	TCAAGCGCCATCTCTTT	958
	F	GCGTGTTGACT			CGGTAATCCACAT

49	RPOC_EC_114_140_F	TAAGAAGCCGGAAACCA	158	RPOC_EC_213_232_R	GGCGCTTGTACTTACCG	831
		TCAACTACCG			CAC

50	RPOC_EC_2178_2196_	TGATTCTGGTGCCCGTG	478	RPOC_EC_2225_2246_R	TTGGCCATCAGGCCACG	1414
	F	GT			CATAC

51	RPOC_EC_2178_2196_	TGATTCCGGTGCCCGTG	477	RPOC_EC_2225_2246_2_R	TTGGCCATCAGACCACG	1413
	2_F	GT			CATAC

52	RPOC_EC_2218_2241_	CTGGCAGGTATGCGTGG	81	RPOC_EC_2313_2337_R	CGCACCGTGGGTTGAGA	790
	F	TCTGATG			TGAAGTAC

53	RPOC_EC_2218_2241_	CTTGCTGGTATGCGTGG	86	RPOC_EC_2313_2337_2_R	CGCACCATGCGTAGAGA	789
	2_F	TCTGATG			TGAAGTAC

54	RPOC_EC_808_833_F	CGTCGGGTGATTAACCG	75	RPOC_EC_865_889_R	GTTTTTCGTTGCGTACG	847
		TAACAACCG			ATGATGTC

55	RPOC_EC_808_833_2_	CGTCGTGTAATTAACCG	76	RPOC_EC_865_891_R	ACGTTTTTCGTTTTGAA	741
	F	TAACAACCG			CGATAATGCT

56	RPOC_EC_993_1019_F	CAAAGGTAAGCAAGGTC	41	RPOC_EC_1036_1059_R	CGAACGGCCTGAGTAGT	785
		GTTTCCGTCA			CAACACG

57	RPOC_EC_993_1019_	CAAAGGTAAGCAAGGAC	40	RPOC_EC_1036_1059_2_R	CGAACGGCCAGAGTAGT	784
	2_F	GTTTCCGTCA			CAACACG

58	SSPE_BA_115_137_F	CAAGCAAACGCACAATC	45	SSPE_BA_197_222_R	TGCACGTCTGTTTCAGT	1201
		AGAAGC			TGCAAATTC

59	TUFB_EC_239_259_F	TAGACTGCCCAGGACAC	204	TUFB_EC_283_303_R	GCCGTCCATCTGAGCAG	815
		GCTG			CACC

60	TUFB_EC_239_259_2_	TTGACTGCCCAGGTCAC	678	TUFB_EC_283_303_2_R	GCCGTCCATTTGAGCAG	816
	F	GCTG			CACC

61	TUFB_EC_976_1000_F	AACTACCGTCCGCAGTT	4	TUFB_EC_1045_1068_R	GTTGTCGCCAGGCATAA	845
		CTACTTCC			CCATTTC

62	TUFB_EC_976_1000_	AACTACCGTCCTCAGTT	5	TUFB_EC_1045_1068_2_R	GTTGTCACCAGGCATTA	844
	2_F	CTACTTCC			CCATTTC

63	TUFB_EC_985_1012_F	CCACAGTTCTACTTCCG	56	TUFB_EC_1033_1062_R	TCCAGGCATTACCATTT	1006
		TACTACTGACG			CTACTCCTTCTGG

66	RPLB_EC_650_679_F	GACCTACAGTAAGAGGT	98	RPLB_EC_739_762_R	TCCAAGTGCTGGTTTAC	999
		TCTGTAATGAACC			CCCATGG

67	RPLB_EC_688_710_F	CATCCACACGGTGGTGG	54	RPLB_EC_736_757_R	GTGCTGGTTTACCCCAT	842
		TGAAGG			GGAGT

68	RPOC_EC_1036_1060_	CGTGTTGACTATTCGGG	78	RPOC_EC_1097_1126_R	ATTCAAGAGCCATTTCT	754
	F	GCGTTCAG			TTTGGTAAACCAC

69	RPOB_EC_3762_3790_	TCAACAACCTCTTGGAG	248	RPOB_EC_3836_3865_R	TTTCTTGAAGAGTATGA	1435
	F	GTAAAGCTCAGT			GCTGCTCCGTAAG

70	RPLB_EC_688_710_F	CATCCACACGGTGGTGG	54	RPLB_EC_743_771_R	TGTTTTGTATCCAAGTG	1356
		TGAAGG			CTGGTTTACCCC

71	VALS_EC_1105_1124_	CGTGGCGGCGTGGTTAT	77	VALS_EC_1195_1218_R	CGGTACGAACTGGATGT	795
	F	CGA			CGCCGTT

72	RPOB_EC_1845_1866_	TATCGCTCAGGCGAACT	233	RPOB_EC_1909_1929_R	GCTGGATTCGCCTTTGC	825
	F	CCAAC			TACG

73	RPLB_EC_669_698_F	TGTAATGAACCCTAATG	623	RPLB_EC_735_761_R	CCAAGTGCTGGTTTACC	767
		ACCATCCACACGG			CCATGGAGTA

74	RPLB_EC_671_700_F	TAATGAACCCTAATGAC	169	RPLB_EC_737_762_R	TCCAAGTGCTGGTTTAC	1000
		CATCCACACGGTG			CCCATGGAG

75	SP101_SPET11_1_29_	AACCTTAATTGGAAAGA	2	SP101_SPET11_92_116_R	CCTACCCAACGTTCACC	779
	F	AACCCAAGAAGT			AAGGGCAG

76	SP101_SPET11_118_	GCTGGTGAAAATAACCC	115	SP101_SPET11_213_238_R	TGTGGCCGATTTCACCA	1340
	147_F	AGATGTCGTCTTC			CCTGCTCCT

77	SP101_SPET11_216_	AGCAGGTGGTGAAATCG	24	SP101_SPET11_308_333_R	TGCCACTTTGACAACTC	1209
	243_F	GCCACATGATT			CTGTTGCTG

78	SP101_SPET11_266_	CTTGTACTTGTGGCTCA	89	SP101_SPET11_355_380_R	GCTGCTTTGATGGCTGA	824
	295_F	CACGGCTGTTTGG			ATCCCCTTC

79	SP101_SPET11_322_	GTCAAAGTGGCACGTTT	132	SP101_SPET11_423_441_R	ATCCCCTGCTTCTGCTG	753
	344_F	ACTGGC			CC

80	SP101_SPET11_358_	GGGGATTCAGCCATCAA	126	SP101_SPET11_448_473_R	CCAACCTTTTCCACAAC	766
	387_F	AGCAGCTATTGAC			AGAATCAGC

81	SP101_SPET11_600_	CCTTACTTCGAACTATG	62	SP101_SPET11_686_714_R	CCCATTTTTTCACGCAT	772
	629_F	AATCTTTTGGAAG			GCTGAAAATATC

82	SP101_SPET11_658_	GGGGATTGATATCACCG	127	SP101_SPET11_756_784_R	GATTGGCGATAAAGTGA	813
	684_F	ATAAGAAGAA			TATTTTCTAAAA

83	SP101_SPET11_776_	TCGCCAATCAAAACTAA	364	SP101_SPET11_871_896_R	GCCCACCAGAAAGACTA	814
	801_F	GGGAATGGC			GCAGGATAA

84	SP101_SPET11_893_	GGGCAACAGCAGCGGAT	123	SP101_SPET11_988_1012_R	CATGACAGCCAAGACCT	763
	921_F	TGCGATTGCGCG			CACCCACC

85	SP101_SPET11_1154_	CAATACCGCAACAGCGG	47	SP101_SPET11_1251_1277_R	GACCCCAACCTGGCCTT	804
	1179_F	TGGCTTGGG			TTGTCGTTGA

86	SP101_SPET11_1314_	CGCAAAAAAATCCAGCT	68	SP101_SPET11_1403_1431_R	AAACTATTTTTTTAGCT	711
	1336_F	ATTAGC			ATACTCGAACAC

87	SP101_SPET11_1408_	CGAGTATAGCTAAAAAA	67	SP101_SPET11_1486_1515_R	GGATAATTGGTCGTAAC	828
	1437_F	ATAGTTTATGACA			AAGGGATAGTGAG

88	SP101_SPET11_1688_	CCTATATTAATCGTTTA	60	SP101_SPET11_1783_1808_R	ATATGATTATCATTGAA	752
	1716_F	CAGAAACTGGCT			CTGCGGCCG

89	SP101_SPET11_1711_	CTGGCTAAAACTTTGGC	82	SP101_SPET11_1808_1835_R	GCGTGACGACCTTCTTG	821
	1733_F	AACGGT			AATTGTAATCA

90	SP101_SPET11_1807_	ATGATTACAATTCAAGA	33	SP101_SPET11_1901_1927_R	TTGGACCTGTAATCAGC	1412
	1835_F	AGGTCGTCACGC			TGAATACTGG

91	SP101_SPET11_1967_	TAACGGTTATCATGGCC	155	SP101_SPET11_2062_2083_R	ATTGCCCAGAAATCAAA	755
	1991_F	CAGATGGG			TCATC

92	SP101_SPET11_2260_	CAGAGACCGTTTTATCC	50	SP101_SPET11_2375_2397_R	TCTGGGTGACCTGGTGT	1131
	2283_F	TATCAGC			TTTAGA

93	SP101_SPET11_2375_	TCTAAAACACCAGGTCA	390	SP101_SPET11_2470_2497_R	AGCTGCTAGATGAGCTT	747
	2399_F	CCCAGAAG			CTGCCATGGCC

94	SP101_SPET11_2468_	ATGGCCATGGCAGAAGC	35	SP101_SPET11_2543_2570_R	CCATAAGGTCACCGTCA	770
	2487_F	TCA			CCATTCAAAGC

95	SP101_SPET11_2961_	ACCATGACAGAAGGCAT	15	SP101_SPET11_3023_3045_R	GGAATTTACCAGCGATA	827
	2984_F	TTTGACA			GACACC

96	SP101_SPET11_3075_	GATGACTTTTTAGCTAA	108	SP101_SPET11_3168_3196_R	AATCGACGACCATCTTG	715
	3103_F	TGGTCAGGCAGC			GAAAGATTTCTC

97	SP101_SPET11_3386_	AGCGTAAAGGTGAACCT	25	SP101_SPET11_3480_3506_R	CCAGCAGTTACTGTCCC	769
	3403_F	T			CTCATCTTTG

98	SP101_SPET11_3511_	GCTTCAGGAATCAATGA	116	SP101_SPET11_3605_3629_R	GGGTCTACACCTGCACT	832
	3535_F	TGGAGCAG			TGCATAAC

111	RPOB_EC_3775_3803_	CTTGGAGGTAAGTCTCA	87	RPOB_EC_3829_3858_R	CGTATAAGCTGCACCAT	797
	F	TTTTGGTGGGCA			AAGCTTGTAATGC

112	VALS_EC_1833_1850_	CGACGCGCTGCGCTTCA	65	VALS_EC_1920_1943_R	GCGTTCCACAGCTTGTT	822
	F	C			GCAGAAG

113	RPOB_EC_1336_1353_	GACCACCTCGGCAACCG	97	RPOB_EC_1438_1455_R	TTCGCTCTCGGCCTGGC	1386
	F	T			C

114	TUFB_EC_225_251_F	GCACTATGCACACGTAG	111	TUFB_EC_284_309_R	TATAGCACCATCCATCT	930
		ATTGTCCTGG			GAGCGGCAC

115	DNAK_EC_428_449_F	CGGCGTACTTCAACGAC	72	DNAK_EC_503_522_R	CGCGGTCGGCTCGTTGA	792
		AGCCA			TGA

116	VALS_EC_1920_1943_	CTTCTGCAACAAGCTGT	85	VALS_EC_1948_1970_R	TCGCAGTTCATCAGCAC	1075
	F	GGAACGC			GAAGCG

117	TUFB_EC_757_774_F	AAGACGACCTGCACGGG	6	TUFB_EC_849_867_R	GCGCTCCACGTCTTCAC	819
		C			GC

118	23S_EC_2646_2667_	CTGTTCTTAGTACGAGA	84	23S_EC_2745_2765_R	TTCGTGCTTAGATGCTT	1389
	F	GGACC			TCAG

119	16S_EC_969_985_1P_	ACGCGAAGAACCTTACp	19	16S_EC_1061_1078_2P_R	ACGACACGAGCpTpGAC	733
	F	C			GAC

120	16S_EC_972_985_2P_	CGAAGAACpCpTTACC	63	16S_EC_1064_1075_2P_R	ACACGAGCpTpGAC	727
	F

121	16S_EC_972_985_F	CGAAGAACCTTACC	63	16S_EC_1064_1075_R	ACACGAGCTGAC	727

122	TRNA_ILE-	CCTGATAAGGGTGAGGT	61	23S_EC_40_59_R	ACGTCCTTCATCGCCTC	740
	RRNH_EC_32_50.2_F	CG			TGA

123	23S_EC_-7_15_F	GTTGTGAGGTTAAGCGA	140	23S_EC_430_450_R	CTATCGGTCAGTCAGGA	799
		CTAAG			GTAT

124	23S_EC_-7_15_F	GTTGTGAGGTTAAGCGA	141	23S_EC_891_910_R	TTGCATCGGGTTGGTAA	1403
		CTAAG			GTC

125	23S_EC_430_450_F	ATACTCCTGACTGACCG	30	23S_EC_1424_1442_R	AACATAGCCTTCTCCGT	712
		ATAG			CC

126	23S_EC_891_910_F	GACTTACCAACCCGATG	100	23S_EC_1908_1931_R	TACCTTAGGACCGTTAT	893
		CAA			AGTTACG

127	23S_EC_1424_1442_F	GGACGGAGAAGGCTATG	117	23S_EC_2475_2494_R	CCAAACACCGCCGTCGA	765
		TT			TAT

128	23S_EC_1908_1931_F	CGTAACTATAACGGTCC	73	23S_EC_2833_2852_R	GCTTACACACCCGGCCT	826
		TAAGGTA			ATC

129	23S_EC_2475_2494_F	ATATCGACGGCGGTGTT	31	TRNA_ASP-	GCGTGACAGGCAGGTAT	820
		TGG		RRNH_EC_23_41.2_R	TC

131	16S_EC_−60_−39_F	AGTCTCAAGAGTGAACA	28	16S_EC_508_525_R	GCTGCTGGCACGGAGTT	823
		CGTAA			A

132	16S_EC_326_345_F	GACACGGTCCAGACTCC	95	16S_EC_1041_1058_R	CCATGCAGCACCTGTCT	771
		TAC			C

133	16S_EC_705_724_F	GATCTGGAGGAATACCG	107	16S_EC_1493_1512_R	ACGGTTACCTTGTTACG	739
		GTG			ACT

134	16S_EC_1268_1287_F	GAGAGCAAGCGGACCTC	101	TRNA_ALA-	CCTCCTGCGTGCAAAGC	780
		ATA		RRNH_EC_30_46.2_R

135	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1061_1078.2_R	ACAACACGAGCTGACGA	719
					C

137	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1061_1078.2_I14_R	ACAACACGAGCTGICGA	721
					C

138	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1061_1078.2_I12_R	ACAACACGAGCIGACGA	718
					C

139	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1061_1078.2_I11_R	ACAACACGAGITGACGA	722
					C

140	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1061_1078.2_I16_R	ACAACACGAGCTGACIA	720
					C

141	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1061_1078.2_2I_R	ACAACACGAICTIACGA	723
					C

142	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1061_1078.2_3I_R	ACAACACIAICTIACGA	724
					C

143	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1061_1078.2_4I_R	ACAACACIAICTIACIA	725
					C

147	23S_EC_2652_2669_	CTAGTACGAGAGGACCG	79	23S_EC_2741_2760_R	ACTTAGATGCTTTCAGC	743
	F	G			GGT

158	16S_EC_683_700_F	GTGTAGCGGTGAAATGC	137	16S_EC_880_894_R	CGTACTCCCCAGGCG	796
		G

159	16S_EC_1100_1116_F	CAACGAGCGCAACCCTT	42	16S_EC_1174_1188_R	TCCCCACCTTCCTCC	1019

215	SSPE_BA_121_137_F	AACGCACAATCAGAAGC	3	SSPE_BA_197_216_R	TCTGTTTCAGTTGCAAA	1132
					TTC

220	GROL_EC_941_959_F	TGGAAGATCTGGGTCAG	544	GROL_EC_1039_1060_R	CAATCTGCTGACGGATC	759
		GC			TGAGC

221	INFB_EC_1103_1124_	GTCGTGAAAACGAGCTG	133	INFB_EC_1174_1191_R	CATGATGGTCACAACCG	764
	F	GAAGA			G

222	HFLB_EC_1082_1102_	TGGCGAACCTGGTGAAC	569	HFLB_EC_1144_1168_R	CTTTCGCTTTCTCGAAC	802
	F	GAAGC			TCAACCAT

223	INFB_EC_1969_1994_	CGTCAGGGTAAATTCCG	74	INFB_EC_2038_2058_R	AACTTCGCCTTCGGTCA	713
	F	TGAAGTTAA			TGTT

224	GROL_EC_219_242_F	GGTGAAAGAAGTTGCCT	128	GROL_EC_328_350_R	TTCAGGTCCATCGGGTT	1377
		CTAAAGC			CATGCC

225	VALS_EC_1105_1124_	CGTGGCGGCGTGGTTAT	77	VALS_EC_1195_1214_R	ACGAACTGGATGTCGCC	732
	F	CGA			GTT

226	16S_EC_556_575_F	CGGAATTACTGGGCGTA	70	16S_EC_683_700_R	CGCATTTCACCGCTACA	791
		AAG			C

227	RPOC_EC_1256_1277_	ACCCAGTGCTGCTGAAC	16	RPOC_EC_1295_1315_R	GTTCAAATGCCTGGATA	843
	F	CGTGC			CCCA

228	16S_EC_774_795_F	GGGAGCAAACAGGATTA	122	16S_EC_880_894_R	CGTACTCCCCAGGCG	796
		GATAC

229	RPOC_EC_1584_1604_	TGGCCCGAAAGAAGCTG	567	RPOC_EC_1623_1643_R	ACGCGGGCATGCAGAGA	737
	F	AGCG			TGCC

230	16S_EC_1082_1100_F	ATGTTGGGTTAAGTCCC	37	16S_EC_1177_1196_R	TGACGTCATCCCCACCT	1158
		GC			TCC

231	16S_EC_1389_1407_F	CTTGTACACACCGCCCG	88	16S_EC_1525_1541_R	AAGGAGGTGATCCAGCC	714
		TC

232	16S_EC_1303_1323_F	CGGATTGGAGTCTGCAA	71	16S_EC_1389_1407_R	GACGGGCGGTGTGTACA	808
		CTCG			AG

233	23S_EC_23_37_F	GGTGGATGCCTTGGC	129	23S_EC_115_130_R	GGGTTTCCCCATTCGG	833

234	23S_EC_187_207_F	GGGAACTGAAACATCTA	121	23S_EC_242_256_R	TTCGCTCGCCGCTAC	1385
		AGTA

235	23S_EC_1602_1620_F	TACCCCAAACCGACACA	184	23S_EC_1686_1703_R	CCTTCTCCCGAAGTTACG	782
		GG

236	23S_EC_1685_1703_F	CCGTAACTTCGGGAGAA	58	23S_EC_1828_1842_R	CACCGGGCAGGCGTC	760
		GG

237	23S_EC_1827_1843_F	GACGCCTGCCCGGTGC	99	23S_EC_1929_1949_R	CCGACAAGGAATTTCGC	775
					TACC

238	23S_EC_2434_2456_F	AAGGTACTCCGGGGATA	9	23S_EC_2490_2511_R	AGCCGACATCGAGGTGC	746
		ACAGGC			CAAAC

239	23S_EC_2599_2616_F	GACAGTTCGGTCCCTAT	96	23S_EC_2653_2669_R	CCGGTCCTCTCGTACTA	777
		C

240	23S_EC_2653_2669_F	TAGTACGAGAGGACCGG	227	23S_EC_2737_2758_R	TTAGATGCTTTCAGCAC	1369
					TTATC

241	23S_BS_-68_-44_F	AAACTAGATAACAGTAG	1	23S_BS_5_21_R	GTGCGCCCTTTCTAACT	841
		ACATCAC			T

242	16S_EC_8_27_F	AGAGTTTGATCATGGCT	23	16S_EC_342_358_R	ACTGCTGCCTCCCGTAG	742
		CAG

243	16S_EC_314_332_F	CACTGGAACTGAGACAC	48	16S_EC_556_575_R	CTTTACGCCCAGTAATT	801
		GG			CCG

244	16S_EC_518_536_F	CCAGCAGCCGCGGTAAT	57	16S_EC_774_795_R	GTATCTAATCCTGTTTG	839
		AC			CTCCC

245	16S_EC_683_700_F	GTGTAGCGGTGAAATGC	137	16S_EC_967_985_R	GGTAAGGTTCTTCGCGT	835
		G			TG

246	16S_EC_937_954_F	AAGCGGTGGAGCATGTG	7	16S_EC_1220_1240_R	ATTGTAGCACGTGTGTA	757
		G			GCCC

247	16S_EC_1195_1213_F	CAAGTCATCATGGCCCT	46	16S_EC_1525_1541_R	AAGGAGGTGATCCAGCC	714
		TA

248	16S_EC_8_27_F	AGAGTTTGATCATGGCT	23	16S_EC_1525_1541_R	AAGGAGGTGATCCAGCC	714
		CAG

249	23S_EC_1831_1849_F	ACCTGCCCAGTGCTGGA	18	23S_EC_1919_1936_R	TCGCTACCTTAGGACCG	1080
		AG			T

250	16S_EC_1387_1407_F	GCCTTGTACACACCTCC	112	16S_EC_1494_1513_R	CACGGCTACCTTGTTAC	761
		CGTC			GAC

251	16S_EC_1390_1411_F	TTGTACACACCGCCCGT	693	16S_EC_1486_1505_R	CCTTGTTACGACTTCAC	783
		CATAC			CCC

252	16S_EC_1367_1387_F	TACGGTGAATACGTTCC	191	16S_EC_1485_1506_R	ACCTTGTTACGACTTCA	731
		CGGG			CCCCA

253	16S_EC_804_822_F	ACCACGCCGTAAACGAT	14	16S_EC_909_929_R	CCCCCGTCAATTCCTTT	773
		GA			GAGT

254	16S_EC_791_812_F	GATACCCTGGTAGTCCA	106	16S_EC_886_904_R	GCCTTGCGACCGTACTC	817
		CACCG			CC

255	16S_EC_789_810_F	TAGATACCCTGGTAGTC	206	16S_EC_882_899_R	GCGACCGTACTCCCCAG	818
		CACGC			G

256	16S_EC_1092_1109_F	TAGTCCCGCAACGAGCG	228	168_EC_1174_1195_R	GACGTCATCCCCACCTT	810
		C			CCTCC

257	23S_EC_2586_2607_F	TAGAACGTCGCGAGACA	203	23S_EC_2658_2677_R	AGTCCATCCCGGTCCTC	749
		GTTCG			TCG

258	RNASEP_SA_31_49_F	GAGGAAAGTCCATGCTC	103	RNASEP_SA_358_379_R	ATAAGCCATGTTCTGTT	750
		AC			CCATC

258	RNASEP_SA_31_49_F	GAGGAAAGTCCATGCTC	103	RNASEP_EC_345_362_R	ATAAGCCGGGTTCTGTC	751
		AC			G

258	RNASEP_SA_31_49_F	GAGGAAAGTCCATGCTC	103	RNASEP_BS_363_384_R	GTAAGCCATGTTTTGTT	838
		AC			CCATC

258	RNASEP_BS_43_61_F	GAGGAAAGTCCATGCTC	104	RNASEP_SA_358_379_R	ATAAGCCATGTTCTGTT	750
		GC			CCATC

258	RNASEP_BS_43_61_F	GAGGAAAGTCCATGCTC	104	RNASEP_EC_345_362_R	ATAAGCCGGGTTCTGTC	751
		GC			G

258	RNASEP_BS_43_61_F	GAGGAAAGTCCATGCTC	104	RNASEP_BS_363_384_R	GTAAGCCATGTTTTGTT	838
		GC			CCATC

258	RNASEP_EC_61_77_F	GAGGAAAGTCCGGGCTC	105	RNASEP_SA_358_379_R	ATAAGCCATGTTCTGTT	750
					CCATC

258	RNASEP_EC_61_77_F	GAGGAAAGTCCGGGCTC	105	RNASEP_EC_345_362_R	ATAAGCCGGGTTCTGTC	751
					G

258	RNASEP_EC_61_77_F	GAGGAAAGTCCGGGCTC	105	RNASEP_BS_363_384_R	GTAAGCCATGTTTTGTT	838
					CCATC

259	RNASEP_BS_43_61_F	GAGGAAAGTCCATGCTC	104	RNASEP_BS_363_384_R	GTAAGCCATGTTTTGTT	838
		GC			CCATC

260	RNASEP_EC_61_77_F	GAGGAAAGTCCGGGCTC	105	RNASEP_EC_345_362_R	ATAAGCCGGGTTCTGTC	751
					G

262	RNASEP_SA_31_49_F	GAGGAAAGTCCATGCTC	103	RNASEP_SA_358_379_R	ATAAGCCATGTTCTGTT	750
		AC			CCATC

263	16S_EC_1082_1100_F	ATGTTGGGTTAAGTCCC	37	16S_EC_1525_1541_R	AAGGAGGTGATCCAGCC	714
		GC

264	16S_EC_556_575_F	CGGAATTACTGGGCGTA	70	16S_EC_774_795_R	GTATCTAATCCTGTTTG	839
		AAG			CTCCC

265	16S_EC_1082_1100_F	ATGTTGGGTTAAGTCCC	37	16S_EC_1177_1196_10G_R	TGACGTCATGCCCACCT	1160
		GC			TCC

266	16S_EC_1082_1100_F	ATGTTGGGTTAAGTCCC	37	16S_EC_1177_1196_10G_	TGACGTCATGGCCACCT	1161
		GC		11G_R	TCC

268	YAED_EC_513_532_F_	GGTGTTAAATAGCCTGG	130	TRNA_ALA-	AGACCTCCTGCGTGCAA	744
	MOD	CAG		RRNH_EC_30_49_F_MOD	AGC

269	16S_EC_1082_1100_	ATGTTGGGTTAAGTCCC	37	16S_EC_1177_1196_R_MOD	TGACGTCATCCCCACCT	1158
	F_MOD	GC			TCC

270	23S_EC_2586_2607_	TAGAACGTCGCGAGACA	203	23S_EC_2658_2677_R_MOD	AGTCCATCCCGGTCCTC	749
	F_MOD	GTTCG			TCG

272	16S_EC_969_985_F	ACGCGAAGAACCTTACC	19	16S_EC_1389_1407_R	GACGGGCGGTGTGTACA	807
					AG

273	16S_EC_683_700_F	GTGTAGCGGTGAAATGC	137	16S_EC_1303_1323_R	CGAGTTGCAGACTGCGA	788
		G			TCCG

274	16S_EC_49_68_F	TAACACATGCAAGTCGA	152	16S_EC_880_894_R	CGTACTCCCCAGGCG	796
		ACG

275	16S_EC_49_68_F	TAACACATGCAAGTCGA	152	16S_EC_1061_1078_R	ACGACACGAGCTGACGA	734
		ACG			C

277	CYA_BA_1349_1370_F	ACAACGAAGTACAATAC	12	CYA_BA_1426_1447_R	CTTCTACATTTTTAGCC	800
		AAGAC			ATCAC

278	16S_EC_1090_1111_	TTAAGTCCCGCAACGAG	650	16S_EC_1175_1196_R	TGACGTCATCCCCACCT	1159
	2_F	CGCAA			TCCTC

279	16S_EC_405_432_F	TGAGTGATGAAGGCCTT	464	16S_EC_507_527_R	CGGCTGCTGGCACGAAG	793
		AGGGTTGTAAA			TTAG

280	GROL_EC_496_518_F	ATGGACAAGGTTGGCAA	34	GROL_EC_577_596_R	TAGCCGCGGTCGAATTG	914
		GGAAGG			CAT

281	GROL_EC_511_536_F	AAGGAAGGCGTGATCAC	8	GROL_EC_571_593_R	CCGCGGTCGAATTGCAT	776
		CGTTGAAGA			GCCTTC

288	RPOB_EC_3802_3821_	CAGCGTTTCGGCGAAAT	51	RPOB_EC_3862_3885_R	CGACTTGACGGTTAACA	786
	F	GGA			TTTCCTG

289	RPOB_EC_3799_3821_	GGGCAGCGTTTCGGCGA	124	RPOB_EC_3862_3888_R	GTCCGACTTGACGGTCA	840
	F	AATGGA			ACATTTCCTG

290	RPOC_EC_2146_2174_	CAGGAGTCGTTCAACTC	52	RPOC_EC_2227_2245_R	ACGCCATCAGGCCACGC	736
	F	GATCTACATGAT			AT

291	ASPS_EC_405_422_F	GCACAACCTGCGGCTGC	110	ASPS_EC_521_538_R	ACGGCACGAGGTAGTCG	738
		G			C

292	RPOC_EC_1374_1393_	CGCCGACTTCGACGGTG	69	RPOC_EC_1437_1455_R	GAGCATCAGCGTGCGTG	811
	F	ACC			CT

293	TUFB_EC_957_979_F	CCACACGCCGTTCTTCA	55	TUFB_EC_1034_1058_R	GGCATCACCATTTCCTT	829
		ACAACT			GTCCTTCG

294	16S_EC_7_33_F	GAGAGTTTGATCCTGGC	102	16S_EC_101_122_R	TGTTACTCACCCGTCTG	1345
		TCAGAACGAA			CCACT

295	VALS_EC_610_649_F	ACCGAGCAAGGAGACCA	17	VALS_EC_705_727_R	TATAACGCACATCGTCA	929
		GC			GGGTGA

344	16S_EC_971_990_F	GCGAAGAACCTTACCAG	113	16S_EC_1043_1062_R	ACAACCATGCACCACCT	726
		GTC			GTC

346	16S_EC_713_732_	TAGAACACCGATGGCGA	202	16S_EC_789_809_TMOD_R	TCGTGGACTACCAGGGT	1110
	TMOD_F	AGGC			ATCTA

347	16S_EC_785_806_	TGGATTAGAGACCCTGG	560	16S_EC_880_897_TMOD_R	TGGCCGTACTCCCCAGG	1278
	TMOD_F	TAGTCC			CG

348	16S_EC_960_981_	TTTCGATGCAACGCGAA	706	16S_EC_1054_1073_TMOD_R	TACGAGCTGACGACAGC	895
	TMOD_F	GAACCT			CATG

349	23S_EC_1826_1843_	TCTGACACCTGCCCGGT	401	23S_EC_1906_1924_TMOD_R	TGACCGTTATAGTTACG	1156
	TMOD_F	GC			GCC

350	CAPC_BA_274_303_	TGATTATTGTTATCCTG	476	CAPC_BA_349_376_TMOD_R	TGTAACCCTTGTCTTTG	1314
	TMOD_F	TTATGCCATTTGAG			AATTGTATTTGC

351	CYA_BA_1353_1379_	TCGAAGTACAATACAAG	355	CYA_BA_1448_1467_TMOD_R	TTGTTAACGGCTTCAAG	1423
	TMOD_F	ACAAAAGAAGG			ACCC

352	INFB_EC_1365_1393_	TTGCTCGTGGTGCACAA	687	INFB_EC_1439_1467_TMOD_R	TTGCTGCTTTCGCATGG	1411
	TMOD_F	GTAACGGATATTA			TTAATTGCTTCAA

353	LEF_BA_756_781_	TAGCTTTTGCATATTAT	220	LEF_BA_843_872_TMOD_R	TTCTTCCAAGGATAGAT	1394
	TMOD_F	ATCGAGCCAC			TTATTTCTTGTTCG

354	RPOC_EC_2218_2241_	TCTGGCAGGTATGCGTG	405	RPOC_EC_2313_2337_TMOD_R	TCGCACCGTGGGTTGAG	1072
	TMOD_F	GTCTGATG			ATGAAGTAC

355	SSPE_BA_115_137_	TCAAGCAAACGCACAAT	255	SSPE_BA_197_222_TMOD_R	TTGCACGTCTGTTTCAG	1402
	TMOD_F	CAGAAGC			TTGCAAATTC

356	RPLB_EC_650_679_	TGACCTACAGTAAGAGG	448	RPLB_EC_739_762_TMOD_R	TTCCAAGTGCTGGTTTA	1380
	TMOD_F	TTCTGTAATGAACC			CCCCATGG

357	RPLB_EC_688_710_	TCATCCACACGGTGGTG	296	RPLB_EC_736_757_TMOD_R	TGTGCTGGTTTACCCCA	1337
	TMOD_F	GTGAAGG			TGGAGT

358	VALS_EC_1105_1124_	TCGTGGCGGCGTGGTTA	385	VALS_EC_1195_1218_TMOD_R	TCGGTACGAACTGGATG	1093
	TMOD_F	TCGA			TCGCCGTT

359	RPOB_EC_1845_1866_	TTATCGCTCAGGCGAAC	659	RPOB_EC_1909_1929_TMOD_R	TGCTGGATTCGCCTTTG	1250
	TMOD_F	TCCAAC			CTACG

360	23S_EC_2646_2667_	TCTGTTCTTAGTACGAG	409	23S_EC_2745_2765_TMOD_R	TTTCGTGCTTAGATGCT	1434
	TMOD_F	AGGACC			TTCAG

361	16S_EC_1090_1111_	TTTAAGTCCCGCAACGA	697	16S_EC_1175_1196_TMOD_R	TTGACGTCATCCCCACC	1398
	2_TMOD_F	GCGCAA			TTCCTC

362	RPOB_EC_3799_3821_	TGGGCAGCGTTTCGGCG	581	RPOB_EC_3862_3888_TMOD_R	TGTCCGACTTGACGGTC	1325
	TMOD_F	AAATGGA			AACATTTCCTG

363	RPOC_EC_2146_2174_	TCAGGAGTCGTTCAACT	284	RPOC_EC_2227_2245_TMOD_R	TACGCCATCAGGCCACG	898
	TMOD_F	CGATCTACATGAT			CAT

364	RPOC_EC_1374_1393_	TCGCCGACTTCGACGGT	367	RPOC_EC_1437_1455_TMOD_R	TGAGCATCAGCGTGCGT	1166
	TMOD_F	GACC			GCT

367	TUFB_EC_957_979_	TCCACACGCCGTTCTTC	308	TUFB_EC_1034_1058_TMOD_R	TGGCATCACCATTTCCT	1276
	TMOD_F	AACAACT			TGTCCTTCG

423	SP101_SPET11_893_	TGGGCAACAGCAGCGGA	580	SP101_SPET11_988_1012_	TCATGACAGCCAAGACC	990
	921_TMOD_F	TTGCGATTGCGCG		TMOD_R	TCACCCACC

424	SP101_SPET11_1154_	TCAATACCGCAACAGCG	258	SP101_SPET11_1251_1277_	TGACCCCAACCTGGCCT	1155
	1179_TMOD_F	GTGGCTTGGG		TMOD_R	TTTGTCGTTGA

425	SP101_SPET11_118_	TGCTGGTGAAAATAACC	528	SP101_SPET11_213_238_	TTGTGGCCGATTTCACC	1422
	147_TMOD_F	CAGATGTCGTCTTC		TMOD_R	ACCTGCTCCT

426	SP101_SPET11_1314_	TCGCAAAAAAATCCAGC	363	SP101_SPET11_1403_1431_	TAAACTATTTTTTTAGC	849
	1336_TMOD_F	TATTAGC		TMOD_R	TATACTCGAACAC

427	SP101_SPET11_1408_	TCGAGTATAGCTAAAAA	359	SP101_SPET11_1486_1515_	TGGATAATTGGTCGTAA	1268
	1437_TMOD_F	AATAGTTTATGACA		TMOD_R	CAAGGGATAGTGAG

428	SP101_SPET11_1688_	TCCTATATTAATCGTTT	334	SP101_SPET11_1783_1808_	TATATGATTATCATTGA	932
	1716_TMOD_F	ACAGAAACTGGCT		TMOD_R	ACTGCGGCCG

429	SP101_SPET11_1711_	TCTGGCTAAAACTTTGG	406	SP101_SPET11_1808_1835_	TGCGTGACGACCTTCTT	1239
	1733_TMOD_F	CAACGGT		TMOD_R	GAATTGTAATCA

430	SP101_SPET11_1807_	TATGATTACAATTCAAG	235	SP101_SPET11_1901_1927_	TTTGGACCTGTAATCAG	1439
	1835_TMOD_F	AAGGTCGTCACGC		TMOD_R	CTGAATACTGG

431	SP101_SPET11_1967_	TTAACGGTTATCATGGC	649	SP101_SPET11_2062_2083_	TATTGCCCAGAAATCAA	940
	1991_TMOD_F	CCAGATGGG		TMOD_R	ATCATC

432	SP101_SPET11_216_	TAGCAGGTGGTGAAATC	210	SP101_SPET11_308_333_	TTGCCACTTTGACAACT	1404
	243_TMOD_F	GGCCACATGATT		TMOD_R	CCTGTTGCTG

433	SP101_SPET11_2260_	TCAGAGACCGTTTTATC	272	SP101_SPET11_2375_2397_	TTCTGGGTGACCTGGTG	1393
	2283_TMOD_F	CTATCAGC		TMOD_R	TTTTAGA

434	SP101_SPET11_2375_	TTCTAAAACACCAGGTC	675	SP101_SPET11_2470_2497_	TAGCTGCTAGATGAGCT	918
	2399_TMOD_F	ACCCAGAAG		TMOD_R	TCTGCCATGGCC

435	SP101_SPET11_2468_	TATGGCCATGGCAGAAG	238	SP101_SPET11_2543_2570_	TCCATAAGGTCACCGTC	1007
	2487_TMOD_F	CTCA		TMOD_R	ACCATTCAAAGC

436	SP101_SPET11_266_	TCTTGTACTTGTGGCTC	417	SP101_SPET11_355_380_	TGCTGCTTTGATGGCTG	1249
	295_TMOD_F	ACACGGCTGTTTGG		TMOD_R	AATCCCCTTC

437	SP101_SPET11_2961_	TACCATGACAGAAGGCA	183	SP101_SPET11_3023_3045_	TGGAATTTACCAGCGAT	1264
	2984_TMOD_F	TTTTGACA		TMOD_R	AGACACC

438	SP101_SPET11_3075_	TGATCACTTTTTAGCTA	473	SP101_SPET11_3168_3196_	TAATCGACGACCATCTT	875
	3103_TMOD_F	ATGGTCAGGCAGC		TMOD_R	GGAAAGATTTCTC

439	SP101_SPET11_322_	TGTCAAAGTGGCACGTT	631	SP101_SPET11_423_441_	TATCCCCTGCTTCTGCT	934
	344_TMOD_F	TACTGGC		TMOD_R	GCC

440	SP101_SPET11_3386_	TAGCGTAAAGGTGAACC	215	SP101_SPET11_3480_3506_	TCCAGCAGTTACTGTCC	1005
	3403_TMOD_F	TT		TMOD_R	CCTCATCTTTG

441	SP101_SPET11_3511_	TGCTTCAGGAATCAATG	531	SP101_SPET11_3605_3629_	TGGGTCTACACCTGCAC	1294
	3535_TMOD_F	ATGGAGCAG		TMOD_R	TTGCATAAC

442	SP101_SPET11_358_	TGGGGATTCAGCCATCA	588	SP101_SPET11_448_473_	TCCAACCTTTTCCACAA	998
	387_TMOD_F	AAGCAGCTATTGAC		TMOD_R	CAGAATCAGC

443	SP101_SPET11_600_	TCCTTACTTCGAACTAT	348	SP101_SPET11_686_714_	TCCCATTTTTTCACGCA	1018
	629_TMOD_F	GAATCTTTTGGAAG		TMOD_R	TGCTGAAAATATC

444	SP101_SPET11_658_	TGGGGATTGATATCACC	589	SP101_SPET11_756_784_	TGATTGGCGATAAAGTG	1189
	684_TMOD_F	GATAAGAAGAA		TMOD_R	ATATTTTCTAAAA

445	SP101_SPET11_776_	TTCGCCAATCAAAACTA	673	SP101_SPET11_871_896_	TGCCCACCAGAAAGACT	1217
	801_TMOD_F	AGGGAATGGC		TMOD_R	AGCAGGATAA

446	SP101_SPET11_1_29_	TAACCTTAATTGGAAAG	154	SP101_SPET11_92_116_	TCCTACCCAACGTTCAC	1044
	TMOD_F	AAACCCAAGAAGT		TMOD_R	CAAGGGCAG

447	SP101_SPET11_364_	TCAGCCATCAAAGCAGC	276	SP101_SPET11_448_471_R	TACCTTTTCCACAACAG	894
	385_F	TATTG			AATCAGC

448	SP101_SPET11_3085_	TAGCTAATGGTCAGGCA	216	SP101_SPET11_3170_3194_R	TCGACGACCATCTTGGA	1066
	3104_F	GCC			AAGATTTC

449	RPLB_EC_690_710_F	TCCACACGGTGGTGGTG	309	RPLB_EC_737_758_R	TGTGCTGGTTTACCCCA	1336
		AAGG			TGGAG

481	BONTA_X52066_538_	TATGGCTCTACTCAA	239	BONTA_X52066_647_660_R	TGTTACTGCTGGAT	1346
	552_F

482	BONTA_X52066_538_	TATpGGCTpCpTp	143	BONTA_X52066_647_660P_R	TGTpTpACpTpG*	1146
	552P_F	ACpTp*CpAA			Cp*TpGGAT

483	BONTA_X52066_701_	GAATAGCAATTAATCCA	94	BONTA_X52066_759_775_R	TTACTTCTAACCCACTC	1367
	720_F	AAT

484	BONTA_X52066_701_	GAATpAGCpAATp	91	BONTA_X52066_759_775P_R	TTACpTpTpCp*T	1359
	720P_F	TpAATpCp*CpAAAT			pAACpCpCpACp*TpC

485	BONTA_X52066_450_	TCTAGTAATAATAGGAC	393	BONTA_X52066_517_539_R	TAACCATTTCGCGTAAG	859
	473_F	CCTCAGC			ATTCAA

486	BONTA_X52066_450_	TCpTpAGTAATAATA	142	BONTA_X52066_517_539P_R	TAACCATpTpTp	857
	473P_F	GGACpCpCpTp*			CpGCGTAAGATpTp*
		CpAGC			CpAA

487	BONTA_X52066_591_	TGAGTCACTTGAAGTTG	463	BONTA_X52066_644_671_R	TCATGTGCTAATGTTAC	992
	620_F	ATACAAATCCTCT			TGCTGGATCTG

608	SSPE_BA_156_168P_F	TGGTpGCpTpAGCpATT	616	SSPE_BA_243_255P_R	TGCpAGCpTGATpTpGT	1241

609	SSPE_BA_75_89P_F	TACpAGAGTpTpTpGCp	192	SSPE_BA_163_177P_R	TGTGCTpTpTpGAATpG	1338
		GAC			CpT

610	SSPE_BA_150_168P_F	TGCTTCTGGTpGCpTpA	533	SSPE_BA_243_264P_R	TGATTGTTTTGCpAGCp	1191
		GCpATT			TGATpTpGT

611	SSPE_BA_72_89P_F	TGGTACpAGAGTpTpTp	602	SSPE_BA_163_182P_R	TCATTTGTGCTpTpTpG	995
		GCpGAC			AATpGCpT

612	SSPE_BA_114_137P_F	TCAAGCAAACGCACAAT	255	SSPE_BA_196_222P_R	TTGCACGTCpTpGTTTC	1401
		pCpAGAAGC			AGTTGCAAATTC

699	SSPE_BA_123_153_F	TGCACAATCAGAAGCTA	488	SSPE_BA_202_231_R	TTTCACAGCATGCACGT	1431
		AGAAAGCGCAAGCT			CTGTTTCAGTTGC

700	SSPE_BA_156_168_F	TGGTGCTAGCATT	612	SSPE_BA_243_255_R	TGCAGCTCATTGT	1202

701	SSPE_BA_75_89_F	TACAGAGTTTGCGAC	179	SSPE_BA_163_177_R	TGTGCTTTGAATGCT	1338

702	SSPE_BA_150_168_F	TGCTTCTGGTGCTAGCA	533	SSPE_BA_243_264_R	TGATTGTTTTGCAGCTG	1190
		TT			ATTGT

703	SSPE_BA_72_89_F	TGGTACAGAGTTTGCGA	600	SSPE_BA_163_182_R	TCATTTGTGCTTTGAAT	995
		C			GCT

704	SSPE_BA_146_168_F	TGCAAGCTTCTGGTGCT	484	SSPE_BA_242_267_R	TTGTGATTGTTTTGCAG	1421
		AGCATT			CTGATTGTG

705	SSPE_BA_63_89_F	TGCTAGTTATGGTACAG	518	SSPE_BA_163_191_R	TCATAACTAGCATTTGT	986
		AGTTTGCGAC			GCTTTGAATGCT

706	SSPE_BA_114_137_F	TCAAGCAAACGCACAAT	255	SSPE_BA_196_222_R	TTGCACGTCTGTTTCAG	1402
		CAGAAGC			TTGCAAATTC

770	PLA_AF053945_7377_	TGACATCCGGCTCACGT	442	PLA_AF053945_7434_7462_R	TGTAAATTCCGCAAAGA	1313
	7402_F	TATTATGGT			CTTTGGCATTAG

771	PLA_AF053945_7382_	TCCGGCTCACGTTATTA	327	PLA_AF053945_7482_7502_R	TGGTCTGAGTACCTCCT	1304
	7404_F	TGGTAC			TTGC

772	PLA_AF053945_7481_	TGCAAAGGAGGTACTCA	481	PLA_AF053945_7539_7562_R	TATTGGAAATACCGGCA	943
	7503_F	GACCAT			GCATCTC

773	PLA_AF053945_7186_	TTATACCGGAAACTTCC	657	PLA_AF053945_7257_7280_R	TAATGCGATACTGGCCT	879
	7211_F	CGAAAGGAG			GCAAGTC

774	CAF1_AF053947_	TCAGTTCCGTTATCGCC	292	CAF1_AF053947_33494_	TGCGGGCTGGTTCAACA	1235
	33407_33430_F	ATTGCAT		33514_R	AGAG

775	CAF1_AF053947_	TCACTCTTACATATAAG	270	CAF1_AF053947_33595_	TCCTGTTTTATAGCCGC	1053
	33515_33541_F	GAAGGCGCTC		33621_R	CAAGAGTAAG

776	CAF1_AF053947_	TGGAACTATTGCAACTG	542	CAF1_AF053947_33499_	TGATGCGGGCTGGTTCA	1183
	33435_33457_F	CTAATG		33517_R	AC

777	CAF1_AF053947_	TCAGGATGGAAATAACC	286	CAF1_AF053947_33755_	TCAAGGTTCTCACCGTT	962
	33687_33716_F	ACCAATTCACTAC		33782_R	TACCTTAGGAG

778	INV_U22457_515_	TGGCTCCTTGGTATGAC	573	INV_U22457_571_598_R	TGTTAAGTGTGTTGCGG	1343
	539_F	TCTGCTTC			CTGTCTTTATT

779	INV_U22457_699_	TGCTGAGGCCTGGACCG	525	INV_U22457_753_776_R	TCACGCGACGAGTGCCA	976
	724_F	ATTATTTAC			TCCATTG

780	INV_U22457_834_	TTATTTACCTGCACTCC	664	INV_U22457_942_966_R	TGACCCAAAGCTGAAAG	1154
	858_F	CACAACTG			CTTTACTG

781	INV_U22457_1558_	TGGTAACAGAGCCTTAT	597	INV_U22457_1619_1643_R	TTGCGTTGCAGATTATC	1408
	1581_F	AGGCGCA			TTTACCAA

782	LL_NC003143_	TGTAGCCGCTAAGCACT	627	LL_NC003143_2367073_	TCTCATCCCGATATTAC	1123
	2366996_2367019_F	ACCATCC		2367097_R	CGCCATGA

783	LL_NC003143_	TGGACGGCATCACGATT	550	LL_NC003143_2367249_	TGGCAACAGCTCAACAC	1272
	2367172_2367194_F	CTCTAC		2367271_R	CTTTGG

874	RPLB_EC_649_679_F	TGICCIACIGTIIGIGG	620	RPLB_EC_739_762_TMOD_R	TTCCAAGTGCTGGTTTA	1380
		TTCTGTAATGAACC			CCCCATGG

875	RPLB_EC_642_679P_F	TpCpCpTpTpGITpGIC	646	RPLB_EC_739_762_TMOD_R	TTCCAAGTGCTGGTTTA	1380
		CIACIGTIIGIGGTTCT			CCCCATGG
		GTAATGAACC

876	MECIA_Y14051_3315_	TTACACATATCGTGAGC	653	MECIA_Y14051_3367_3393_R	TGTGATATGGAGGTGTA	1333
	3341_F	AATGAACTGA			GAAGGTGTTA

877	MECA_Y14051_3774_	TAAAACAAACTACGGTA	144	MECA_Y14051_3828_3854_R	TCCCAATCTAACTTCCA	1015
	3802_F	ACATTGATCGCA			CATACCATCT

878	MECA_Y14051_3645_	TGAAGTAGAAATGACTG	434	MECA_Y14051_3690_3719_R	TGATCCTGAATGTTTAT	1181
	3670_F	AACGTCCGA			ATCTTTAACGCCT

879	MECA_Y14051_4507_	TCAGGTACTGCTATCCA	288	MECA_Y14051_4555_4581_R	TGGATAGACGTCATATG	1269
	4530_F	CCCTCAA			AAGGTGTGCT

880	MECA_Y14051_4510_	TGTACTGCTATCCACCC	626	MECA_Y14051_4586_4610_R	TATTCTTCGTTACTCAT	939
	4530_F	TCAA			GCCATACA

881	MECA_Y14051_4669_	TCACCAGGTTCAACTCA	262	MECA_Y14051_4765_4793_R	TAACCACCCCAAGATTT	858
	4698_F	AAAAATATTAACA			ATCTTTTTGCCA

882	MECA_Y14051_4520_	TCpCpACpCpCpTpCpA	389	MECA_Y14051_4590_4600P_R	TpACpTpCpATpGCpCp	1357
	4530P_F	A			A

883	MECA_Y14051_4520_	TCpCpACpCpCpTpCpA	389	MECA_Y14051_4600_4610P_R	TpATpTpCpTpTpCpGT	1358
	4530P_F	A			pT

902	TRPE_AY094355_	ATGTCGATTGCAATCCG	36	TRPE_AY094355_1569_	TGCGCGAGCTTTTATTT	1231
	1467_1491_F	TACTTGTG		1592_R	GGGTTTC

903	TRPE_AY094355_	TGGATGGCATGGTGAAA	557	TRPE_AY094355_1551_	TATTTGGGTTTCATTCC	944
	1445_1471_F	TGGATATGTC		1580_R	ACTCAGATTCTGG

904	TRPE_AY094355_	TCAAATGTACAAGGTGA	247	TRPE_AY094355_1392_	TCCTCTTTTCACAGGCT	1048
	1278_1303_F	AGTGCGTGA		1418_R	CTACTTCATC

905	TRPE_AY094355_	TCGACCTTTGGCAGGAA	357	TRPE_AY094355_1171_	TACATCGTTTCGCCCAA	885
	1064_1086_F	CTAGAC		1196_R	GATCAATCA

906	TRPE_AY094355_666_	GTGCATGCGGATACAGA	135	TRPE_AY094355_769_791_R	TTCAAAATGCGGAGGCG	1372
	688_F	GCAGAG			TATGTG

907	TRPE_AY094355_757_	TGCAAGCGCGACCACAT	483	TRPE_AY094355_864_883_R	TGCCCAGGTACAACCTG	1218
	776_F	ACG			CAT

908	RECA_AF251469_43_	TGGTACATGTGCCTTCA	601	RECA_AF251469_140_163_R	TTCAAGTGCTTGCTCAC	1375
	68_F	TTGATGCTG			CATTGTC

909	RECA_AF251469_169_	TGACATGCTTGTCCGTT	446	RECA_AF251469_277_300_R	TGGCTCATAAGACGCGC	1280
	190_F	CAGGC			TTGTAGA

910	PARC_X95819_87_	TGGTGACTCGGCATGTT	609	PARC_X95819_201_222_R	TTCGGTATAACGCATCG	1387
	110_F	ATGAAGC			CAGCA

911	PARC_X95819_87_	TGGTGACTCGGCATGTT	609	PARC_X95819_192_219_R	GGTATAACGCATCGCAG	836
	110_F	ATGAAGC			CAAAAGATTTA

912	PARC_X95819_123_	GGCTCAGCCATTTAGTT	120	PARC_X95819_232_260_R	TCGCTCAGCAATAATTC	1081
	147_F	ACCGCTAT			ACTATAAGCCGA

913	PARC_X95819_43_	TCAGCGCGTACAGTGGG	277	PARC_X95819_143_170_R	TTCCCCTGACCTTCGAT	1383
	63_F	TGAT			TAAAGGATAGC

914	OMPA_AY485227_272_	TTACTCCATTATTGCTT	655	OMPA_AY485227_364_388_R	GAGCTGCGCCAACGAAT	812
	301_F	GGTTACACTTTCC			AAATCGTC

915	OMPA_AY485227_379_	TGCGCAGCTCTTGGTAT	509	OMPA_AY485227_492_519_R	TGCCGTAACATAGAAGT	1223
	401_F	CGAGTT			TACCGTTGATT

916	OMPA_AY485227_311_	TACACAACAATGGCGGT	178	OMPA_AY485227_424_453_R	TACGTCGCCTTTAACTT	901
	335_F	AAAGATGG			GGTTATATTCAGC

917	OMPA_AY485227_415_	TGCCTCGAAGCTGAATA	506	OMPA_AY485227_514_546_R	TCGGGCGTAGTTTTTAG	1092
	441_F	TAACCAAGTT			TAATTAAATCAGAAGT

918	OMPA_AY485227_494_	TCAACGGTAACTTCTAT	252	OMPA_AY485227_569_596_R	TCGTCGTATTTATAGTG	1108
	520_F	GTTACTTCTG			ACCAGCACCTA

919	OMPA_AY485227_551_	TCAAGCCGTACGTATTA	257	OMPA_AY485227_658_680_R	TTTAAGCGCCAGAAAGC	1425
	577_F	TTAGGTGCTG			ACCAAC

920	OMPA_AY485227_555_	TCCGTACGTATTATTAG	328	OMPA_AY485227_635_662_R	TCAACACCAGCGTTACC	954
	581_F	GTGCTGGTCA			TAAAGTACCTT

921	OMPA_AY485227_556_	TCGTACGTATTATTAGG	379	OMPA_AY485227_659_683_R	TCGTTTAAGCGCCAGAA	1114
	583_F	TGCTGGTCACT			AGCACCAA

922	OMPA_AY485227_657_	TGTTGGTGCTTTCTGGC	645	OMPA_AY485227_739_765_R	TAAGCCAGCAAGAGCTG	871
	679_F	GCTTAA			TATAGTTCCA

923	OMPA_AY485227_660_	TGGTGCTTTCTGGCGCT	613	OMPA_AY485227_786_807_R	TACAGGAGCAGCAGGCT	884
	683_F	TAAACGA			TCAAG

924	GYRA_AF100557_4_	TCTGCCCGTGTCGTTGG	402	GYRA_AF100557_119_142_R	TCGAACCGAAGTTACCC	1063
	23_F	TGA			TGACCAT

925	GYRA_AF100557_70_	TCCATTGTTCGTATGGC	316	GYRA_AF100557_178_201_R	TGCCAGCTTAGTCATAC	1211
	94_F	TCAAGACT			GGACTTC

926	GYRB_AB008700_19_	TCAGGTGGCTTACACGG	289	GYRB_AB008700_111_140_R	TATTGCGGATCACCATG	941
	40_F	CGTAG			ATGATATTCTTGC

927	GYRB_AB008700_265_	TCTTTCTTGAATGCTGG	420	GYRB_AB008700_369_395_R	TCGTTGAGATGGTTTTT	1113
	292_F	TGTACGTATCG			ACCTTCGTTG

928	GYRB_AB008700_368_	TCAACGAAGGTAAAAAC	251	GYRB_AB008700_466_494_R	TTTGTGAAACAGCGAAC	1440
	394_F	CATCTCAACG			ATTTTCTTGGTA

929	GYRB_AB008700_477_	TGTTCGCTGTTTCACAA	641	GYRB_AB008700_611_632_R	TCACGCGCATCATCACC	977
	504_F	ACAACATTCCA			AGTCA

930	GYRB_AB008700_760_	TACTTACTTGAGAATCC	198	GYRB_AB008700_862_888_R	ACCTGCAATATCTAATG	729
	787_F	ACAAGCTGCAA			CACTCTTACG

931	WAAA_Z96925_2_29_F	TCTTGCTCTTTCGTGAG	416	WAAA_Z96925_115_138_R	CAAGCGGTTTGCCTCAA	758
		TTCAGTAAATG			ATAGTCA

932	WAAA_Z96925_286_	TCGATCTGGTTTCATGC	360	WAAA_Z96925_394_412_R	TGGCACGAGCCTGACCT	1274
	311_F	TGTTTCAGT			GT

939	RPOB_EC_3798_3821_	TGGGCAGCGTTTCGGCG	581	RPOB_EC_3862_3889_R	TGTCCGACTTGACGGTC	1326
	F	AAATGGA			AGCATTTCCTG

940	RPOB_EC_3798_3821_	TGGGCAGCGTTTCGGCG	581	RPOB_EC_3862_3889_2_R	TGTCCGACTTGACGGTT	1327
	F	AAATGGA			AGCATTTCCTG

941	TUFB_EC_275_299_F	TGATCACTGGTGCTGCT	468	TUFB_EC_337_362_R	TGGATGTGCTCACGAGT	1271
		CAGATGGA			CTGTGGCAT

942	TUFB_EC_251_278_F	TGCACGCCGACTATGTT	493	TUFB_EC_337_360_R	TATGTGCTCACGAGTTT	937
		AAGAACATGAT			GCGGCAT

949	GYRB_AB008700_760_	TACTTACTTGAGAATCC	198	GYRB_AB008700_862_888_	TCCTGCAATATCTAATG	1050
	787_F	ACAAGCTGCAA		2_R	CACTCTTACG

958	RPOC_EC_2223_2243_	TGGTATGCGTGGTCTGA	605	RPOC_EC_2329_2352_R	TGCTAGACCTTTACGTG	1243
	F	TGGC			CACCGTG

959	RPOC_EC_918_938_F	TCTGGATAACGGTCGTC	404	RPOC_EC_1009_1031_R	TCCAGCAGGTTCTGACG	1004
		GCGG			GAAACG

960	RPOC_EC_2334_2357_	TGCTCGTAAGGGTCTGG	523	RPOC_EC_2380_2403_R	TACTAGACGACGGGTCA	905
	F	CGGATAC			GGTAACC

961	RPOC_EC_917_938_F	TATTGGACAACGGTCGT	242	RPOC_EC_1009_1034_R	TTACCGAGCAGGTTCTG	1362
		CGCGG			ACGGAAACG

962	RPOB_EC_2005_2027_	TCGTTCCTGGAACACGA	387	RPOB_EC_2041_2064_R	TTGACGTTGCATGTTCG	1399
	F	TGACGC			AGCCCAT

963	RPOB_EC_1527_1549_	TCAGCTGTCGCAGTTCA	282	RPOB_EC_1630_1649_R	TCGTCGCGGACTTCGAA	1104
	F	TGGACC			GCC

964	INFB_EC_1347_1367_	TGCGTTTACCGCAATGC	515	INFB_EC_1414_1432_R	TCGGCATCACGCCGTCG	1090
	F	GTGC			TC

965	VALS_EC_1128_1151_	TATGCTGACCGACCAGT	237	VALS_EC_1231_1257_R	TTCGCGCATCCAGGAGA	1384
	F	GGTACGT			AGTACATGTT

978	RPOC_EC_2145_2175_	TCAGGAGTCGTTCAACT	285	RPOC_EC_2228_2247_R	TTACGCCATCAGGCCAC	1363
	F	CGATCTACATGATG			GCA

1045	CJST_CJ_1668_	TGCTCGAGTGATTGACT	522	CJST_CJ_1774_1799_R	TGAGCGTGTGGAAAAGG	1170
	1700_F	TTGCTAAATTTAGAGA			ACTTGGATG

1046	CJST_CJ_2171_	TCGTTTGGTGGTGGTAG	388	CJST_CJ_2283_2313_R	TCTCTTTCAAAGCACCA	1126
	2197_F	ATGAAAAAGG			TTGCTCATTATAGT

1047	CJST_CJ_584_616_F	TCCAGGACAAATGTATG	315	CJST_CJ_663_692_R	TTCATTTTCTGGTCCAA	1379
		AAAAATGTCCAAGAAC			AGTAAGCAGTATC

1048	CJST_CJ_360_394_F	TCCTGTTATCCCTGAAG	346	CJST_CJ_442_476_R	TCAACTGGTTCAAAAAC	955
		TAGTTAATCAAGTTTGT			ATTAAGTTGTAATTGTC
		T			C

1049	CJST_CJ_2636_	TGCCTAGAAGATCTTAA	504	CJST_CJ_2753_2777_R	TTGCTGCCATAGCAAAG	1409
	2668_F	AAATTTCCGCCAACTT			CCTACAGC

1050	CJST_CJ_1290_	TGGCTTATCCAAATTTA	575	CJST_CJ_1406_1433_R	TTTGCTCATGATCTGCA	1437
	1320_F	GATCGTGGTTTTAC			TGAAGCATAAA

1051	CJST_CJ_3267_	TTTGATTTTACGCCGTC	707	CJST_CJ_3356_3385_R	TCAAAGAACCCGCACCT	951
	3293_F	CTCCAGGTCG			AATTCATCATTTA

1052	CJST_CJ_5_39_F	TAGGCGAAGATATACAA	222	CJST_CJ_104_137_R	TCCCTTATTTTTCTTTC	1029
		AGAGTATTAGAAGCTAG			TACTACCTTCGGATAAT
		A

1053	CJST_CJ_1080_	TTGAGGGTATGCACCGT	681	CJST_CJ_1166_1198_R	TCCCCTCATGTTTAAAT	1022
	1110_F	CTTTTTGATTCTTT			GATCAGGATAAAAAGC

1054	CJST_CJ_2060_	TCCCGGACTTAATATCA	323	CJST_CJ_2148_2174_R	TCGATCCGCATCACCAT	1068
	2090_F	ATGAAAATTGTGGA			CAAAAGCAAA

1055	CJST_CJ_2869_	TGAAGCTTGTTCTTTAG	432	CJST_CJ_2979_3007_R	TCCTCCTTGTGCCTCAA	1045
	2895_F	CAGGACTTCA			AACGCATTTTTA

1056	CJST_CJ_1880_	TCCCAATTAATTCTGCC	317	CJST_CJ_1981_2011_R	TGGTTCTTACTTGCTTT	1309
	1910_F	ATTTTTCCAGGTAT			GCATAAACTTTCCA

1057	CJST_CJ_2185_	TAGATGAAAAGGGCGAA	208	CJST_CJ_2283_2316_R	TGAATTCTTTCAAAGCA	1152
	2212_F	GTGGCTAATGG			CCATTGCTCATTATAGT

1058	CJST_CJ_1643_	TTATCGTTTGTGGAGCT	660	CJST_CJ_1724_1752_R	TGCAATGTGTGCTATGT	1198
	1670_F	AGTGCTTATGC			CAGCAAAAAGAT

1059	CJST_CJ_2165_	TGCGGATCGTTTGGTGG	511	CJST_CJ_2247_2278_R	TCCACACTGGATTGTAA	1002
	2194_F	TTGTAGATGAAAA			TTTACCTTGTTCTTT

1060	CJST_CJ_599_632_F	TGAAAAATGTCCAAGAA	424	CJST_CJ_711_743_R	TCCCGAACAATGAGTTG	1024
		GCATAGCAAAAAAAGCA			TATCAACTATTTTTAC

1061	CJST_CJ_360_393_F	TCCTGTTATCCCTGAAG	345	CJST_CJ_443_477_R	TACAACTGGTTCAAAAA	882
		TAGTTAATCAAGTTTGT			CATTAAGCTGTAATTGT
					C

1062	CJST_CJ_2678_	TCCCCAGGACACCCTGA	321	CJST_CJ_2760_2787_R	TGTGCTTTTTTTGCTGC	1339
	2703_F	AATTTCAAC			CATAGCAAAGC

1063	CJST_CJ_1268_	AGTTATAAACACGGCTT	29	CJST_CJ_1349_1379_R	TCGGTTTAAGCTCTACA	1096
	1299_F	TCCTATGGCTTATCC			TGATCGTAAGGATA

1064	CJST_CJ_1680_	TGATTTTGCTAAATTTA	479	CJST_CJ_1795_1822_R	TATGTGTAGTTGAGCTT	938
	1713_F	GAGAAATTGCGGATGAA			ACTACATGAGC

1065	CJST_CJ_2857_	TGGCATTTCTTATGAAG	565	CJST_CJ_2965_2998_R	TGCTTCAAAACGCATTT	1253
	2887_F	CTTGTTCTTTAGCA			TTACATTTTCGTTAAAG

1070	RNASEP_BKM_580_	TGCGGGTAGGGAGCTTG	512	RNASEP_BKM_665_686_R	TCCGATAAGCCGGATTC	1034
	599_F	AGC			TGTGC

1071	RNASEP_BKM_616_	TCCTAGAGGAATGGCTG	333	RNASEP_BKM_665_687_R	TGCCGATAAGCCGGATT	1222
	637_F	CCACG			CTGTGC

1072	RNASEP_BDP_574_	TGGCACGGCCATCTCCG	561	RNASEP_BDP_616_635_R	TCGTTTCACCCTGTCAT	1115
	592_F	TG			GCCG

1073	23S_BRM_1110_1129_	TGCGCGGAAGATGTAAC	510	23S_BRM_1176_1201_R	TCGCAGGCTTACAGAAC	1074
	F	GGG			GCTCTCCTA

1074	23S_BRM_515_536_F	TGCATACAAACAGTCGG	496	23S_BRM_616_635_R	TCGGACTCGCTTTCGCT	1088
		AGCCT			ACG

1075	RNASEP_CLB_459_	TAAGGATAGTGCAACAG	162	RNASEP_CLB_498_526_R	TGCTCTTACCTCACCGT	1247
	487_F	AGATATACCGCC			TCCACCCTTACC

1076	RNASEP_CLB_459_	TAAGGATAGTGCAACAG	162	RNASEP_CLB_498_522_R	TTTACCTCGCCTTTCCA	1426
	487_F	AGATATACCGCC			CCCTTACC

1077	ICD_CXB_93_120_F	TCCTGACCGACCCATTA	343	ICD_CXB_172_194_R	TAGGATTTTTCCACGGC	921
		TTCCCTTTATC			GGCATC

1078	ICD_CXB_92_120_F	TTCCTGACCGACCCATT	671	ICD_CXB_172_194_R	TAGGATTTTTCCACGGC	921
		ATTCCCTTTATC			GGCATC

1079	ICD_CXB_176_198_F	TCGCCGTGGAAAAATCC	369	ICD_CXB_224_247_R	TAGCCTTTTCTCCGGCG	916
		TACGCT			TAGATCT

1080	IS1111A_NC002971_	TCAGTATGTATCCACCG	290	IS1111A_NC002971_6928_	TAAACGTCCGATACCAA	848
	6866_6891_F	TAGCCAGTC		6954_R	TGGTTCGCTC

1081	IS1111A_NC002971_	TGGGTGACATTCATCAA	594	IS1111A_NC002971_7529_	TCAACAACACCTCCTTA	952
	7456_7483_F	TTTCATCGTTC		7554_R	TTCCCACTC

1082	RNASEP_RKP_419_	TGGTAAGAGCGCACCGG	599	RNASEP_RKP_542_565_R	TCAAGCGATCTACCCGC	957
	448_F	TAAGTTGGTAACA			ATTACAA

1083	RNASEP_RKP_422_	TAAGAGCGCACCGGTAA	159	RNASEP_RKP_542_565_R	TCAAGCGATCTACCCGC	957
	443_F	GTTGG			ATTACAA

1084	RNASEP_RKP_466_	TCCACCAAGAGCAAGAT	310	RNASEP_RKP_542_565_R	TCAAGCGATCTACCCGC	957
	491_F	CAAATAGGC			ATTACAA

1085	RNASEP_RKP_264_	TCTAAATGGTCGTGCAG	391	RNASEP_RKP_295_321_R	TCTATAGAGTCCGGACT	1119
	287_F	TTGCGTG			TTCCTCGTGA

1086	RNASEP_RKP_426_	TGCATACCGGTAAGTTG	497	RNASEP_RKP_542_565_R	TCAAGCGATCTACCCGC	957
	448_F	GCAACA			ATTACAA

1087	OMPB_RKP_860_890_F	TTACAGGAAGTTTAGGT	654	OMPB_RKP_972_996_R	TCCTGCAGCTCTACCTG	1051
		GGTAATCTAAAAGG			CTCCATTA

1088	OMPB_RKP_1192_	TCTACTGATTTTGGTAA	392	OMPB_RKP_1288_1315_R	TAGCAgCAAAAGTTATC	910
	1221_F	TCTTGCAGCACAG			ACACCTGCAGT

1089	OMPB_RKP_3417_	TGCAAGTGGTACTTCAA	485	OMPB_RKP_3520_3550_R	TGGTTGTAGTTCCTGTA	1310
	3440_F	CATGGGG			GTTGTTGCATTAAC

1090	GLTA_RKP_1043_	TGGGACTTGAAGCTATC	576	GLTA_RKP_1138_1162_R	TGAACATTTGCGACGGT	1147
	1072_F	GCTCTTAAAGATG			ATACCCAT

1091	GLTA_RKP_400_428_F	TCTTCTCATCCTATGGC	413	GLTA_RKP_499_529_R	TGGTGGGTATCTTAGCA	1305
		TATTATGCTTGC			ATCATTCTAATAGC

1092	GLTA_RKP_1023_	TCCGTTCTTACAAATAG	330	GLTA_RKP_1129_1156_R	TTGGCGACGGTATACCC	1415
	1055_F	CAATAGAACTTGAAGC			ATAGCTTTATA

1093	GLTA_RKP_1043_	TGGAGCTTGAAGCTATC	553	GLTA_RKP_1138_1162_R	TGAACATTTGCGACGGT	1147
	1072_2_F	GCTCTTAAAGATG			ATACCCAT

1094	GLTA_RKP_1043_	TGGAACTTGAAGCTCTC	543	GLTA_RKP_1138_1164 R	TGTGAACATTTGCGACG	1330
	1072_3_F	GCTCTTAAAGATG			GTATACCCAT

1095	GLTA_RKP_400_428_F	TCTTCTCATCCTATGGC	413	GLTA_RKP_505_534_R	TGCGATGGTAGGTATCT	1230
		TATTATGCTTGC			TAGCAATCATTCT

1096	CTXA_VBC_117_142_F	TCTTATGCCAAGAGGAC	410	CTXA_VBC_194_218_R	TGCCTAACAAATCCCGT	1226
		AGAGTGAGT			CTGAGTTC

1097	CTXA_VBC_351_377_F	TGTATTAGGGGCATACA	630	CTXA_VBC_441_466_R	TGTCATCAAGCACCCCA	1324
		GTCCTCATCC			AAATGAACT

1098	RNASEP_VBC_331_	TCCGCGGAGTTGACTGG	325	RNASEP_VBC_388_414_R	TGACTTTCCTCCCCCTT	1163
	349_F	GT			ATCAGTCTCC

1099	TOXR_VBC_135_158_F	TCGATTAGGCAGCAACG	362	TOXR_VBC_221_246_R	TTCAAAACCTTGCTCTC	1370
		AAAGCCG			GCCAAACAA

1100	ASD_FRT_1_29_F	TTGCTTAAAGTTGGTTT	690	ASD_FRT_86_116_R	TGAGATGTCGAAAAAAA	1164
		TATTGGTTGGCG			CGTTGGCAAAATAC

1101	ASD_FRT_43_76_F	TCAGTTTTAATGTCTCG	295	ASD_FRT_129_156_R	TCCATATTGTTGCATAA	1009
		TATGATCGAATCAAAAG			AACCTGTTGGC

1102	GALE_FRT_168_199_F	TTATCAGCTAGACCTTT	658	GALE_FRT_241_269_R	TCACCTACAGCTTTAAA	973
		TAGGTAAAGCTAAGC			GCCAGCAAAATG

1103	GALE_FRT_834_865_F	TCAAAAAGCCCTAGGTA	245	GALE_FRT_901_925_R	TAGCCTTGGCAACATCA	915
		AAGAGATTCCATATC			GCAAAACT

1104	GALE_FRT_308_339_F	TCCAAGGTACACTAAAC	306	GALE_FRT_390_422_R	TCTTCTGTAAAGGGTGG	1136
		TTACTTGAGCTAATG			TTTATTATTCATCCCA

1105	IPAH_SGF_258_277_F	TGAGGACCGTGTCGCGC	458	IPAH_SGF_301_327_R	TCCTTCTGATGCCTGAT	1055
		TCA			GGACCAGGAG

1106	IPAH_SGF_113_134_F	TCCTTGACCGCCTTTCC	350	IPAH_SGF_172_191_R	TTTTCCAGCCATGCAGC	1441
		GATAC			GAC

1107	IPAH_SGF_462_486_F	TCAGACCATGCTCGCAG	271	IPAH_SGF_522_540_R	TGTCACTCCCGACACGC	1322
		AGAAACTT			CA

1111	RNASEP_BRM_461_	TAAACCCCATCGGGAGC	147	RNASEP_BRM_542_561_R	TGCCTCGCGCAACCTAC	1227
	488_F	AAGACCGAATA			CCG

1112	RNASEP_BRM_325_	TACCCCAGGGAAAGTGC	185	RNASEP_BRM_402_428_R	TCTCTTACCCCACCCTT	1125
	347_F	CACAGA			TCACCCTTAC

1128	HUPB_CJ_113_134_F	TAGTTGCTCAAACAGCT	230	HUPB_CJ_157_188_R	TCCCTAATAGTAGAAAT	1028
		GGGCT			AACTGCATCAGTAGC

1129	HUPB_CJ_76_102_F	TCCCGGAGCTTTTATGA	324	HUPB_CJ_157_188_R	TCCCTAATAGTAGAAAT	1028
		CTAAAGCAGAT			AACTGCATCAGTAGC

1130	HUPB_CJ_76_102_F	TCCCGGAGCTTTTATGA	324	HUPB_CJ_114_135_R	TAGCCCAGCTGTTTGAG	913
		CTAAAGCAGAT			CAACT

1151	AS_MLST-11-	TGAGATTGCTGAACATT	454	AB_MLST-11-	TTGTACATTTGAAACAA	1418
	OIF007_62_91_F	TAATGCTGATTGA		OIF007_169_203_R	TATGCATGACATGTGAA
					T

1152	AB_MLST-11-	TATTGTTTCAAATGTAC	243	AB_MLST-11-	TCACAGGTTCTACTTCA	969
	OIF007_185_214_F	AAGGTGAAGTGCG		OIF007_291_324_R	TCAATAATTTCCATTGC

1153	AB_MLST-11-	TGGAACGTTATCAGGTG	541	AB_MLST-11-	TTGCAATCGACATATCC	1400
	OIF007_260_289_F	CCCCAAAAATTCG		OIF007_364_393_R	ATTTCACCATGCC

1154	AB_MLST-11-	TGAAGTGCGTGATGATA	436	AB_MLST-11-	TCCGCCAAAAACTCCCC	1036
	OIF007_206_239_F	TCGATGCACTTGATGTA		OIF007_318_344_R	TTTTCACAGG

1155	AB_MLST-11-	TCGGTTTAGTAAAAGAA	378	AB_MLST-11-	TTCTGCTTGAGGAATAG	1392
	OIF007_522_552_F	CGTATTGCTCAACC		OIF007_587_610_R	TGCGTGG

1156	AB_MLST-11-	TCAACCTGACTGCGTGA	250	AB_MLST-11-	TACGTTCTACGATTTCT	902
	OIF007_547_571_F	ATGGTTGT		OIF007_656_686_R	TCATCAGGTACATC

1157	AB_MLST-11-	TCAAGCAGAAGCTTTGG	256	AB_MLST-11-	TACAACGTGATAAACAC	881
	OIF007_601_627_F	AAGAAGAAGG		OIF007_710_736_R	GACCAGAAGC

1158	AB_MLST-11-	TCGTGCCCGCAATTTGC	384	AB_MLST-11-	TAATGCCGGGTAGTGCA	878
	OIF007_1202_1225_F	ATAAAGC		OIF007_1266_1296_R	ATCCATTCTTCTAG

1159	AB_MLST-11-	TCGTGCCCGCAATTTGC	384	AB_MLST-11-	TGCACCTGCGGTCGAGC	1199
	OIF007_1202_1225_F	ATAAAGC		OIF007_1299_1316_R	G

1160	AB_MLST-11-	TTGTAGCACAGCAAGGC	694	AB_MLST-11-	TGCCATCCATAATCACG	1215
	OIF007_1234_1264_F	AAATTTCCTGAAAC		OIF007_1335_1362_R	CCATACTGACG

1161	AB_MLST-11-	TAGGTTTACGTCAGTAT	225	AB_MLST-11-	TGCCAGTTTCCACATTT	1212
	OIF007_1327_1356_F	GGCGTGATTATGG		OIF007_1422_1448_R	CACGTTCGTG

1162	AB_MLST-11-	TCGTGATTATGGATGGC	383	AB_MLST-11-	TCGCTTGAGTGTAGTCA	1083
	OIF007_1345_1369_F	AACGTGAA		OIF007_1470_1494_R	TGATTGCG

1163	AB_MLST-11-	TTATGGATGGCAACGTG	662	AB_MLST-11-	TCGCTTGAGTGTAGTCA	1083
	OIF007_1351_1375_F	AAACGCGT		OIF007_1470_1494_R	TGATTGCG

1164	AB_MLST-11-	TCTTTGCCATTGAAGAT	422	AB_MLST-11-	TCGCTTGAGTGTAGTCA	1083
	OIF007_1387_1412_F	GACTTAAGC		OIF007_1470_1494_R	TGATTGCG

1165	AB_MLST-11-	TACTAGCGGTAAGCTTA	194	AB_MLST-11-	TGAGTCGGGTTCACTTT	1173
	OIF007_1542_1569_F	AACAAGATTGC		OIF007_1656_1680_R	ACCTGGCA

1166	AB_MLST-11-	TTGCCAATGATATTCGT	684	AB_MLST-11-	TGAGTCGGGTTCACTTT	1173
	OIF007_1566_1593_F	TGGTTAGCAAG		OIF007_1656_1680_R	ACCTGGCA

1167	AB_MLST-11-	TCGGCGAAATCCGTATT	375	AB_MLST-11-	TACCGGAAGCACCAGCG	890
	OIF007_1611_1638_F	CCTGAAAATGA		OIF007_1731_1757_R	ACATTAATAG

1168	AB_MLST-11-	TACCACTATTAATGTCG	182	AB_MLST-11-	TGCAACTGAATAGATTG	1195
	OIF007_1726_1752_F	CTGGTGCTTC		OIF007_1790_1821_R	CAGTAAGTTATAAGC

1169	AB_MLST-11-	TTATAACTTACTGCAAT	656	AB_MLST-11-	TGAATTATGCAAGAAGT	1151
	OIF007_1792_1826_F	CTATTCAGTTGCTTGGT		OIF007_1876_1909_R	GATCAATTTTCTCACGA
		G

1170	AB_MLST-11-	TTATAACTTACTGCAAT	656	AB_MLST-11-	TGCCGTAACTAACATAA	1224
	OIF007_1792_1826_F	CTATTCAGTTGCTTGGT		OIF007_1895_1927_R	GAGAATTATGCAAGAA
		G

1171	AB_MLST-11-	TGGTTATGTACCAAATA	618	AB_MLST-11-	TGACGGCATCGATACCA	1157
	OIF007_1970_2002_F	CTTTGTCTGAAGATGG		OIF007_2097_2118_R	CCGTC

1172	RNASEP_BRM_461_	TAAACCCCATCGGGAGC	147	RNASEP_BRM_542_561_2_R	TGCCTCGTGCAACCCAC	1228
	488_F	AAGACCGAATA			CCG

2000	CTXB_NC002505_46_	TCAGCGTATGCACATGG	278	CTXB_NC002505_132_162_R	TCCGGCTAGAGATTCTG	1039
	70_F	AACTCCTC			TATACGACAATATC

2001	FUR_NC002505_87_	TGAGTGCCAACATATCA	465	FUR_NC002505_205_228_R	TCCGCCTTCAAAATGGT	1037
	113_F	GTGCTGAAGA			GGCGAGT

2002	FUR_NC002505_87_	TGAGTGCCAACATATCA	465	FUR_NC002505_178_205_R	TCACGATACCTGCATCA	974
	113_F	GTGCTGAAGA			TCAAATTGGTT

2003	GAPA_NC002505_533_	TCGACAACACCATTATC	356	GAPA_NC002505_646_671_R	TCAGAATCGATGCCAAA	980
	560_F	TATGGTGTGAA			TGCGTCATC

2004	GAPA_NC002505_694_	TCAATGAACGACCAACA	259	GAPA_NC002505_769_798_R	TCCTCTATGCAACTTAG	1046
	721_F	AGTGATTGATG			TATCAACAGGAAT

2005	GAPA_NC002505_753_	TGCTAGTCAATCTATCA	517	GAPA_NC0002505_856_881_R	TCCATCGCAGTCACGTT	1011
	782_F	TTCCGGTTGATAC			TACTGTTGG

2006	GYRB_NC002505_2_	TGCCGGACAATTACGAT	501	GYRB_NC002505_109_134_R	TCCACCACCTCAAAGAC	1003
	32_F	TCATCGAGTATTAA			CATGTGGTG

2007	GYRB_NC002505_123_	TGAGGTGGTGGATAACT	460	GYRB_NC002505_199_225_R	TCCGTCATCGCTGACAG	1042
	152_F	CAATTGATGAAGC			AAACTGAGTT

2008	GYRB_NC002505_768_	TATGCAGTGGAACGATG	236	GYRB_NC002505_832_860_R	TGGAAACCGGCTAAGTG	1262
	794_F	GTTTCCAAGA			AGTACCACCATC

2009	GYRB_NC002505_837_	TGGTACTCACTTAGCGG	603	GYRB_NC002505_937_957_R	TCCTTCACGCGCATCAT	1054
	860_F	GTTTCCG			CACC

2010	GYRB_NC002505_934_	TCGGGTGATGATGCGCG	377	GYRB_NC002505_982_1007_R	TGGCTTGAGAATTTAGG	1283
	956_F	TGAAGG			ATCCGGCAC

2011	GYRB_NC002505_	TAAAGCCCGTGAAATGA	148	GYRB_NC002505_1255_	TGAGTCACCCTCCACAA	1172
	1161_1190_F	CTCGTCGTAAAGG		1284_R	TGTATAGTTCAGA

2012	OMPU_NC002505_85_	TACGCTGACGGAATCAA	190	OMPU_NC002505_154_180_R	TGCTTCAGCACGGCCAC	1254
	110_F	CCAAAGCGG			CAACTTCTAG

2013	OMPU_NC002505_258_	TGACGGCCTATACGGTG	451	OMPU_NC002505_346_369_R	TCCGAGACCAGCGTAGG	1033
	283_F	TTGGTTTCT			TGTAACG

2014	OMPU_NC002505_431_	TCACCGATATCATGGCT	266	OMPU_NC002505_544_567_R	TCGGTCAGCAAAACGGT	1094
	455_F	TACCACGG			AGCTTGC

2015	OMPU_NC002505_	TAGGCGTGAAAGCAAGC	223	OMPU_NC002505_625_651_R	TAGAGAGTAGCCATCTT	908
	533_557_F	TACCGTTT			CACCGTTGTC

2016	OMPU_NC002505_689_	TAGGTGCTGGTTACGCA	224	OMPU_NC002505_725_751_R	TGGGGTAAGACGCGGCT	1291
	713_F	GATCAAGA			AGCATGTATT

2017	OMPU_NC002505_727_	TACATGCTAGCCGCGTC	181	OMPU_NC002505_811_835_R	TAGCAGCTAGCTCGTAA	911
	747_F	TTAC			CCAGTGTA

2018	OMPU_NC002505_931_	TACTACTTCAAGCCGAA	193	OMPU_NC002505_1033_	TTAGAAGTCGTAACGTG	1368
	953_F	CTTCCG		1053_R	GACC

2019	OMPU_NC002505_927_	TACTTACTACTTCAAGC	197	OMPU_NC002505_1033_	TGGTTAGAAGTCGTAAC	1307
	953_F	CGAACTTCCG		1054_R	GTGGACC

2020	TCPA_NC002505_48_	TCACGATAAGAAAACCG	269	TCPA_NC002505_148_170_R	TTCTGCGAATCAATCGC	1391
	73_F	GTCAAGAGG			ACGCTG

2021	TDH_NC004605_265_	TGGCTGACATCCTACAT	574	TDH_NC004605_357_386_R	TGTTGAAGCTGTACTTG	1351
	289_F	GACTGTGA			ACCTGATTTTACG

2022	VVHA_NC004460_772_	TCTTATTCCAACTTCAA	412	VVHA_NC004460_862_886_R	TACCAAAGCGTGCACGA	887
	802_F	ACCGAACTATGACG			TAGTTGAG

2023	23S_EC_2643_2667_F	TGCCTGTTCTTAGTACG	508	23S_EC_2746_2770_R	TGGGTTTCGCGCTTAGA	1297
		AGAGGACC			TGCTTTCA

2024	16S_EC_713_732_	TAGAACACCGATGGCGA	202	16S_EC_789_811_R	TGCGTGGACTACCAGGG	1240
	TMOD_F	AGGC			TATCTA

2025	16S_EC_784_806_F	TGGATTAGAGACCCTGG	560	16S_EC_880_897_TMOD_R	TGGCCGTACTCCCCAGG	1278
		TAGTCC			CG

2026	16S_EC_959_981_F	TGTCGATGCAACGCGAA	634	16S_EC_1052_1074_R	TACGAGCTGACGACAGC	896
		GAACCT			CATGCA

2027	TUFB_EC_956_979_F	TGCACACGCCGTTCTTC	489	TUFB_EC_1034_1058_2_R	TGCATCACCATTTCCTT	1204
		AACAACT			GTCCTTCG

2028	RPOC_EC_2146_2174_	TCAGGAGTCGTTCAACT	284	RPOC_EC_2227_2249_R	TGCTAGGCCATCAGGCC	1244
	TMOD_F	CGATCTACATGAT			ACGCAT

2029	RPOB_EC_1841_1866_	TGGTTATCGCTCAGGCG	617	RPOB_EC_1909_1929_TMOD_R	TGCTGGATTCGCCTTTG	1250
	F	AACTCCAAC			CTACG

2030	RPLB_EC_650_679_	TGACCTACAGTAAGAGG	449	RPLB_EC_739_763_R	TGCCAAGTGCTGGTTTA	1208
	TMOD_F	TTCTGTAATGAACC			CCCCATGG

2031	RPLB_EC_690_710_F	TCCACACGGTGGTGGTG	309	RPLB_EC_737_760_R	TGGGTGCTGGTTTACCC	1295
		AAGG			CATGGAG

2032	INFB_EC_1366_1393_	TCTCGTGGTGCACAAGT	397	INFB_EC_1439_1469_R	TGTGCTGCTTTCGCATG	1335
	F	AACGGATATTA			GTTAATTGCTTCAA

2033	VALS_EC_1105_1124_	TCGTGGCGGCGTGGTTA	385	VALS_EC_1195_1219_R	TGGGTACGAACTGGATG	1292
	TMOD_F	TCGA			TCGCCGTT

2034	SSPE_BA_113_137_F	TGCAAGCAAACGCACAA	482	SSPE_BA_197_222_TMOD_R	TTGCACGTCTGTTTCAG	1402
		TCAGAAGC			TTGCAAATTC

2035	RPOC_EC_2218_2241_	TCTGGCAGGTATGCGTG	405	RPOC_EC_2313_2338_R	TGGCACCGTGGGTTGAG	1273
	TMOD_F	GTCTGATG			ATGAAGTAC

2056	MECI-R_NC003923-	TTTACACATATCGTGAG	698	MECI-R_NC003923-41798-	TTGTGATATGGAGGTGT	1420
	41798-41609_33_60_	CAATGAACTGA		41609_86_113_R	AGAAGGTGTTA
	F

2057	AGR-III_NC003923-	TCACCAGTTTGCCACGT	263	AGR-III_NC003923-	ACCTGCATCCCTAAACG	730
	2108074-	ATCTTCAA		2108074-2109507_56_79_R	TACTTGC
	2109507_1_23_F

2058	AGR-III_NC003923-	TGAGCTTTTAGTTGACT	457	AGR-III_NC003923-	TACTTCAGCTTCGTCCA	906
	2108074-	TTTTCAACAGC		2108074-2109507_622_	ATAAAAAATCACAAT
	2109507_569_596_F			653_R

2059	AGR-III_NC003923-	TTTCACACAGCGTGTTT	701	AGR-III_NC003923-	TGTAGGCAAGTGCATAA	1319
	2108074-2109507_	ATAGTTCTACCA		2108074-2109507_1070_	GAAATTGATACA
	1024_1052_F			1098_R

2060	AGR-I_AJ617706_	TGGTGACTTCATAATGG	610	AGR-I_AJ617706_694_726_R	TCCCCATTTAATAATTC	1021
	622_651_F	ATGAAGTTGAAGT			CACCTACTATCACACT

2061	AGR-I_AJ617706_	TGGCATTTTAAAAAACA	579	AGR-I_AJ617706_626_655_R	TGGTACTTCAACTTCAT	1302
	580_611_F	TTGGTAACATCGCAC			CCATTATGAAGTC

2062	AGR-II_NC002745-	TCTTGCAGCAGTTTATT	415	AGR-II_NC002745-2079448-	TTGTTTATTGTTTCCAT	1424
	2079448-2080879_	TGATGAACCTAAAGT		2080879_700_731_R	ATGCTACACACTTTC
	620_651_F

2063	AGR-II_NC002745-	TGTACCCGCTGAATTAA	624	AGR-II_NC002745-2079448-	TCGCCATAGCTAAGTTG	1077
	2079448-2080879_	CGAATTTATACGAC		2080879_715_745_R	TTTATTGTTTCCAT
	649_679_F

2064	AGR-IV_AJ617711_	TGGTATTCTATTTTGCT	606	AGR-IV_AJ617711_1004_	TGCGCTATCAACGATTT	1233
	931_961_F	GATAATGACCTCGC		1035_R	TGACAATATATGTGA

2065	AGR-IV_AJ617711_	TGGCACTCTTGCCTTTA	562	AGR-IV_AJ617711_309_	TCCCATACCTATGGCGA	1017
	250_283_F	ATATTAGTAAACTATCA		335_R	TAACTGTCAT

2066	BLAZ_NC002952	TCCACTTATCGCAAATG	312	BLAZ_NC002952	TGGCCACTTTTATCAGC	1277
	(1913827 . . .	GAAAATTAAGCAA		(1913827 . . . 1914672)_	AACCTTACAGTC
	1914672)_68_68_F			68_68_R

2067	BLAZ_NC002952	TGCACTTATCGCAAATG	494	BLAZ_NC002952	TAGTCTTTTGGAACACC	926
	(1913827 . . .	GAAAATTAAGCAA		(1913827 . . . 1914672)_	GTCTTTAATTAAAGT
	1914672)_68_68_2_F			68_68_2_R

2068	BLAZ_NC002952	TGATACTTCAACGCCTG	467	BLAZ_NC002952	TGGAACACCGTCTTTAA	1263
	(1913827 . . .	CTGCTTTC		(1913827 . . . 1914672)_	TTAAAGTATCTCC
	1914672)_68_68_3_F			68_68_3_R

2069	BLAZ_NC002952	TATACTTCAACGCCTGC	232	BLAZ_NC002952	TCTTTTCTTTGCTTAAT	1145
	(1913827 . . .	TGCTTTC		(1913827 . . . 1914672)_	TTTCCATTTGCGAT
	1914672)_68_68_4_F			68_68_4_R

2070	BLAZ_NC002952	TGCAATTGCTTTAGTTT	487	BLAZ_NC0002952	TTACTTCCTTACCACTT	1366
	(1913827 . . .	TAAGTGCATGTAATTC		(1913827 . . . 1914672)_	TTAGTATCTAAAGCATA
	1914672)_1_33_F			34_67_R

2071	BLAZ_NC002952	TCCTTGCTTTAGTTTTA	351	BLAZ_NC0002952	TGGGGACTTCCTTACCA	1289
	(1913827 . . .	AGTGCATGTAATTCAA		(1913827 . . . 1914672)_	CTTTTAGTATCTAA
	1914672)_3_34_F			40_68_R

2072	BSA-A_NC003923-	TAGCGAATGTGGCTTTA	214	BSA-A_NC003923-1304065-	TGCAAGGGAAACCTAGA	1197
	1304065-	CTTCACAATT		1303589_165_193_R	ATTACAAACCCT
	1303589_99_125_F

2073	BSA-A_NC003923-	ATCAATTTGGTGGCCAA	32	BSA-A_NC003923-1304065-	TGCATAGGGAAGGTAAC	1203
	1304065-	GAACCTGG		1303589_253_278_R	ACCATAGTT
	1303589_194_218_F

2074	BSA-A_NC003923-	TTGACTGCGGCACAACA	679	BSA-A_NC003923-1304065-	TAACAACGTTACCTTCG	856
	1304065-	CGGAT		1303589_388_415_R	CGATCCACTAA
	1303589_328_349_F

2075	BSA-A_NC003923-	TGCTATGGTGTTACCTT	519	BSA-A_NC003923-1304065-	TGTTGTGCCGCAGTCAA	1353
	1304065-	CCCTATGCA		1303589_317_344_R	ATATCTAAATA
	1303589_253_278_F

2076	BSA-B_NC003923-	TAGCAACAAATATATCT	209	BSA-B_NC003923-1917149-	TGTGAAGAACTTTCAAA	1331
	1917149-	GAAGCAGCGTACT		1914156_1011_1039_R	TCTGTGAATCCA
	1914156_953_982_F

2077	BSA-B_NC003923-	TGAAAAGTATGGATTTG	426	BSA-B_NC003923-1917149-	TCTTCTTGAAAAATTGT	1138
	1917149-	AACAACTCGTGAATA		1914156_1109_1136_R	TGTCCCGAAAC
	1914156_1050_1081_
	F

2078	BSA-B_NC003923-	TCATTATCATGCGCCAA	300	BSA-B_NC003923-1917149-	TGGACTAATAACAATGA	1267
	1917149-	TGAGTGCAGA		1914156_1323_1353_R	GCTCATTGTACTGA
	1914156_1260_1286_
	F

2079	BSA-B_NC003923-	TTTCATCTTATCGAGGA	703	BSA-B_NC003923-1917149-	TGAATATGTAATGCAAA	1148
	1917149-	CCCGAAATCGA		1914156_2186_2216_R	CCAGTCTTTGTCAT
	1914156_2126_2153_
	F

2080	ERMA_NC002952-	TCGCTATCTTATCGTTG	372	ERMA_NC002952-55890-	TGAGTCTACACTTGGCT	1174
	55890-	AGAAGGGATT		56621_487_513_R	TAGGATGAAA
	56621_366_392_F

2081	ERMA_NC002952-	TAGCTATCTTATCGTTG	217	ERMA_NC002952-55890-	TGAGCATTTTTATATCC	1167
	55890-	AGAAGGGATTTGC		56621_438_465_R	ATCTCCACCAT
	56621_366_395_F

2082	ERMA_NC002952-	TGATCGTTGAGAAGGGA	470	ERMA_NC002952-55890-	TCTTGGCTTAGGATGAA	1143
	55890-	TTTGCGAAAAGA		56621_473_504_R	AATATAGTGGTGGTA
	56621_374_402_F

2083	ERMA_NC002952-	TGCAAAATCTGCAACGA	480	ERMA_NC002952-55890-	TCAATACAGAGTCTACA	964
	55890-	GCTTTGG		56621_491_520_R	CTTGGCTTAGGAT
	56621_404_427_F

2084	ERMA_NC002952-	TCATCCTAAGCCAAGTG	297	ERMA_NC002952-55890-	TGGACGATATTCACGGT	1266
	55890-	TAGACTCTGTA		56621_586_615_R	TTACCCACTTATA
	56621_489_516_F

2085	ERMA_NC002952-	TATAAGTGGGTAAACCG	231	ERMA_NC002952-55890-	TTGACATTTGCATGCTT	1397
	55890-	TGAATATCGTGT		56621_640_665_R	CAAAGCCTG
	56621_586_614_F

2086	ERMC_NC005908-	TCTGAACATGATAATAT	399	ERMC_NC005908-2004-	TCCGTAGTTTTGCATAA	1041
	2004-2738_85_116_F	CTTTGAAATCGGCTC		2738_173_206_R	TTTATGGTCTATTTCAA

2087	ERMC_NC005908-	TCATGATAATATCTTTG	298	ERMC_NC005908-2004-	TTTATGGTCTATTTCAA	1429
	2004-2738_90_120_F	AAATCGGCTCAGGA		2738_160_189_R	TGGCAGTTACGAA

2088	ERMC_NC005908-	TCAGGAAAAGGGCATTT	283	ERMC_NC005908-2004-	TATGGTCTATTTCAATG	936
	2004-2738_115_139_	TACCCTTG		2738_161_187_R	GCAGTTACGA
	F

2089	ERMC_NC005908-	TAATCGTGGAATACGGG	168	ERMC_NC005908-2004-	TCAACTTCTGCCATTAA	956
	2004-2738_374_397_	TTTGCTA		2738_425_452_R	AAGTAATGCCA
	F

2090	ERMC_NC005908-	TCTTTGAAATCGGCTCA	421	ERMC_NC005908-2004-	TGATGGTCTATTTCAAT	1185
	2004-2738_101_125_	GGAAAAGG		2738_159_188_R	GGCAGTTACGAAA
	F

2091	ERMB_Y13600-625-	TGTTGGGAGTATTCCTT	644	ERMB_Y13600-625-	TCAACAATCAGATAGAT	953
	1362_291_321_F	ACCATTTAAGCACA		1362_352_380_R	GTCAGACGCATG

2092	ERMB_Y13600-625-	TGGAAAGCCATGCGTCT	536	ERMB_Y13600-625-	TGCAAGAGCAACCCTAG	1196
	1362_344_367_F	GACATCT		1362_415_437_R	TGTTCG

2093	ERMB_Y13600-625-	TGGATATTCACCGAACA	556	ERMB_Y13600-625-	TAGGATGAAAGCATTCC	919
	1362_404_429_F	CTAGGGTTG		1362_471_493_R	GCTGGC

2094	ERMB_Y13600-625-	TAAGCTGCCAGCGGAAT	161	ERMB_Y13600-625-	TCATCTGTGGTATGGCG	989
	1362_465_487_F	GCTTTC		1362_521_545_R	GGTAAGTT

2095	PVLUK_NC003923-	TGAGCTGCATCAACTGT	456	PVLUK_NC003923-1529595-	TGGAAAACTCATGAAAT	1261
	1529595-	ATTGGATAG		1531285_775_804_R	TAAAGTGAAAGGA
	1531285_688_713_F

2096	PVLUK_NC003923-	TGGAACAAAATAGTCTC	539	PVLUK_NC003923-1529595-	TCATTAGGTAAAATGTC	993
	1529595-	TCGGATTTTGACT		1531285_1095_1125_R	TGGACATGATCCAA
	1531285_1039_1068_
	F

2097	PVLUK_NC003923-	TGAGTAACATCCATATT	461	PVLUK_NC003923-1529595-	TCTCATGAAAAAGGCTC	1124
	1529595-	TCTGCCATACGT		1531285_950_978_R	AGGAGATACAAG
	1531285_908_936_F

2098	PVLUK_NC003923-	TCGGAATCTGATGTTGC	373	PVLUK_NC003923-1529595-	TCACACCTGTAAGTGAG	968
	1529595-	AGTTGTT		1531285_654_682_R	AAAAAGGTTGAT
	1531285_610_633_F

2099	SA442_NC003923-	TGTCGGTACACGATATT	635	SA442_NC003923-2538576-	TTTCCGATGCAACGTAA	1433
	2538576-	CTTCACGA		2538831_98_124_R	TGAGATTTCA
	2538831_11_35_F

2100	SA442_NC003923-	TGAAATCTCATTACGTT	427	SA442_NC003923-2538576-	TCGTATGACCAGCTTCG	1098
	2538576-	GCATCGGAAA		2538831_163_188_R	GTACTACTA
	2538831_98_124_F

2101	SA442_NC003923-	TCTCATTACGTTGCATC	395	SA442_NC003923-2538576-	TTTATGACCAGCTTCGG	1428
	2538576-	GGAAACA		2538831_161_187_R	TACTACTAAA
	2538831_103_126_F

2102	SA442_NC003923-	TAGTACCGAAGCTGGTC	226	SA442_NC003923-2538576-	TGATAATGAAGGGAAAC	1179
	2538576-	ATACGA		2538831_231_257_R	CTTTTTCACG
	2538831_166_188_F

2103	SEA_NC003923-	TGCAGGGAACAGCTTTA	495	SEA_NC003923-2052219-	TCGATCGTGACTCTCTT	1070
	2052219-	GGCA		2051456_173_200_R	TATTTTCAGTT
	2051456_115_135_F

2104	SEA_NC003923-	TAACTCTGATGTTTTTG	156	SEA_NC003923-2052219-	TGTAATTAACCGAAGGT	1315
	2052219-	ATGGGAAGGT		2051456_621_651_R	TCTGTAGAAGTATG
	2051456_572_598_F

2105	SEA_NC003923-	TGTATGGTGGTGTAACG	629	SEA_NC003923-2052219-	TAACCGTTTCCAAAGGT	861
	2052219-	TTACATGATAATAATC		2051456_464_492_R	ACTGTATTTTGT
	2051456_382_414_F

2106	SEA_NC003923-	TTGTATGTATGGTGGTG	695	SEA_NC003923-2052219-	TAACCGTTTCCAAAGGT	862
	2052219-	TAACGTTACATGA		2051456_459_492_R	ACTGTATTTTGTTTACC
	2051456_377_406_F

2107	SEB_NC002758-	TTTCACATGTAATTTTG	702	SEB_NC002758-2135540-	TCATCTGGTTTAGGATC	988
	2135540-	ATATTCGCACTGA		2135140_273_298_R	TGGTTGACT
	2135140_208_237_F

2108	SEB_NC002758-	TATTTCACATGTAATTT	244	SEB_NC002758-2135540-	TGCAACTCATCTGGTTT	1194
	2135540-	TGATATTCGCACT		2135140_281_304_R	AGGATCT
	2135140_206_235_F

2109	SEB_NC002758-	TAACAACTCGCCTTATG	151	SEB_NC002758-2135540-	TGTGCAGGCATCATGTC	1334
	2135540-	AAACGGGATATA		2135140_402_402_R	ATACCAA
	2135140_402_402_F

2110	SEB_NC002758-	TTGTATGTATGGTGGTG	696	SEB_NC002758-2135540-	TTACCATCTTCAAATAC	1361
	2135540-	TAACTGAGCA		2135140_402_402_2_R	CCGAACAGTAA
	2135140_402_402_2_F

2111	SEC_NC003923-	TTAACATGAAGGAAACC	648	SEC_NC003923-651678-	TGAGTTTGCACTTCAAA	1177
	851678-	ACTTTGATAATGG		852768_620_647_R	AGAAATTGTGT
	852768_546_575_F

2112	SEC_NC003923-	TGGAATAACAAAACATG	546	SEC_NC003923-851678-	TCAGTTTGCACTTCAAA	985
	851678-	AAGGAAACCACTT		852768_619_647_R	AGAAATTGTGTT
	852768_537_566_F

2113	SEC_NC003923-	TGAGTTTAACAGTTCAC	466	SEC_NC003923-851678-	TCGCCTGGTGCAGGCAT	1078
	851678-	CATATGAAACAGG		852768_794_815_R	CATAT
	852768_720_749_F

2114	SEC_NC003923-	TGGTATGATATGATGCC	604	SEC_NC003923-851678-	TCTTCACACTTTTAGAA	1133
	851678-	TGCACCA		852768_853_886_R	TCAACCGTTTTATTGTC
	852768_787_810_F

2115	SED_M28521_657_	TGGTGGTGAAATAGATA	615	SED_M28521_741_770_R	TGTACACCATTTATCCA	1318
	682_F	GGACTGCTT			CAAATTGATTGGT

2116	SED_M28521_690_	TGGAGGTGTCACTCCAC	554	SED_M28521_739_770_R	TGGGCACCATTTATCCA	1288
	711_F	ACGAA			CAAATTGATTGGTAT

2117	SED_M28521_833_	TTGCACAAGCAAGGCGC	683	SED_M28521_888_911_R	TCGCGCTGTATTTTTCC	1079
	854_F	TATTT			TCCGAGA

2118	SED_M28521_962_	TGGATGTTAAGGGTGAT	559	SED_M28521_1022_1048_R	TGTCAATATGAAGGTGC	1320
	987_F	TTTCCCGAA			TCTGTGGATA

2119	SEA-SEE_NC002952-	TTTACACTACTTTTATT	699	SEA-SEE_NC002952-	TCATTTATTTCTTCGCT	994
	2131289-	CATTGCCCTAACG		2131289-2130703_71_98_R	TTTCTCGCTAC
	2130703_16_45_F

2120	SEA-SEE_NC002952-	TGATCATCCGTGGTATA	469	SEA-SEE_NC002952-	TAAGCACCATATAAGTC	870
	2131289-	ACGATTTATTAGT		2131289-2130703_314_	TACTTTTTTCCCTT
	2130703_249_278_F			344_R

2121	SEE_NC002952-	TGACATGATAATAACCG	445	SEE_NC002952-2131289-	TCTATAGGTACTGTAGT	1120
	2131289-	ATTGACCGAAGA		2130703_465_494_R	TTGTTTTCCGTCT
	2130703_409_437_F

2122	SEE_NC002952-	TGTTCAAGAGCTAGATC	640	SEE_NC002952-2131289-	TTTGCACCTTACCGCCA	1436
	2131289-	TTCAGGCAA		2130703_586_586_R	AAGCT
	2130703_525_550_F

2123	SEE_NC002952-	TGTTCAAGAGCTAGATC	639	SEE_NC002952-2131289-	TACCTTACCGCCAAAGC	892
	2131289-	TTCAGGCA		2130703_586_586_2_R	TGTCT
	2130703_525_549_F

2124	SEE_NC002952-	TCTGGAGGCACACCAAA	403	SEE_NC002952-2131289-	TCCGTCTATCCACAAGT	1043
	2131289-	TAAAACA		2130703_444_471_R	TAATTGGTACT
	2130703_361_384_F

2125	SEG_NC002758-	TGCTCAACCCGATCCTA	520	SEG_NC002758-1955100-	TAACTCCTCTTCCTTCA	863
	1955100-	AATTAGACGA		1954171_321_346_R	ACAGGTGGA
	1954171_225_251_F

2126	SEG_NC002758-	TGGACAATAGACAATCA	548	SEG_NC002758-1955100-	TGCTTTGTAATCTAGTT	1260
	1955100-	CTTGGATTTACA		1954171_671_702_R	CCTGAATAGTAACCA
	1954171_623_651_F

2127	SEG_NC002758-	TGGAGGTTGTTGTATGT	555	SEG_NC002758-1955100-	TGTCTATTGTCGATTGT	1329
	1955100-	ATGGTGGT		1954171_607_635_R	TACCTGTACAGT
	1954171_540_564_F

2128	SEG_NC002758-	TACAAAGCAAGACACTG	173	SEG_NC002758-1955100-	TGATTCAAATGCAGAAC	1187
	1955100-	GCTCACTA		1954171_735_762_R	CATCAAACTCG
	1954171_694_718_F

2129	SEH_NC002953-	TTGCAACTGCTGATTTA	682	SEH_NC002953-60024-	TAGTGTTGTACCTCCAT	927
	60024-	GCTCAGA		60977_547_576_R	ATAGACATTCAGA
	60977_449_472_F

2130	SEH_NC002953-	TAGAAATCAAGGTGATA	201	SEH_NC002953-60024-	TTCTGAGCTAAATCAGC	1390
	60024-	GTGGCAATGA		60977_450_473_R	AGTTGCA
	60977_408_434_F

2131	SEH_NC002953-	TCTGAATGTCTATATGG	400	SEH_NC002953-60024-	TACCATCTACCCAAACA	888
	60024-	AGGTACAACACTA		60977_608_634_R	TTAGCACCAA
	60977_547_576_F

2132	SEH_NC002953-	TTCTGAATGTCTATATG	677	SEH_NC002953-60024-	TAGCACCAATCACCCTT	909
	60024-	GAGGTACAACACT		60977_594_616_R	TCCTGT
	60977_546_575_F

2133	SEI_NC002758-	TCAACTCGAATTTTCAA	253	SEI_NC002758-1957830-	TCACAAGGACCATTATA	966
	1957830-	CAGGTACCA		1956949_419_446_R	ATCAATGCCAA
	1956949_324_349_F

2134	SEI_NC002758-	TTCAACAGGTACCAATG	666	SEI_NC002758-1957830-	TGTACAAGGACCATTAT	1316
	1957830-	ATTTGATCTCA		1956949_420_447_R	AATCAATGCCA
	1956949_336_363_F

2135	SEI_NC002758-	TGATCTCAGAATCTAAT	471	SEI_NC002758-1957830-	TCTGGCCCCTCCATACA	1129
	1957830-	AATTGGGACGAA		1956949_449_474_R	TGTATTTAG
	1956949_356_384_F

2136	SEI_NC002758-	TCTCAAGGTGATATTGG	394	SEI_NC002758-1957830-	TGGGTAGGTTTTTATCT	1293
	1957830-	TGTAGGTAACTTAA		1956949_290_316_R	GTGACGCCTT
	1956949_223_253_F

2137	SEJ_AF053140_1307_	TGTGGAGTAACACTGCA	637	SEJ_AF053140_1381_1404_R	TCTAGCGGAACAACAGT	1118
	1332_F	TGAAAACAA			TCTGATG

2138	SEJ_AF053140_1378_	TAGCATCAGAACTGTTG	211	SEJ_AF053140_1429_1458_R	TCCTGAAGATCTAGTTC	1049
	1403_F	TTCCGCTAG			TTGAATGGTTACT

2139	SEJ_AF053140_1431_	TAACCATTCAAGAACTA	153	SEJ_AF053140_1500_1531_R	TAGTCCTTTCTGAATTT	925
	1459_F	GATCTTCAGGCA			TACCATCAAAGGTAC

2140	SEJ_AF053140_1434_	TCATTCAAGAACTAGAT	301	SEJ_AF053140_1521_1549_R	TCAGGTATGAAACACGA	984
	1461_F	CTTCAGGCAAG			TTAGTCCTTTCT

2141	TSST_NC002758-	TGGTTTAGATAATTCCT	619	TSST_NC002758-2137564-	TGTAAAAGCAGGGCTAT	1312
	2137564-	TAGGATCTATGCGT		2138293_278_305_R	AATAAGGACTC
	2138293_206_236_F

2142	TSST_NC002758-	TGCGTATAAAAAACACA	514	TSST_NC002758-2137564-	TGCCCTTTTGTAAAAGC	1221
	2137564-	GATGGCAGCA		2138293_289_313_R	AGGGCTAT
	2138293_232_258_F

2143	TSST_NC002758-	TCCAAATAAGTGGCGTT	304	TSST_NC002758-2137564-	TACTTTAAGGGGCTATC	907
	2137564-	ACAAATACTGAA		2138293_448_478_R	TTTACCATGAACCT
	2138293_382_410_F

2144	TSST_NC002758-	TCTTTTACAAAAGGGGA	423	TSST_NC002758-2137564-	TAAGTTCCTTCGCTAGT	874
	2137564-	AAAAGTTGACTT		2138293_347_373_R	ATGTTGGCTT
	2138293_297_325_F

2145	ARCC_NC003923-	TCGCCGGCAATGCCATT	368	ARCC_NC003923-2725050-	TGAGTTAAAATGCGATT	1175
	2725050-	GGATA		2724595_97_128_R	GATTTCAGTTTCCAA
	2724595_37_58_F

2146	ARCC_NC003923-	TGAATAGTGATAGAACT	437	ARCC_NC003923-2725050-	TCTTCTTCTTTCGTATA	1137
	2725050-	GTAGGCACAATCGT		2724595_214_245_R	AAAAGGACCAATTGG
	2724595_131_161_F

2147	ARCC_NC003923-	TTGGTCCTTTTTATACG	691	ARCC_NC003923-2725050-	TGGTGTTCTAGTATAGA	1306
	2725050-	AAAGAAGAAGTTGAA		2724595_322_353_R	TTGAGGTAGTGGTGA
	2724595_218_249_F

2148	AROE_NC003923-	TTGCGAATAGAACGATG	686	AROE_NC003923-1674726-	TCGAATTCAGCTAAATA	1064
	1674726-	GCTCGT		1674277_435_464_R	CTTTTCAGCATCT
	1674277_371_393_F

2149	AROE_NC003923-	TGGGGCTTTAAATATTC	590	AROE_NC003923-1674726-	TACCTGCATTAATCGCT	891
	1674726-	CAATTGAAGATTTTCA		1674277_155_181_R	TGTTCATCAA
	1674277_30_62_F

2150	AROE_NC003923-	TGATGGCAAGTGGATAG	474	AR0E_NC003923-1674726-	TAAGCAATACCTTTACT	869
	1674726-	GGTATAATACAG		1674277_308_335_R	TGCACCACCTG
	1674277_204_232_F

2151	GLPF_NC003923-	TGCACCGGCTATTAAGA	491	GLPF_NC003923-1296927-	TGCAACAATTAATGCTC	1193
	1296927-	ATTACTTTGCCAACT		1297391_382_414_R	CGACAATTAAAGGATT
	1297391_270_301_F

2152	GLPF_NC003923-	TGGATGGGGATTAGCGG	558	GLPF_NC003923-1296927-	TAAAGACACCGCTGGGT	850
	1296927-	TTACAATG		1297391_81_108_R	TTAAATGTGCA
	1297391_27_51_F

2153	GLPF_NC003923-	TAGCTGGCGCGAAATTA	218	GLPF_NC003923-1296927-	TCACCGATAAATAAAAT	972
	1296927-	GGTGT		1297391_323_359_R	ACCTAAAGTTAATGCCA
	1297391_239_260_F				TTG

2154	GMK_NC003923-	TACTTTTTTAAAACTAG	200	GMK_NC003923-1190906-	TGATATTGAACTGGTGT	1180
	1190906-	GGATGCGTTTGAAGC		1191334_166_197_R	ACCATAATAGTTGCC
	1191334_91_122_F

2155	GMK_NC003923-	TGAAGTAGAAGGTGCAA	435	GMK_NC003923-1190906-	TCGCTCTCTCAAGTGAT	1082
	1190906-	AGCAAGTTAGA		1191334_305_333_R	CTAAACTTGGAG
	1191334_240_267_F

2156	GMK_NC003923-	TCACCTCCAAGTTTAGA	268	GMK_NC003923-1190906-	TGGGACGTAATCGTATA	1284
	1190906-	TCACTTGAGAGA		1191334_403_432_R	AATTCATCATTTC
	1191334_301_329_F

2157	PTA_NC003923-	TCTTGTTTATGCTGGTA	418	PTA_NC003923-628885-	TGGTACACCTGGTTTCG	1301
	628885-	AAGCAGATGG		629355_314_345_R	TTTTGATGATTTGTA
	629355_237_263_F

2158	PTA_NC003923-	TGAATTAGTTCAATCAT	439	PTA_NC003923-628885-	TGCATTGTACCGAAGTA	1207
	628885-	TTGTTGAACGACGT		629355_211_239_R	GTTCACATTGTT
	629355_141_171_F

2159	PTA_NC003923-	TCCAAACCAGGTGTATC	303	PTA_NC003923-628885-	TGTTCTGGATTGATTGC	1349
	628885-	AAGAACATCAGG		629355_393_422_R	ACAATCACCAAAG
	629355_328_356_F

2160	TPI_NC003923-	TGCAAGTTAAGAAAGCT	486	TPI_NC003923-830671-	TGAGATGTTGATGATTT	1165
	830671-	GTTGCAGGTTTAT		831072_209_239_R	ACCAGTTCCGATTG
	831072_131_160_F

2161	TPI_NC003923-	TCCCACGAAACAGATGA	318	TPI_NC003923-830671-	TGGTACAACATCGTTAG	1300
	830671-	AGAAATTAACAAAAAAG		831072_97_129_R	CTTTACCACTTTCACG
	831072_1_34_F

2162	TPI_NC003923-	TCAAACTGGGCAATCGG	246	TPI_NC003923-830671-	TGGCAGCAATAGTTTGA	1275
	830671-	AACTGGTAAATC		831072_253_286_R	CGTACAAATGCACACAT
	831072_199_227_F

2163	YQI_NC003923-	TGAATTGCTGCTATGAA	440	YQI_NC003923-378916-	TCGCCAGCTAGCACGAT	1076
	378916-	AGGTGGCTT		379431_259_284_R	GTCATTTTC
	379431_142_167_F

2164	YQI_NC003923-	TACAACATATTATTAAA	175	YQI_NC003923-378916-	TTCGTGCTGGATTTTGT	1388
	378916-	GAGACGGGTTTGAATCC		379431_120_145_R	CCTTGTCCT
	379431_44_77_F

2165	YQI_NC003923-	TCCAGCACGAATTGCTG	314	YQI_NC003923-378916-	TCCAACCCAGAACCACA	997
	378916-	CTATGAAAG		379431_193_221_R	TACTTTATTCAC
	379431_135_160_F

2166	YQI_NC003923-	TAGCTGGCGGTATGGAG	219	YQI_NC003923-378916-	TCCATCTGTTAAACCAT	1013
	378916-	AATATGTCT		379431_364_396_R	CATATACCATGCTATC
	379431_275_300_F

2167	BLAZ_	TCCACTTATCGCAAATG	312	BLAZ_	TGGCCACTTTTATCAGC	1277
	(1913827 . . .	GAAAATTAAGCAA		(1913827 . . . 1914672)_	AACCTTACAGTC
	1914672)_546_575_F			655_683_R

2168	BLAZ_	TGCACTTATCGCAAATG	494	BLAZ_	TAGTCTTTTGGAACACC	926
	(1913827 . . .	GAAAATTAAGCAA		(1913827 . . . 1914672)_	GTCTTTAATTAAAGT
	1914672)_546_575_—			628_659_R
	2_F

2169	BLAZ_	TGATACTTCAACGCCTG	467	BLAZ_	TGGAACACCGTCTTTAA	1263
	(1913827 . . .	CTGCTTTC		(1913827 . . . 1914672)_	TTAAAGTATCTCC
	1914672)_507_531_F			622_651_R

2170	BLAZ_	TATACTTCAACGCCTGC	232	BLAZ_	TCTTTTCTTTGCTTAAT	1145
	(1913827 . . .	TGCTTTC		(1913827 . . . 1914672)_	TTTCCATTTGCGAT
	1914672)_508_531_F			553_583_R

2171	BLAZ_	TGCAATTGCTTTAGTTT	487	BLAZ_	TTACTTCCTTACCACTT	1366
	(1913827 . . .	TAAGTGCATGTAATTC		(1913827 . . . 1914672)_	TTAGTATCTAAAGCATA
	1914672)_24_56_F			121_154_R

2172	BLAZ_	TCCTTGCTTTAGTTTTA	351	BLAZ_	TGGGGACTTCCTTACCA	1289
	(1913827 . . .	AGTGCATGTAATTCAA		(1913827 . . . 1914672)_	CTTTTAGTATCTAA
	1914672)_26_58_F			127_157_R

2173	BLAZ_NC002952-	TCCACTTATCGCAAATG	312	BLAZ_NC002952-1913827-	TGGCCACTTTTATCAGC	1277
	1913827-	GAAAATTAAGCAA		1914672_655_683_R	AACCTTACAGTC
	1914672_546_575_F

2174	BLAZ_NC002952-	TGCACTTATCGCAAATG	494	BLAZ_NC002952-1913827-	TAGTCTTTTGGAACACC	926
	1913827-	GAAAATTAAGCAA		1914672_628_659_R	GTCTTTAATTAAAGT
	1914672_546_575_
	2_F

2175	BLAZ_NC002952-	TGATACTTCAACGCCTG	467	BLAZ_NC002952-1913827-	TGGAACACCGTCTTTAA	1263
	1913827-	CTGCTTTC		1914672_622_651_R	TTAAAGTATCTCC
	1914672_507_531_F

2176	BLAZ_NC002952-	TATACTTCAACGCCTGC	232	BLAZ_NC002952-1913827-	TCTTTTCTTTGCTTAAT	1145
	1913827-	TGCTTTC		1914672_553_583_R	TTTCCATTTGCGAT
	1914672_508_531_F

2177	BLAZ_NC002952-	TGCAATTGCTTTAGTTT	487	BLAZ_NC002952-1913827-	TTACTTCCTTACCACTT	1366
	1913827-	TAAGTGCATGTAATTC		1914672_121_154_R	TTAGTATCTAAAGCATA
	1914672_24_56_F

2178	BLAZ_NC002952-	TCCTTGCTTTAGTTTTA	351	BLAZ_NC002952-1913827-	TGGGGACTTCCTTACCA	1289
	1913827-	AGTGCATGTAATTCAA		1914672_127_157_R	CTTTTAGTATCTAA
	1914672_26_58_F

2247	TUFB_NC002758-	TGTTGAACGTGGTCAAA	643	TUFB_NC002758-615038-	TGTCACCAGCTTCAGCG	1321
	615038-	TCAAAGTTGGTG		616222_793_820_R	TAGTCTAATAA
	616222_693_721_F

2248	TUFB_NC002758-	TCGTGTTGAACGTGGTC	386	TUFB_NC002758-615038-	TGTCACCAGCTTCAGCG	1321
	615038-	AAATCAAAGT		616222_793_820_R	TAGTCTAATAA
	616222_690_716_F

2249	TUFB_NC002758-	TGAACGTGGTCAAATCA	430	TUFB_NC002758-615038-	TGTCACCAGCTTCAGCG	1321
	615038-	AAGTTGGTGAAGA		616222_793_820_R	TAGTCTAATAA
	616222_696_725_F

2250	TUFB_NC002758-	TCCCAGGTGACGATGTA	320	TUFB_NC002758-615038-	TGGTTTGTCAGAATCAC	1311
	615038-	CCTGTAATC		616222_601_630_R	GTTCTGGAGTTGG
	616222_488_513_F

2251	TUFB_NC002758-	TGAAGGTGGACGTCACA	433	TUFB_NC002758-615038-	TAGGCATAACCATTTCA	922
	615038-	CTCCATTCTTC		616222_1030_1060_R	GTACCTTCTGGTAA
	616222_945_972_F

2252	TUFB_NC002758-	TCCAATGCCACAAACTC	307	TUFB_NC002758-615038-	TTCCATTTCAACTAATT	1382
	615038-	GTGAACA		616222_424_459_R	CTAATAATTCTTCATCG
	616222_333_356_F				TC

2253	NUC_NC002758-	TCCTGAAGCAAGTGCAT	342	NUC_NC002758-894288-	TACGCTAAGCCACGTCC	899
	894288-	TTACGA		894974_483_509_R	ATATTTATCA
	894974_402_424_F

2254	NUC_NC002758-	TCCTTATAGGGATGGCT	349	NUC_NC002758-894288-	TGTTTGTGATGCATTTG	1354
	894288-	ATCAGTAATGTT		894974_165_189_R	CTGAGCTA
	894974_53_81_F

2255	NUC_NC002758-	TCAGCAAATGCATCACA	273	NUC_NC002758-894288-	TAGTTGAAGTTGCACTA	928
	894288-	AACAGATAA		894974_222_250_R	TATACTGTTGGA
	894974_169_194_F

2256	NUC_NC002758-	TACAAAGGTCAACCAAT	174	NUC_NC002758-894288-	TAAATGCACTTGCTTCA	853
	894288-	GACATTCAGACTA		894974_396_421_R	GGGCCATAT
	894974_316_345_F

2270	RPOB_EC_3798_3821_	TGGCCAGCGCTTCGGTG	566	RPOB_EC_3868_3895_R	TCACGTCGTCCGACTTC	979
	1_F	AAATGGA			ACGGTCAGCAT

2271	RPOB_EC_3789_3812_	TCAGTTCGGCGGTCAGC	294	RPOB_EC_3860_3890_R	TCGTCGGACTTAACGGT	1107
	F	GCTTCGG			CAGCATTTCCTGCA

2272	RPOB_EC_3789_3812_	TCAGTTCGGCGGTCAGC	294	RPOB_EC_3860_3890_2_R	TCGTCCGACTTAACGGT	1102
	F	GCTTCGG			CAGCATTTCCTGCA

2273	RPOB_EC_3789_3812_	TCAGTTCGGCGGTCAGC	294	RPOB_EC_3862_3890_R	TCGTCGGACTTAACGGT	1106
	F	GCTTCGG			CAGCATTTCCTG

2274	RPOB_EC_3789_3812_	TCAGTTCGGCGGTCAGC	294	RPOB_EC_3862_3890_2_R	TCGTCCGACTTAACGGT	1101
	F	GCTTCGG			CAGCATTTCCTG

2275	RPOB_EC_3793_3812_	TTCGGCGGTCAGCGCTT	674	RPOB_EC_3865_3890_R	TCGTCGGACTTAACGGT	1105
	F	CGG			CAGCATTTC

2276	RPOB_EC_3793_3812_	TTCGGCGGTCAGCGCTT	674	RPOB_EC_3865_3890_2_R	TCGTCCGACTTAACGGT	1100
	F	CGG			CAGCATTTC

2309	MUPR_X75439_1658_	TCCTTTGATATATTATG	352	MUPR_X75439_1744_1773_R	TCCCTTCCTTAATATGA	1030
	1689_F	CGATGGAAGGTTGGT			GAAGGAAACCACT

2310	MUPR_X75439_1330_	TTCCTCCTTTTGAAAGC	669	MUPR_X75439_1413_1441_R	TGAGCTGGTGCTATATG	1171
	1353_F	GACGGTT			AACAATACCAGT

2312	MUPR_X75439_1314_	TTTCCTCCTTTTGAAAG	704	MUPR_X75439_1381_1409_R	TATATGAACAATACCAG	931
	1338_F	CGACGGTT			TTCCTTCTGAGT

2313	MUPR_X75439_2486_	TAATTGGGCTCTTTCTC	172	MUPR_X75439_2548_2574_R	TTAATCTGGCTGCGGAA	1360
	2516_F	GCTTAAACACCTTA			GTGAAATCGT

2314	MUPR_X75439_2547_	TACGATTTCACTTCCGC	188	MUPR_X75439_2605_2630_R	TCGTCCTCTCGAATCTC	1103
	2572_F	AGCCAGATT			CGATATACC

2315	MUPR_X75439_2666_	TGCGTACAATACGCTTT	513	MUPR_X75439_2711_2740_R	TCAGATATAAATGGAAC	981
	2696_F	ATGAAATTTTAACA			AAATGGAGCCACT

2316	MUPR_X75439_2813_	TAATCAAGCATTGGAAG	165	MUPR_X75439_2867_2890_R	TCTGCATTTTTGCGAGC	1127
	2843_F	ATGAAATGCATACC			CTGTCTA

2317	MUPR_X75439_884_	TGACATGGACTCCCCCT	447	MUPR_X75439_977_1007_R	TGTACAATAAGGAGTCA	1317
	914_F	ATATAACTCTTGAG			CCTTATGTCCCTTA

2318	CTXA_NC002505-	TGGTCTTATGCCAAGAG	608	CTXA_NC002505-1568114-	TCGTGCCTAACAAATCC	1109
	1568114-	GACAGAGTGAGT		1567341_194_221_R	CGTCTGAGTTC
	1567341_114_142_F

2319	CTXA_NC002505-	TCTTATGCCAAGAGGAC	411	CTXA_NC002505-1568114-	TCGTGCCTAACAAATCC	1109
	1568114-	AGAGTGAGTACT		1567341_194_221_R	CGTCTGAGTTC
	1567341_117_145_F

2320	CTXA_NC002505-	TGGTCTTATGCCAAGAG	608	CTXA_NC002505-1568114-	TAACAAATCCCGTCTGA	855
	1568114-	GACAGAGTGAGT		1567341_186_214_R	GTTCCTCTTGCA
	1567341_114_142_F

2321	CTXA_NC002505-	TCTTATGCCAAGAGGAC	411	CTXA_NC002505-1568114-	TAACAAATCCCGTCTGA	855
	1568114-	AGAGTGAGTACT		1567341_186_214_R	GTTCCTCTTGCA
	1567341_117_145_F

2322	CTXA_NC002505-	AGGACAGAGTGAGTACT	27	CTXA_NC002505-1568114-	TCCCGTCTGAGTTCCTC	1027
	1568114-	TTGACCGAGGT		1567341_180_207_R	TTGCATGATCA
	1567341_129_156_F

2323	CTXA_NC002505-	TGCCAAGAGGACAGAGT	500	CTXA_NC002505-1568114-	TAACAAATCCCGTCTGA	855
	1568114-	GAGTACTTTGA		1567341_186_214_R	GTTCCTCTTGCA
	1567341_122_149_F

2324	INV_U22457-74-	TGCTTATTTACCTGCAC	530	INV_U22457-74-	TGACCCAAAGCTGAAAG	1154
	3772_831_858_F	TCCCACAACTG		3772_942_966_R	CTTTACTG

2325	INV_U22457-74-	TGAATGCTTATTTACCT	438	INV_U22457-74-	TAACTGACCCAAAGCTG	864
	3772_827_857_F	GCACTCCCACAACT		3772_942_970_R	AAAGCTTTACTG

2326	INV_U22457-74-	TGCTGGTAACAGAGCCT	526	INV_U22457-74-	TGGGTTGCGTTGCAGAT	1296
	3772_1555_1581_F	TATAGGCGCA		3772_1619_1647_R	TATCTTTACCAA

2327	INV_U22457-74-	TGGTAACAGAGCCTTAT	598	INV_U22457-74-	TCATAAGGGTTGCGTTG	987
	3772_1558_1585_F	AGGCGCATATG		3772_1622_1652_R	CAGATTATCTTTAC

2328	ASD_NC006570-	TGAGGGTTTTATGCTTA	459	ASD_NC006570-439714-	TGATTCGATCATACGAG	1188
	439714-	AAGTTGGTTTTATTGGT		438608_54_84_R	ACATTAAAACTGAG
	438608_3_37_F	T

2329	ASD_NC006570-	TAAAGTTGGTTTTATTG	149	ASD_NC006570-439714-	TCAAAATCTTTTGATTC	948
	439714-	GTTGGCGCGGA		438608_66_95_R	GATCATACGAGAC
	438608_18_45_F

2330	ASD_NC006570-	TTAAAGTTGGTTTTATT	647	ASD_NC006570-439714-	TCCCAATCTTTTGATTC	1016
	439714-	GGTTGGCGCGGA		438608_67_95_R	GATCATACGAGA
	438608_17_45_F

2331	ASD_NC006570-	TTTTATGCTTAAAGTTG	709	ASD_NC006570-439714-	TCTGCCTGAGATGTCGA	1128
	439714-	GTTTTATTGGTTGGC		438608_107_134_R	AAAAAACGTTG
	438608_9_40_F

2332	GALE_AF513299_171_	TCAGCTAGACCTTTTAG	280	GALE_AF513299_241_271_R	TCTCACCTACAGCTTTA	1122
	200_F	GTAAAGCTAAGCT			AAGCCAGCAAAATG

2333	GALE_AF513299_168_	TTATCAGCTAGACCTTT	658	GALE_AF513299_245_271_R	TCTCACCTACAGCTTTA	1121
	199_F	TAGGTAAAGCTAAGC			AAGCCAGCAA

2334	GALE_AF513299_168_	TTATCAGCTAGACCTTT	658	GALE_AF513299_233_264_R	TACAGCTTTAAAGCCAG	883
	199_F	TAGGTAAAGCTAAGC			CAAAATGAATTACAG

2335	GALE_AF513299_169_	TCCCAGCTAGACCTTTT	319	GALE_AF513299_252_279_R	TTCAACACTCTCACCTA	1374
	198_F	AGGTAAAGCTAAG			CAGCTTTAAAG

2336	PLA_AF053945_7371_	TTGAGAAGACATCCGGC	680	PLA_AF053945_7434_7468_R	TACGTATGTAAATTCC	900
	7403_F	TCACGTTATTATGGTA			GCAAAGACTTTGGCAT
					TAG

2337	PLA_AF053945_7377_	TGACATCCGGCTCACGT	443	PLA_AF053945_7428_7455_R	TCCGCAAAGACTTTGGC	1035
	7403_F	TATTATGGTA			ATTAGGTGTGA

2338	PLA_AF053945_7377_	TGACATCCGGCTCACGT	444	PLA_AF053945_7430_7460_R	TAAATTCCGCAAAGACT	854
	7404_F	TATTATGGTAC			TTGGCATTAGGTGT

2339	CAF_AF053947_	TCCGTTATCGCCATTGC	329	CAF_AF053947_33498_	TAAGAGTGATGCGGGCT	866
	33412_33441_F	ATTATTTGGAACT		33523_R	GGTTCAACA

2340	CAF_AF053947_	TGCATTATTTGGAACTA	499	CAF_AF053947_33483_	TGGTTCAACAAGAGTTG	1308
	33426_33458_F	TTGCAACTGCTAATGC		33507_R	CCGTTGCA

2341	CAF_AF053947_	TCAGTTCCGTTATCGCC	291	CAF_AF053947_33483_	TTCAACAAGAGTTGCCG	1373
	33407_33429_F	ATTGCA		33504_R	TTGCA

2342	CAF_AF053947_	TCAGTTCCGTTATCGCC	293	CAF_AF053947_33494_	TGATGCGGGCTGGTTCA	1184
	33407_33431_F	ATTGCATT		33517_R	ACAAGAG

2344	GAPA_NC_002505_1_	TCAATGAACGATCAACA	260	GAPA_NC_002505_29_58_R_1	TCCTTTATGCAACTTGG	1060
	28_F_1	AGTGATTGATG			TATCAACAGGAAT

2472	OMPA_NC000117_68_	TGCCTGTAGGGAATCCT	507	OMPA_NC000117_145_167_R	TCACACCAAGTAGTGCA	967
	89_F	GCTGA			AGGATC

2473	OMPA_NC000117_798_	TGATTACCATGAGTGGC	475	OMPA_NC000117_865_893_R	TCAAAACTTGCTCTAGA	947
	821_F	AAGCAAG			CCATTTAACTCC

2474	OMPA_NC000117_645_	TGCTCAATCTAAACCTA	521	OMPA_NC000117_757_777_R	TGTCGCAGCATCTGTTC	1328
	671_F	AAGTCGAAGA			CTGC

2475	OMPA_NC000117_947_	TAACTGCATGGAACCCT	157	OMPA_NC000117_1011_	TGACAGGACACAATCTG	1153
	973_F	TCTTTACTAG		1040_R	CATGAAGTCTGAG

2476	OMPA_NC000117_774_	TACTGGAACAAAGTCTG	196	OMPA_NC000117_871_894_R	TTCAAAAGTTGCTCGAG	1371
	795_F	CGACC			ACCATTG

2477	OMPA_NC000117_457_	TTCTATCTCGTTGGTTT	676	OMPA_NC000117_511_534_R	TAAAGAGACGTTTGGTA	851
	483_R	ATTCGGAGTT			GTTCATTTGC

2478	OMPA_NC000117_687_	TAGCCCAGCACAATTTG	212	OMPA_NC000117_787_816_R	TTGCCATTCATGGTATT	1406
	710_F	TGATTCA			TAAGTGTAGCAGA

2479	OMPA_NC000117_540_	TGGCGTAGTAGAGCTAT	571	OMPA_NC000117_649_672_R	TTCTTGAACGCGAGGTT	1395
	566_F	TTACAGACAC			TCGATTG

2480	OMPA_NC000117_338_	TGCACGATGCGGAATGG	492	OMPA_NC000117_417_444_R	TCCTTTAAAATAACCGC	1058
	360_F	TTCACA			TAGTAGCTCCT

2481	OMP2_NC000117_18_	TATGACCAAACTCATCA	234	OMP2_NC000117_71_91_R	TCCCGCTGGCAAATAAA	1025
	40_F	GACGAG			CTCG

2482	OMP2_NC000117_354_	TGCTACGGTAGGATCTC	516	OMP2_NC000117_445_471_R	TGGATCACTGCTTACGA	1270
	382_F	CTTATCCTATTG			ACTCAGCTTC

2483	OMP2_NC000117_	TGGAAAGGTGTTGCAGC	537	OMP2_NC000117_1396_	TACGTTTGTATCTTCTG	903
	1297_1319_F	TACTCA		1419_R	CAGAACC

2484	OMP2_NC000117_	TCTGGTCCAACAAAAGG	407	OMP2_NC000117_1541_	TCCTTTCAATGTTACAG	1062
	1465_1493_F	AACGATTACAGG		1569_R	AAAACTCTACAG

2485	OMP2_NC000117_44_	TGACGATCTTCGCGGTG	450	OMP2_NC000117_120_148_R	TGTCAGCTAAGCTAATA	1323
	66_F	ACTAGT			ACGTTTGTAGAG

2486	OMP2_NC000117_166_	TGACAGCGAAGAAGGTT	441	OMP2_NC000117_240_261_R	TTGACATCGTCCCTCTT	1396
	190_F	AGACTTGTCC			CACAG

2487	GYRA_NC000117_514_	TCAGGCATTGCGGTTGG	287	GYRA_NC000117_640_660_R	TGCTGTAGGGAAATCAG	1251
	536_F	GATGGC			GGCC

2488	GYRA_NC000117_801_	TGTGAATAAATCACGAT	636	GYRA_NC000117_871_893_R	TTGTCAGACTCATCGCG	1419
	827_F	TGATTGAGCA			AACATC

2489	GYRA_NC002952_219_	TGTCATGGGTAAATATC	632	GYRA_NC002952_319_345_R	TCCATCCATAGAACCAA	1010
	242_F	ACCCTCA			AGTTACCTTG

2490	GYRA_NC002952_964_	TACAAGCACTCCCAGCT	176	GYRA_NC002952_1024_	TCGCAGCGTGCGTGGCA	1073
	983_F	GCA		1041_R	C

2491	GYRA_NC002952_	TCGCCCGCGAGGACGT	366	GYRA_NC002952_1546_	TTGGTGCGCTTGGCGTA	1416
	1505_1520_F			1562_R

2492	GYRA_NC002952_59_	TCAGCTACATCGACTAT	279	GYRA_NC002952_124_143_R	TGGCGATGCACTGGCTT	1279
	81_F	GCGATG			GAG

2493	GYRA_NC002952_216_	TGACGTCATCGGTAAGT	452	GYRA_NC002952_313_333_R	TCCGAAGTTGCCCTGGC	1032
	239_F	ACCACCC			CGTC

2494	GYRA_NC002952_219_	TGTACTCGGTAAGTATC	625	GYRA_NC002952_308_330_R	TAAGTTACCTTGCCCGT	873
	242_2_F	ACCCGCA			CAACCA

2495	GYRA_NC002952_115_	TGAGATGGATTTAAACC	453	GYRA_NC002952_220_242_R	TGCGGGTGATACTTACC	1236
	141_F	TGTTCACCGC			GAGTAC

2496	GYRA_NC002952_517_	TCAGGCATTGCGGTTGG	287	GYRA_NC002952_643_663_R	TGCTGTAGGGAAATCAG	1251
	539_F	GATGGC			GGCC

2497	GYRA_NC002952_273_	TCGTATGGCTCAATGGT	380	GYRA_NC002952_338_360_R	TGCGGCAGCACTATCAC	1234
	293_F	GGAG			CATCCA

2498	GYRA_NC000912_257_	TGAGTAAGTTCCACCCG	462	GYRA_NC000912_346_370_R	TCGAGCCGAAGTTACCC	1067
	278_F	CACGG			TGTCCGTC

2504	ARCC_NC003923-	TAGTpGATpAGAACpTp	229	ARCC_NC003923-2725050-	TCpTpTpTpCpGTATAA	1116
	2725050-	GTAGGCpACpAATpCpG		2724595_214_239P_R	AAAGGACpCpAATpTpG
	2724595_135_161P_F	T			G

2505	PTA_NC003923-	TCTTGTpTpTpATGCpT	417	PTA_NC003923-628885-	TACpACpCpTGGTpTpT	904
	628885-	pGGTAAAGCAGATGG		629355_314_342P_R	pCpGTpTpTpTpGATGA
	629355_237_263P_F				TpTpTpGTA

2517	CJMLST_ST1_1852_	TTTGCGGATGAAGTAGG	708	CJMLST_ST1_1945_1977_R	TGTTTTATGTGTAGTTG	1355
	1883_F	TGCCTATCTTTTTGC			AGCTTACTACATGAGC

2518	CJMLST_ST1_2963_	TGAAATTGCTACAGGCC	428	CJMLST_ST1_3073_3097_R	TCCCCATCTCCGCAAAG	1020
	2992_F	CTTTAGGACAAGG			ACAATAAA

2519	CJMLST_ST1_2350_	TGCTTTTGATGGTGATG	535	CJMLST_ST1_2447_2481_R	TCTACAACACTTGATTG	1117
	2378_F	CAGATCGTTTGG			TAATTTGCCTTGTTCTT
					T

2520	CJMLST_ST1_654_	TATGTCCAAGAAGCATA	240	CJMLST_ST1_725_756_R	TCGGAAACAAAGAATTC	1084
	684_F	GCAAAAAAAGCAAT			ATTTTCTGGTCCAAA

2521	CJMLST_ST1_360_	TCCTGTTATTCCTGAAG	347	CJMLST_ST1_454_487_R	TGCTATATGCTACAACT	1245
	395_F	TAGTTAATCAAGTTTGT			GGTTCAAAAACATTAAG
		TA

2522	CJMLST_ST1_1231_	TGGCAGTTTTACAAGGT	564	CJMLST_ST1_1312_1340_R	TTTAGCTACTATTCTAG	1427
	1258_F	GCTGTTTCATC			CTGCCATTTCCA

2523	CJMLST_ST1_3543_	TGCTGTAGCTTATCGCG	529	CJMLST_ST1_3656_3685_R	TCAAAGAACCAGCACCT	950
	3574_F	AAATGTCTTTGATTT			AATTCATCATTTA

2524	CJMLST_ST1_1_17_F	TAAAACTTTTGCCGTAA	145	CJMLST_ST1_55_84_R	TGTTCCAATAGCAGTTC	1348
		TGATGGGTGAAGATAT			CGCCCAAATTGAT

2525	CJMLST_ST1_1312_	TGGAAATGGCAGCTAGA	538	CJMLST_ST1_1383_1417_R	TTTCCCCGATCTAAATT	1432
	1342_F	ATAGTAGCTAAAAT			TGGATAAGCCATAGGAA
					A

2526	CJMLST_ST1_2254_	TGGGCCTAATGGGCTTA	582	CJMLST_ST1_2352_2379_R	TCCAAACGATCTGCATC	996
	2286_F	ATATCAATGAAAATTG			ACCATCAAAAG

2527	CJMLST_ST1_1380_	TGCTTTCCTATGGCTTA	534	CJMLST_ST1_1486_1520_R	TGCATGAAGCATAAAAA	1205
	1411_F	TCCAAATTTAGATCG			CTGTATCAAGTGCTTTT
					A

2528	CJMLST_ST1_3413_	TTGTAAATGCCGGTGCT	692	CJMLST_ST1_3511_3542_R	TGCTTGCTCAAATCATC	1257
	3437_F	TCAGATCC			ATAAACAATTAAAGC

2529	CJMLST_ST1_1130_	TACGCGTCTTGAAGCGT	189	CJMLST_ST1_1203_1230_R	TAGGATGAGCATTATCA	920
	1156_F	TTCGTTATGA			GGGAAAGAATC

2530	CJMLST_ST1_2840_	TGGGGCTTTGCTTTATA	591	CJMLST_ST1_2940_2973_R	TAGCGATTTCTACTCCT	917
	2872_F	GTTTTTTACATTTAAG			AGAGTTGAAATTTCAGG

2531	CJMLST_ST1_2058_	TATTCAAGGTGGTCCTT	241	CJMLST_ST1_2131_2162_R	TTGGTTCTTACTTGTTT	1417
	2084_F	TGATGCATGT			TGCATAAACTTTCCA

2532	CJMLST_ST1_553_	TCCTGATGCTCAAAGTG	344	CJMLST_ST1_655_685_R	TATTGCTTTTTTTGCTA	942
	585_F	CTTTTTTAGATCCTTT			TGCTTCTTGGACAT

2564	GLTA_NC002163-	TCATGTTGAGCTTAAAC	299	GLTA_NC002163-1604930-	TTTTGCTCATGATCTGC	1443
	1604930-	CTATAGAAGTAAAAGC		1604529_352_380_R	ATGAAGCATAAA
	1604529_306_338_F

2565	UNCA_NC002163-	TCCCCCACGCTTTAATT	322	UNCA_NC002163-112166-	TCGACCTGGAGGACGAC	1065
	112166-	GTTTATGATGATTTGAG		112647_146_171_R	GTAAAATCA
	112647_80_113_F

2566	UNCA_NC002163-	TAATGATGAATTAGGTG	170	UNCA_NC002163-112166-	TGGGATAACATTGGTTG	1285
	112166-	CGGGTTCTTT		112647_294_329_R	GAATATAAGCAGAAACA
	112647_233_259_F				TC

2567	PGM_NC002163-	TCTTGATACTTGTAATG	414	PGM_NC002163-327773-	TCCATCGCCAGTTTTTG	1012
	327773-	TGGGCGATAAATATGT		328270_365_396_R	CATAATCGCTAAAAA
	328270_273_305_F

2568	TKT_NC002163-	TTATGAAGCGTGTTCTT	661	TKT_NC002163-1569415-	TCAAAACGCATTTTTAC	946
	1569415-	TAGCAGGACTTCA		1569873_350_383_R	ATCTTCGTTAAAGGCTA
	1569873_255_284_F

2570	GLTA_NC002163-	TCGTCTTTTTGATTCTT	381	GLTA_NC002163-1604930-	TGTTCATGTTTAAATGA	1347
	1604930-	TCCCTGATAATGC		1604529_109_142_R	TCAGGATAAAAAGCACT
	1604529_39_68_F

2571	TKT_NC002163-	TGATCTTAAAAATTTCC	472	TKT_NC002163-1569415-	TGCCATAGCAAAGCCTA	1214
	1569415-	GCCAACTTCATTC		1569903_139_162_R	CAGCATT
	1569903_33_62_F

2572	TKT_NC002163-	TAAGGTTTATTGTCTTT	164	TKT_NC002163-1569415-	TACATCTCCTTCGATAG	886
	1569415-	GTGGAGATGGGGATTT		1569903_313_345_R	AAATTTCATTGCTATC
	1569903_207_239_F

2573	TKT_NC002163-	TAGCCTTTAACGAAAAT	213	TKT_NC002163-1569415-	TAAGACAAGGTTTTGTG	865
	1569415-	GTAAAAATGCGTTTTGA		1569903_449_481_R	GATTTTTTAGCTTGTT
	1569903_350_383_F

2574	TKT_NC002163-	TTCAAAAACTCCAGGCC	665	TKT_NC002163-1569415-	TTGCCATAGCAAAGCCT	1405
	1569415-	ATCCTGAAATTTCAAC		1569903_139_163_R	ACAGCATT
	1569903_60_92_F

2575	GLTA_NC002163-	TCGTCTTTTTGATTCTT	382	GLTA_NC002163-1604930-	TGCCATTTCCATGTACT	1216
	1604930-	TCCCTGATAATGCTC		1604529_139_168_R	CTTCTCTAACATT
	1604529_39_70_F

2576	GLYA_NC002163-	TCAGCTATTTTTCCAGG	281	GLYA_NC002163-367572-	ATTGCTTCTTACTTGCT	756
	367572-	TATCCAAGGTGG		368079_476_508_R	TAGCATAAATTTTCCA
	368079_386_414_F

2577	GLYA_NC002163-	TGGTGCGAGTGCTTATG	611	GLYA_NC002163-367572-	TGCTCACCTGCTACAAC	1246
	367572-	CTCGTATTAT		368079_242_270_R	AAGTCCAGCAAT
	368079_148_174_F

2578	GLYA_NC002163-	TGTAAGCTCTACAACCC	622	GLYA_NC002163-367572-	TTCCACCTTGGATACCT	1381
	367572-	ACAAAACCTTACG		368079_384_416_R	GGAAAAATAGCTGAAT
	368079_298_327_F

2579	GLYA_NC002163-	TGGTGGACATTTAACAC	614	GLYA_NC002163-367572-	TCAAGCTCTACACCATA	961
	367572-	ATGGTGCAAA		368079_52_81_R	AAAAAAGCTCTCA
	368079_1_27_F

2580	PGM_NC002163-	TGAGCAATGGGGCTTTG	455	PGM_NC002163-327746-	TTTGCTCTCCGCCAAAG	1438
	327746-	AAAGAATTTTTAAAT		328270_356_379_R	TTTCCAC
	328270_254_285_F

2581	PGM_NC002163-	TGAAAAGGGTGAAGTAG	425	PGM_NC002163-327746-	TGCCCCATTGCTCATGA	1219
	327746-	CAAATGGAGATAG		328270_241_267_R	TAGTAGCTAC
	328270_153_182_F

2582	PGM_NC002163-	TGGCCTAATGGGCTTAA	568	PGM_NC002163-327746-	TGCACGCAAACGCTTTA	1200
	327746-	TATCAATGAAAATTG		328270_79_102_R	CTTCAGC
	328270_19_50_F

2583	UNCA_NC002163-	TAAGCATGCTGTGGCTT	160	UNCA_NC002163-112166-	TGCCCTTTCTAAAAGTC	1220
	112166-	ATCGTGAAATG		112647_196_225_R	TTGAGTGAAGATA
	112647_114_141_F

2584	UNCA_NC002163-	TGCTTCGGATCCAGCAG	532	UNCA_NC002163-112166-	TGCATGCTTACTCAAAT	1206
	112166-	CACTTCAATA		112647_88_123_R	CATCATAAACAATTAAA
	112647_3_29_F				GC

2585	ASPA_NC002163-	TTAATTTGCCAAAAATG	652	ASPA_NC002163-96692-	TGCAAAAGTAACGGTTA	1192
	96692-	CAACCAGGTAG		97166_403_432_R	CATCTGCTCCAAT
	97166_308_335_F

2586	ASPA_NC002163-	TCGCGTTGCAACAAAAC	370	ASPA_NC002163-96692-	TCATGATAGAACTACCT	991
	96692-	TTTCTAAAGTATGT		97166_316_346_R	GGTTGCATTTTTGG
	97166_228_258_F

2587	GLNA_NC002163-	TGGAATGATGATAAAGA	547	GLNA_NC002163-658085-	TGAGTTTGAACCATTTC	1176
	658085-	TTTCGCAGATAGCTA		657609_340_371_R	AGAGCGAATATCTAC
	657609_244_275_F

2588	TKT_NC002163-	TCGCTACAGGCCCTTTA	371	TKT_NC002163-1569415-	TCCCCATCTCCGCAAAG	1020
	1569415-	GGACAAG		1569903_212_236_R	ACAATAAA
	1569903_107_130_F

2589	TKT_NC002163-	TGTTCTTTAGCAGGACT	642	TKT_NC002163-1569415-	TCCTTGTGCTTCAAAAC	1057
	1569415-	TCACAAACTTGATAA		1569903_361_393_R	GCATTTTTACATTTTC
	1569903_265_296_F

2590	GLYA_NC002163-	TGCCTATCTTTTTGCTG	505	GLYA_NC002163-367572-	TCCTCTTGGGCCACGCA	1047
	367572-	ATATAGCACATATTGC		368095_317_340_R	AAGTTTT
	368095_214_246_F

2591	GLYA_NC002163-	TCCTTTGATGCATGTAA	353	GLYA_NC002163-367572-	TCTTGAGCATTGGTTCT	1141
	367572-	TTGCTGCAAAAGC		368095_485_516_R	TACTTGTTTTGCATA
	368095_415_444_F

2592	PGM_NC002163_21_	TCCTAATGGACTTAATA	332	PGM_NC002163_116_142_R	TCAAACGATCCGCATCA	949
	54_F	TCAATGAAAATTGTGGA			CCATCAAAAG

2593	PGM_NC002163_149_	TAGATGAAAAAGGCGAA	207	PGM_NC002163_247_277_R	TCCCCTTTAAAGCACCA	1023
	176_F	GTGGCTAATGG			TTACTCATTATAGT

2594	GLNA_NC002163-	TGTCCAAGAAGCATAGC	633	GLNA_NC002163-658085-	TCAAAAACAAAGAATTC	945
	658085-	AAAAAAAGCAA		657609_148_179_R	ATTTTCTGGTCCAAA
	657609_79_106_F

2595	ASPA_NC002163-	TCCTGTTATTCCTGAAG	347	ASPA_NC002163-96685-	TCAAGCTATATGCTACA	960
	96685-	TAGTTAATCAAGTTTGT		97196_467_497_R	ACTGGTTCAAAAAC
	97196_367_402_F	TA

2596	ASPA_NC002163-	TGCCGTAATGATAGGTG	502	ASPA_NC002163-96685-	TACAACCTTCGGATAAT	880
	96685-97196_1_33_F	AAGATATACAAAGAGT		97196_95_127_R	CAGGATGAGAATTAAT

2597	ASPA_NC002163-	TGGAACAGGAATTAATT	540	ASPA_NC002163-96685-	TAAGCTCCCGTATCTTG	872
	96685-	CTCATCCTGATTATCC		97196_185_210_R	AGTCGCCTC
	97196_85_117_F

2598	PGM_NC002163-	TGGCAGCTAGAATAGTA	563	PGM_NC002163-327746-	TCACGATCTAAATTTGC	975
	327746-	GCTAAAATCCCTAC		328270_230_261_R	ATAAGCCATAGGAAA
	328270_165_195_F

2599	PGM_NC002163-	TGGGTCGTGGTTTTACA	593	PGM_NC002163-327746-	TTTTGCTCATGATCTGC	1443
	327746-	GAAAATTTCTTATATAT		328270_353_381_R	ATGAAGCATAAA
	328270_252_286_F	G

2600	PGM_NC002163-	TGGGATGAAAAAGCGTT	577	PGM_NC002163-327746-	TGATAAAAAGCACTAAG	1178
	327746-	CTTTTATCCATGA		328270_95_123_R	CGATGAAACAGC
	328270_1_30_F

2601	PGM_NC002163-	TAAACACGGCTTTCCTA	146	PGM_NC002163-327746-	TCAAGTGCTTTTACTTC	963
	327746-	TGGCTTATCCAAAT		328270_314_345_R	TATAGGTTTAAGCTC
	328270_220_250_F

2602	UNCA_NC002163-	TGTAGCTTATCGCGAAA	628	UNCA_NC002163-112166-	TGCTTGCTCTTTCAAGC	1258
	112166-	TGTCTTTGATTTT		112647_199_229_R	AGTCTTGAATGAAG
	112647_123_152_F

2603	UNCA_NC002163-	TCCAGATGGACAAATTT	313	UNCA_NC002163-112166-	TCCGAAACTTGTTTTGT	1031
	112166-	TCTTAGAAACTGATTT		112647_430_461_R	AGCTTTAATTTGAGC
	112647_333_365_F

2734	GYRA_AY291534_237_	TCACCCTCATGGTGATT	265	GYRA_AY291534_268_288_R	TTGCGCCATACGTACCA	1407
	264_F	CAGCTGTTTAT			TCGT

2735	GYRA_AY291534_224_	TAATCGGTAAGTATCAC	167	GYRA_AY291534_256_285_R	TGCCATACGTACCATCG	1213
	252_F	CCTCATGGTGAT			TTTCATAAACAGC

2736	GYRA_AY291534_170_	TAGGAATTACGGCTGAT	221	GYRA_AY291534_268_288_R	TTGCGCCATACGTACCA	1407
	198_F	AAAGCGTATAAA			TCGT

2737	GYRA_AY291534_224_	TAATCGGTAAGTATCAC	167	GYRA_AY291534_319_346_R	TATCGACAGATCCAAAG	935
	252_F	CCTCATGGTGAT			TTACCATGCCC

2738	GYRA_NC002953-	TAAGGTATGACACCGGA	163	GYRA_NC002953-7005-	TCTTGAGCCATACGTAC	1142
	7005-9668_166_195_	TAAATCATATAAA		9668_265_287_R	CATTGC
	F

2739	GYRA_NC002953-	TAATGGGTAAATATCAC	171	GYRA_NC002953-7005-	TATCCATTGAACCAAAG	933
	7005-9668_221_249_	CCTCATGGTGAC		9668_316_343_R	TTACCTTGGCC
	F

2740	GYRA_NC002953-	TAATGGGTAAATATCAC	171	GYRA_NC002953-7005-	TAGCCATACGTACCATT	912
	7005-9668_221_249_	CCTCATGGTGAC		9668_253_283_R	GCTTCATAAATAGA
	F

2741	GYRA_NC002953-	TCACCCTCATGGTGACT	264	GYRA_NC002953-7005-	TCTTGAGCCATACGTAC	1142
	7005-9668_234_261_	CATCTATTTAT		9668_265_287_R	CATTGC
	F

2842	CAPC_AF188935-	TGGGATTATTGTTATCC	578	CAPC_AF188935-56074-	TGGTAACCCTTGTCTTT	1299
	56074-	TGTTATGCCATTTGAGA		55628_348_378_R	GAATTGTATTTGCA
	55628_271_304_F

2843	CAPC_AF188935-	TGATTATTGTTATCCTG	476	CAPC_AF188935-56074-	TGTAACCCTTGTCTTTG	1314
	56074-	TTATGCpCpATpTpTpG		55628_349_377P_R	AATpTpGTATpTpTpGC
	55628_273_303P_F	AG

2844	CAPC_AF188935-	TCCGTTGATTATTGTTA	331	CAPC_AF188935-56074-	TGTTAATGGTAACCCTT	1344
	56074-	TCCTGTTATGCCATTTG		55628_349_384_R	GTCTTTGAATTGTATTT
	55628_268_303_F	AG			GC

2845	CAPC_AF188935-	TCCGTTGATTATTGTTA	331	CAPC_AF188935-56074-	TAACCCTTGTCTTTGAA	860
	56074-	TCCTGTTATGCCATTTG		55628_337_375_R	TTGTATTTGCAATTAAT
	55628_268_303_F	AG			CCTGG

2846	PARC_X95819_33_58_	TCCAAAAAAATCAGCGC	302	PARC_X95819_121_153_R	TAAAGGATAGCGGTAAC	852
	F	GTACAGTGG			TAAATGGCTGAGCCAT

2847	PARC_X95819_65_92_	TACTTGGTAAATACCAC	199	PARC_X95819_157_178_R	TACCCCAGTTCCCCTGA	889
	F	CCACATGGTGA			CCTTC

2848	PARC_X95819_69_93_	TGGTAAATACCACCCAC	596	PARC_X95819_97_128_R	TGAGCCATGAGTACCAT	1169
	F	ATGGTGAC			GGCTTCATAACATGC

2849	PARC_NC003997-	TTCCGTAAGTCGGCTAA	668	PARC_NC003997-3362578-	TCCAAGTTTGACTTAAA	1001
	3362578-	AACAGTCG		3365001_256_283_R	CGTACCATCGC
	3365001_181_205_F

2850	PARC_NC003997-	TGTAACTATCACCCGCA	621	PARC_NC003997-3362578-	TCGTCAACACTACCATT	1099
	3362578-	CGGTGAT		3365001_304_335_R	ATTACCATGCATCTC
	3365001_217_240_F

2851	PARC_NC003997-	TGTAACTATCACCCGCA	621	PARC_NC003997-3362578-	TGACTTAAACGTACCAT	1162
	3362578-	CGGTGAT		3365001_244_275_R	CGCTTCATATACAGA
	3365001_217_240_F

2852	GYRA_AY642140_-1_	TAAATCTGCCCGTGTCG	150	GYRA_AY642140_71_100_R	TGCTAAAGTCTTGAGCC	1242
	24_F	TTGGTGAC			ATACGAACAATGG

2853	GYRA_AY642140_26_	TAATCGGTAAATATCAC	166	GYRA_AY642140_121_146_R	TCGATCGAACCGAAGTT	1069
	54_F	CCGCATGGTGAC			ACCCTGACC

2854	GYRA_AY642140_26_	TAATCGGTAAATATCAC	166	GYRA_AY642140_58_89_R	TGAGCCATACGAACAAT	1168
	54_F	CCGCATGGTGAC			GGTTTCATAAACAGC

2860	CYA_AF065404_1348_	TCCAACGAAGTACAATA	305	CYA_AF065404_1448_1472_R	TCAGCTGTTAACGGCTT	983
	1379_F	CAAGACAAAAGAAGG			CAAGACCC

2861	LEF_BA_AF065404_	TCGAAAGCTTTTGCATA	354	LEF_BA_AF065404_843_	TCTTTAAGTTCTTCCAA	1144
	751_781_F	TTATATCGAGCCAC		881_R	GGATAGATTTATTTCTT
					GTTCG

2862	LEF_BA_AF065404_	TGCATATTATATCGAGC	498	LEF_BA_AF065404_843_	TCTTTAAGTTCTTCCAA	1144
	762_788_F	CACAGCATCG		881_R	GGATAGATTTATTTCTT
					GTTCG

2917	MUTS_AY698802_106_	TCCGCTGAATCTGTCGC	326	MUTS_AY698802_172_193_R	TGCGGTCTGGCGCATAT	1237
	125_F	CGC			AGGTA

2918	MUTS_AY698802_172_	TACCTATATGCGCCAGA	187	MUTS_AY698802_228_252_R	TCAATCTCGACTTTTTG	965
	192_F	CCGC			TGCCGGTA

2919	MUTS_AY698802_228_	TACCGGCGCAAAAAGTC	186	MUTS_AY698802_314_342_R	TCGGTTTCAGTCATCTC	1097
	252_F	GAGATTGG			CACCATAAAGGT

2920	MUTS_AY698802_315_	TCTTTATGGTGGAGATG	419	MUTS_AY698802_413_433_R	TGCCAGCGACAGACCAT	1210
	342_F	ACTGAAACCGA			CGTA

2921	MUTS_AY698802_394_	TGGGCGTGGAACGTCCA	585	MUTS_AY698802_497_519_R	TCCGGTAACTGGGTCAG	1040
	411_F	C			CTCGAA

2922	AB_MLST-11-OIF007_	TGGGcGATGCTGCgAAA	583	AB_MLST-11-OIF007_1110_	TAGTATCACCACGTACA	923
	991_1018_F	TGGTTAAAAGA		1137_R	CCCGGATCAGT

2927	GAPA_NC002505_694_	TCAATGAACGACCAACA	259	GAPA_NC_002505_29_58_R_1	TCCTTTATGCAACTTGG	1060
	721_F	AGTGATTGATG			TATCAACAGGAAT

2928	GAPA_NC002505_694_	TCGATGAACGACCAACA	361	GAPA_NC002505_769_798_	TCCTTTATGCAACTTGG	1061
	721_2_F	AGTGATTGATG		2_R	TATCAACCGGAAT

2929	GAPA_NC002505_694_	TCGATGAACGACCAACA	361	GAPA_NC002505_769_798_	TCCTTTATGCAACTTAG	1059
	721_2_F	AGTGATTGATG		3_R	TATCAACCGGAAT

2932	INFB_EC_1364_1394_	TTGCTCGTGGTGCACAA	688	INFB_EC_1439_1468_R	TTGCTGCTTTCGCATGG	1410
	F	GTAACGGATATTAC			TTAATCGCTTCAA

2933	INFB_EC_1364_1394_	TTGCTCGTGGTGCAIAA	689	INFB_EC_1439_1468_R	TTGCTGCTTTCGCATGG	1410
	2_F	GTAACGGATATIAC			TTAATCGCTTCAA

2934	INFB_EC_80_110_F	TTGCCCGCGGTGCGGAA	685	INFB_EC_1439_1468_R	TTGCTGCTTTCGCATGG	1410
		GTAACCGATATTAC			TTAATCGCTTCAA

2949	ACS_NC002516-	TCGGCGCCTGCCTGATG	376	ACS_NC002516-970624-	TGGACCACGCCGAAGAA	1265
	970624-	A		971013_364_383_R	CGG
	971013_299_316_F

2950	ARO_NC002516-	TCACCGTGCCGTTCAAG	267	ARO_NC002516-26883-	TGTGTTGTCGCCGCGCA	1341
	26883-27380_4_26_F	GAAGAG		27380_111_128_R	G

2951	ARO_NC002516-26883-	TTTCGAAGGGCCTTTCG	705	ARO_NC002516-26883-	TCCTTGGCATACATCAT	1056
	27380_356_377_F	ACCTG		27380_459_484_R	GTCGTAGCA

2952	GUA_NC002516-	TGGACTCCTCGGTGGTC	551	GUA_NC002516-4226546-	TCGGCGAACATGGCCAT	1091
	4226546-	GC		4226174_127_146_R	CAC
	4226174_23_41_F

2953	GUA_NC002516-	TGACCAGGTGATGGCCA	448	GUA_NC002516-4226546-	TGCTTCTCTTCCGGGTC	1256
	4226546-	TGTTCG		4226174_214_233_R	GGC
	4226174_120_142_F

2954	GUA_NC002516-	TTTTGAAGGTGATCCGT	710	GUA_NC002516-4226546-	TGCTTGGTGGCTTCTTC	1259
	4226546-	GCCAACG		4226174_265_287_R	GTCGAA
	4226174_155_178_F

2955	GUA_NC002516-	TTCCTCGGCCGCCTGGC	670	GUA_NC002516-4226546-	TGCGAGGAACTTCACGT	1229
	4226546-			4226174_288_309_R	CCTGC
	4226174_190_206_F

2956	GUA_NC002516-	TCGGCCGCACCTTCATC	374	GUA_NC002516-4226546-	TCGTGGGCCTTGCCGGT	1111
	4226546-	GAAGT		4226174_355_371_R
	4226174_242_263_F

2957	MUT_NC002516-	TGGAAGTCATCAAGCGC	545	MUT_NC002516-5551158-	TCACGGGCCAGCTCGTC	978
	5551158-	CTGGC		5550717_99_116_R	T
	5550717_5_26_F

2958	MUT_NC002516-	TCGAGCAGGCGCTGCCG	358	MUT_NC002516-5551158-	TCACCATGCGCCCGTTC	971
	5551158-			5550717_256_277_R		ACATA
	5550717_152_168_F

2959	NUO_NC002516-	TCAACCTCGGCCCGAAC	249	NUO_NC002516-2984589-	TCGGTGGTGGTAGCCGA	1095
	2984589-	CA		2984954_97_117_R	TCTC
	2984954_8_26_F

2960	NUO_NC002516-	TACTCTCGGTGGAGAAG	195	NUC_NC002516-2984589-	TTCAGGTACAGCAGGTG	1376
	2984589-	CTCGC		2984954_301_326_R	GTTCAGGAT
	2984954_218_239_F

2961	PPS_NC002516-	TCCACGGTCATGGAGCG	311	PPS_NC002516-1915014-	TCCATTTCCGACACGTC	1014
	1915014-	CTA		1915383_140_165_R	GTTGATCAC
	1915383_44_63_F

2962	PPS_NC002516-	TCGCCATCGTCACCAAC	365	PPS_NC002516-1915014-	TCCTGGCCATCCTGCAG	1052
	1915014-	CG		1915383_341_360_R	GAT
	1915383_240_258_F

2963	TRP_NC002516-	TGCTGGTACGGGTCGAG	527	TRP_NC002516-671831-	TCGATCTCCTTGGCGTC	1071
	671831-	GA		672273_131_150_R	CGA
	672273_24_42_F

2964	TRP_NC002516-	TGCACATCGTGTCCAAC	490	TRP_NC002516-671831-	TGATCTCCATGGCGCGG	1182
	671831-	GTCAC		672273_362_383_R	ATCTT
	672273_261_282_F

2972	AB_MLST-11-	TGGGIGATGCTGCIAAA	592	AB_MLST-11-	TAGTATCACCACGTACI	924
	OIF007_1007_1034_F	TGGTTAAAAGA		OIF007_1126_1153_R	CCIGGATCAGT

2993	OMPU_NC002505-	TTCCCACCGATATCATG	667	OMPU_NC002505_544_567_R	TCGGTCAGCAAAACGGT	1094
	674828-	GCTTACCACGG			AGCTTGC
	675880_428_455_F

2994	GAPA_NC002505-	TCCTCAATGAACGAICA	335	GAPA_NC002505-506780-	TTTTCCCTTTATGCAAC	1442
	506780-	ACAAGTGATTGATG		507937_769_802_R	TTAGTATCAACIGGAAT
	507937_691_721_F

2995	GAPA_NC002505-	TCCTCIATGAACGAICA	339	GAPA_NC002505-506780-	TCCATACCTTTATGCAA	1008
	506780-	ACAAGTGATTGATG		507937_769_803_R	CTTIGTATCAACIGGAA
	507937_691_721_2_F				T

2996	GAPA_NC002505-	TCTCGATGAACGACCAA	396	GAPA_NC002505-506780-	TCGGAAATATTCTTTCA	1085
	506780-	CAAGTGATTGATG		507937_785_817_R	ATACCTTTATGCAACT
	507937_692_721_F

2997	GAPA_NC002505-	TCCTCGATGAACGAICA	337	GAPA_NC002505-506780-	TCGGAAATATTCTTTCA	1085
	506780-	ACAAGTIATTGATG		507937_785_817_R	ATACCTTTATGCAACT
	507937_691_721_3_F

2998	GAPA_NC002505-	TCCTCAATGAATGATCA	336	GAPA_NC002505-506780-	TCGGAAATATTCTTTCA	1087
	506780-	ACAAGTGATTGATG		507937_784_817_R	ATICCTTTITGCAACTT
	507937_691_721_4_F

2999	GAPA_NC002505-	TCCTCIATGAAIGAICA	340	GAPA_NC002505-506780-	TCGGAAATATTCTTTCA	1086
	506780-	ACAAGTIATTGATG		507937_784_817_2_R	ATACCTTTATGCAACTT
	507937_691_721_5_F

3000	GAPA_NC002505-	TCCTCGATGAATGAICA	338	GAPA_NC002505-506780-	TTTCAATACCTTTATGC	1430
	506780-	ACAAGTIATTGATG		507937_769_805_R	AACTTIGTATCAACIGG
	507937_691_721_6_F				AAT

3001	CTXB_NC002505-	TCAGCATATGCACATGG	275	CTXB_NC002505-1566967-	TCCCGGCTAGAGATTCT	1026
	1566967-	AACACCTCA		1567341_139_163_R	GTATACGA
	1567341_46_71_F

3002	CTXB_NC002505-	TCAGCATATGCACATGG	274	CTXB_NC002505-1566967-	TCCGGCTAGAGATTCTG	1038
	1566967-	AACACCTC		1567341_132_162_R	TATACGAAAATATC
	1567341_46_70_F

3003	CTXB_NC002505-	TCAGCATATGCACATGG	274	CTXB_NC002505-1566967-	TGCCGTATACGAAAATA	1225
	1566967-	AACACCTC		1567341_118_150_R	TCTTATCATTTAGCGT
	1567341_46_70_F

3004	TUFB_NC002758-	TACAGGCCGTGTTGAAC	180	TUFB_NC002758-615038-	TCAGCGTAGTCTAATAA	982
	615038-	GTGG		616222_778_809_R	TTTACGGAACATTTC
	616222_684_704_F

3005	TUFB_NC002758-	TGCCGTGTTGAACGTGG	503	TUFB_NC002758-615038-	TGCTTCAGCGTAGTCTA	1255
	615038-	TCAAAT		616222_783_813_R	ATAATTTACGGAAC
	616222_688_710_F

3006	TUFB_NC002758-	TGTGGTCAAATCAAAGT	638	TUFB_NC002758-615038-	TGCGTAGTCTAATAATT	1238
	615038-	TGGTGAAGAA		616222_778_807_R	TACGGAACATTTC
	616222_700_726_F

3007	TUFB_NC002758-	TGGTCAAATCAAAGTTG	607	TUFB_NC002758-615038-	TGCGTAGTCTAATAATT	1238
	615038-	GTGAAGAA		616222_778_807_R	TACGGAACATTTC
	616222_702_726_F

3008	TUFB_NC002758-	TGAACGTGGTCAAATCA	431	TUFB_NC002758-615038-	TCACCAGCTTCAGCGTA	970
	615038-	AAGTTGGTGAAGAA		616222_785_818_R	GTCTAATAATTTACGGA
	616222_696_726_F

3009	TUFB_NC002758-	TCGTGTTGAACGTGGTC	386	TUFB_NC002758-615038-	TCTTCAGCGTAGTCTAA	1134
	615038-	AAATCAAAGT		616222_778_812_R	TAATTTACGGAACATTT
	616222_690_716_F				C

3010	MECI-R_NC003923-	TCACATATCGTGAGCAA	261	MECI-R_NC003923-41798-	TGTGATATGGAGGTGTA	1332
	41798-41609_36_59_	TGAACTG		41609_89_112_R	GAAGGTG
	F

3011	MECI-R_NC003923-	TGGGCGTGAGCAATGAA	584	MECI-R_NC003923-41798-	TGGGATGGAGGTGTAGA	1287
	41798-41609_40_66_	CTGATTATAC		41609_81_110_R	AGGTGTTATCATC
	F

3012	MECI-R_NC003923-	TGGACACATATCGTGAG	549	MECI-R_NC003923-41798-	TGGGATGGAGGTGTAGA	1286
	41798-	CAATGAACTGA		41609_81_110_R	AGGTGTTATCATC
	41609_33_60_2_F

3013	MECI-R_NC003923-	TGGGTTTACACATATCG	595	MECI-R_NC003923-41798-	TGGGGATATGGAGGTGT	1290
	41798-41609_29_60_	TGAGCAATGAACTGA		41609_81_113_R	AGAAGGTGTTATCATC
	F

3014	MUPR_X75439_2490_	TGGGCTCTTTCTCGCTT	587	MUPR_X75439_2548_2570_R	TCTGGCTGCGGAAGTGA	1130
	2514_F	AAACACCT			AATCGT

3015	MUPR_X75439_2490_	TGGGCTCTTTCTCGCTT	586	MUPR_X75439_2547_2568_R	TGGCTGCGGAAGTGAAA	1281
	2513_F	AAACACC			TCGTA

3016	MUPR_X75439_2482_	TAGATAATTGGGCTCTT	205	MUPR_X75439_2551_2573_R	TAATCTGGCTGCGGAAG	876
	2510_F	TCTCGCTTAAAC			TGAAAT

3017	MUPR_X75439_2490_	TGGGCTCTTTCTCGCTT	587	MUPR_X75439_2549_2573_R	TAATCTGGCTGCGGAAG	877
	2514_F	AAACACCT			TGAAATCG

3018	MUPR_X75439_2482_	TAGATAATTGGGCTCTT	205	MUPR_X75439_2559_2589_R	TGGTATATTCGTTAATT	1303
	2510_F	TCTCGCTTAAAC			AATCTGGCTGCGGA

3019	MUPR_X75439_2490_	TGGGCTCTTTCTCGCTT	587	MUPR_X75439_2554_2581_R	TCGTTAATTAATCTGGC	1112
	2514_F	AAACACCT			TGCGGAAGTGA

3020	AROE_NC003923-	TGATGGCAAGTGGATAG	474	AROE_NC003923-1674726-	TAAGCAATACCTTTACT	868
	1674726-	GGTATAATACAG		1674277_309_335_R	TGCACCACCT
	1674277_204_232_F

3021	AROE_NC003923-	TGGCGAGTGGATAGGGT	570	AROE_NC003923-1674726-	TTCATAAGCAATACCTT	1378
	1674726-	ATAATACAG		1674277_311_339_R	TACTTGCACCAC
	1674277_207_232_F

3022	AROE_NC003923-	TGGCpAAGTpGGATpAG	572	AROE_NC003923-1674726-	TAAGCAATACCpTpTpT	867
	1674726-	GGTpATpAATpACpAG		1674277_311_335P_R	pACTpTpGCpACpCpAC
	1674277_207_232P_F

3023	ARCC_NC003923-	TCTGAAATGAATAGTGA	398	ARCC_NC003923-2725050-	TCTTCTTCTTTCGTATA	1137
	2725050-	TAGAACTGTAGGCAC		2724595_214_245_R	AAAAGGACCAATTGG
	2724595_124_155_F

3024	ARCC_NC003923-	TGAATAGTGATAGAACT	437	ARCC_NC003923-2725050-	TCTTCTTTCGTATAAAA	1139
	2725050-	GTAGGCACAATCGT		2724595_212_242_R	AGGACCAATTGGTT
	2724595_131_161_F

3025	ARCC_NC003923-	TGAATAGTGATAGAACT	437	ARCC_NC003923-2725050-	TGCGCTAATTCTTCAAC	1232
	2725050-	GTAGGCACAATCGT		2724595_232_260_R	TTCTTCTTTCGT
	2724595_131_161_F

3026	PTA_NC003923-	TACAATGCTTGTTTATG	177	PTA_NC003923-628885-	TGTTCTTGATACACCTG	1350
	628885-	CTGGTAAAGCAG		629355_322_351_R	GTTTCGTTTTGAT
	629355_231_259_F

3027	PTA_NC003923-	TACAATGCTTGTTTATG	177	PTA_NC003923-628885-	TGGTACACCTGGTTTCG	1301
	628885-	CTGGTAAAGCAG		629355_314_345_R	TTTTGATGATTTGTA
	629355_231_259_F

3028	PTA_NC003923-	TCTTGTTTATGCTGGTA	418	PTA_NC003923-628885-	TGTTCTTGATACACCTG	1350
	628885-	AAGCAGATGG		629355_322_351_R	GTTTCGTTTTGAT
	629355_237_263_F

Primer pair name codes and reference sequences are shown in Table 3. The primer name code typically represents the gene to which the given primer pair is targeted. The primer pair name may include specific coordinates with respect to a reference sequence defined by an extraction of a section of sequence or defined by a GenBank gi number, or the corresponding complementary sequence of the extraction, or the entire GenBank gi number as indicated by the label “no extraction.” Where “no extraction” is indicated for a reference sequence, the coordinates of a primer pair named to the reference sequence are with respect to the GenBank gi listing. Gene abbreviations are shown in bold type in the “Gene Name” column.
To determine the exact primer hybridization coordinates of a given pair of primers on a given bioagent nucleic acid sequence and to determine the sequences, molecular masses and base compositions of an amplification product to be obtained upon amplification of nucleic acid of a known bioagent with known sequence information in the region of interest with a given pair of primers, one with ordinary skill in bioinformatics is capable of obtaining alignments of the primers of the present invention with the GenBank gi number of the relevant nucleic acid sequence of the known bioagent. For example, the reference sequence GenBank gi numbers (Table 3) provide the identities of the sequences which can be obtained from GenBank. Alignments can be done using a bioinformatics tool such as BLASTn provided to the public by NCBI (Bethesda, Md.). Alternatively, a relevant GenBank sequence may be downloaded and imported into custom programmed or commercially available bioinformatics programs wherein the alignment can be carried out to determine the primer hybridization coordinates and the sequences, molecular masses and base compositions of the amplification product. For example, to obtain the hybridization coordinates of primer pair number 2095 (SEQ ID NOs: 456:1261), First the forward primer (SEQ ID NO: 456) is subjected to a BLASTn search on the publicly available NCBI BLAST website. “RefSeq_Genomic” is chosen as the BLAST database since the gi numbers refer to genomic sequences. The BLAST query is then performed. Among the top results returned is a match to GenBank gi number 21281729 (Accession Number NC_—003923). The result shown below, indicates that the forward primer hybridizes to positions 1530282 . . . 1530307 of the genomic sequence of Staphylococcus aureus subsp. aureus MW2 (represented by gi number 21281729).
Staphylococcus aureus subsp. aureus MW2, complete genome Length=2820462
Features in this part of subject sequence:
Panton-Valentine leukocidin chain F precursor
Score=52.0 bits (26), Expect=2e-05
Identities=26/26 (100%), Gaps=0/26 (0%)
Strand=Plus/Plus

Query 1 TGAGCTGCATCAACTGTATTGGATAG 26

||||||||||||||||||||||||||

Sbjct 1530282 TGAGCTGCATCAACTGTATTGGATAG 1530307

The hybridization coordinates of the reverse primer (SEQ ID NO: 1261) can be determined in a similar manner and thus, the bioagent identifying amplicon can be defined in terms of genomic coordinates. The query/subject arrangement of the result would be presented in Strand=Plus/Minus format because the reverse strand hybridizes to the reverse complement of the genomic sequence. The preceding sequence analyses are well known to one with ordinary skill in bioinformatics and thus, Table 3 contains sufficient information to determine the primer hybridization coordinates of any of the primers of Table 2 to the applicable reference sequences described therein.

TABLE 3


Primer Name Codes and Reference Sequence

			Reference
			GenBank gi
Primer name code	Gene Name	Organism	number

16S_EC	16S rRNA (16S ribosomal RNA gene)	Escherichia coli	16127994
23S_EC	23S rRNA (23S ribosomal RNA gene)	Escherichia coli	16127994
CAPC_BA	capC (capsule biosynthesis gene)	Bacillus anthracis	6470151
CYA_BA	cya (cyclic AMP gene)	Bacillus anthracis	4894216
DNAK_EC	dnaK (chaperone dnaK gene)	Escherichia coli	16127994
GROL_EC	groL (chaperonin groL)	Escherichia coli	16127994
HFLB_EC	hflb (cell division protein peptidase	Escherichia coli	16127994
	ftsH)
INFB_EC	infB (protein chain initiation factor	Escherichia coli	16127994
	infB gene)
LEF_BA	lef (lethal factor)	Bacillus anthracis	21392688
PAG_BA	pag (protective antigen)	Bacillus anthracis	21392688
RPLB_EC	rplB (50S ribosomal protein L2)	Escherichia coli	16127994
RPOB_EC	rpoB (DNA-directed RNA polymerase beta	Escherichia coli	6127994
	chain)
RPOC_EC	rpoC (DNA-directed RNA polymerase	Escherichia coli	16127994
	beta′ chain)
SP101ET_SPET_11	Artificial Sequence Concatenation	Artificial	15674250
	comprising:	Sequence* -
	gki (glucose kinase)	partial gene
	gtr (glutamine transporter protein)	sequences of
	murI (glutamate racemase)	Streptococcus
	mutS (DNA mismatch repair protein)	pyogenes
	xpt (xanthine phosphoribosyl
	transferase)
	yqiL (acetyl-CoA-acetyl transferase)
	tkt (transketolase)
SSPE_BA	sspE (small acid-soluble spore	Bacillus anthracis	30253828
	protein)
TUFB_EC	tufB (Elongation factor Tu)	Escherichia coli	16127994
VALS_EC	valS (Valyl-tRNA synthetase)	Escherichia coli	16127994
ASPS_EC	aspS (Aspartyl-tRNA synthetase)	Escherichia coli	16127994
CAF1_AF053947	caf1 (capsular protein caf1)	Yersinia pestis	2996286
INV_U22457	inv (invasin)	Yersinia pestis	1256565
LL_NC003143	Y. pestis specific chromosomal genes -	Yersinia pestis	16120353
	difference region
BONTA_X52066	BoNT/A (neurotoxin type A)	Clostridium	40381
		botulinum
MECA_Y14051	mecA methicillin resistance gene	Staphylococcus	2791983
		aureus
TRPE_AY094355	trpE (anthranilate synthase (large	Acinetobacter	20853695
	component))	baumanii
RECA_AF251469	recA (recombinase A)	Acinetobacter	9965210
		baumanii
GYRA_AF100557	gyrA (DNA gyrase subunit A)	Acinetobacter	4240540
		baumanii
GYRB_AB008700	gyrB (DNA gyrase subunit B)	Acinetobacter	4514436
		baumanii
WAAA_Z96925	waaA (3-deoxy-D-manno-octulosonic-acid	Acinetobacter	2765828
	transferase)	baumanii
CJST_CJ	Artificial Sequence Concatenation	Artificial	15791399
	comprising:	Sequence* -
	tkt (transketolase)	partial gene
	glyA (serine hydroxymethyltransferase)	sequences of
	gltA (citrate synthase)	Campylobacter
	aspA (aspartate ammonia lyase)	jejuni
	glnA (glutamine synthase)
	pgm (phosphoglycerate mutase)
	uncA (ATP synthetase alpha chain)
RNASEP_BDP	RNase P (ribonuclease P)	Bordetella	33591275
		pertussis
RNASEP_BKM	RNase P (ribonuclease P)	Burkholderia	53723370
		mallei
RNASEP_BS	RNase P (ribonuclease P)	Bacillus subtilis	16077068
RNASEP_CLB	RNase P (ribonuclease P)	Clostridium	18308982
		perfringens
RNASEP_EC	RNase P (ribonuclease P)	Escherichia coli	16127994
RNASEP_RKP	RNase P (ribonuclease P)	Rickettsia	15603881
		prowazekii
RNASEP_SA	RNase P (ribonuclease P)	Staphylococcus	15922990
		aureus
RNASEP_VBC	RNase P (ribonuclease P)	Vibrio cholerae	15640032
ICD_CXB	icd (isocitrate dehydrogenase)	Coxiella burnetii	29732244
IS1111A	multi-locus IS1111A insertion element	Acinetobacter	29732244
		baumannii
OMPA_AY485227	ompA (outer membrane protein A)	Rickettsia	40287451
		prowazekii
OMPB_RKP	ompB (outer membrane protein B)	Rickettsia	15603881
		prowazekii
GLTA_RKP	gltA (citrate synthase)	Vibrio cholerae	15603881
TOXR_VBC	toxR (transcription regulator toxR)	Francisella	15640032
		tularensis
ASD_FRT	asd (Aspartate semialdehyde	Francisella	56707187
	dehydrogenase)	tularensis
GALE_FRT	galE (UDP-glucose 4-epimerase)	Shigella flexneri	56707187
IPAH_SGF	ipaH (invasion plasmid antigen)	Campylobacter	30061571
		jejuni
HUPB_CJ	hupB (DNA-binding protein Hu-beta)	Coxiella burnetii	15791399
AB_MLST	Artificial Sequence Concatenation	Artificial	Sequenced
	comprising:	Sequence* -	in-house
	trpE (anthranilate synthase component	partial gene	(SEQ ID
	I))	sequences of	NO: 1444)
	adk (adenylate kinase)	Acinetobacter
	mutY (adenine glycosylase)	baumannii
	fumC (fumarate hydratase)
	efp (elongation factor p)
	ppa (pyrophosphate phospho-
	hydratase
MUPR_X75439	mupR (mupriocin resistance gene)	Staphylococcus	438226
		aureus
PARC_X95819	parC (topoisomerase IV)	Acinetobacter	1212748
		baumannii
SED_M28521	sed (enterotoxin D)	Staphylococcus	1492109
		aureus
PLA_AF053945	pla (plasminogen activator)	Yersinia pestis	2996216
SEJ_AF053140	sej (enterotoxin J)	Staphylococcus	3372540
		aureus
GYRA_NC000912	gyrA (DNA gyrase subunit A)	Mycoplasma	13507739
		pneumoniae
ACS_NC002516	acsA (Acetyl CoA Synthase)	Pseudomonas	15595198
		aeruginosa
ARO_NC002516	aroE (shikimate 5-dehydrogenase	Pseudomonas	15595198
		aeruginosa
GUA_NC002516	guaA (GMP synthase)	Pseudomonas	15595198
		aeruginosa
MUT_NC002516	mutL (DNA mismatch repair protein)	Pseudomonas	15595198
		aeruginosa
NUO_NC002516	nuoD (NADH dehydrogenase I chain C, D)	Pseudomonas	15595198
		aeruginosa
PPS_NC002516	ppsA (Phosphoenolpyruvate synthase)	Pseudomonas	15595198
		aeruginosa
TRP_NC002516	trpE (Anthranilate synthetase	Pseudomonas	15595198
	component I)	aeruginosa
OMP2_NC000117	ompB (outer membrane protein B)	Chlamydia	15604717
		trachomatis
OMPA_NC000117	ompA (outer membrane protein B)	Chlamydia	15604717
		trachomatis
GYRA_NC000117	gyrA (DNA gyrase subunit A)	Chlamydia	15604717
		trachomatis
CTXA_NC002505	ctxA (Cholera toxin A subunit)	Vibrio cholerae	15640032
CTXB_NC002505	ctxB (Cholera toxin B subunit)	Vibrio cholerae	15640032
FUR_NC002505	fur (ferric uptake regulator protein)	Vibrio cholerae	15640032
GAPA_NC_002505	gapA (glyceraldehyde-3-phosphate	Vibrio cholerae	15640032
	dehydrogenase)
GYRB_NC002505	gyrB (DNA gyrase subunit B)	Vibrio cholerae	15640032
OMPU_NC002505	ompU (outer membrane protein)	Vibrio cholerae	15640032
TCPA_NC002505	tcpA (toxin-coregulated pilus)	Vibrio cholerae	15640032
ASPA_NC002163	aspA (aspartate ammonia lyase)	Campylobacter	15791399
		jejuni
GLNA_NC002163	glnA (glutamine synthetase)	Campylobacter	15791399
		jejuni
GLTA_NC002163	gltA (glutamate synthase)	Campylobacter	15791399
		jejuni
GLYA_NC002163	glyA (serine hydroxymethyltransferase)	Campylobacter	15791399
		jejuni
PGM_NC002163	pgm (phosphoglyceromutase)	Campylobacter	15791399
		jejuni
TKT_NC002163	tkt (transketolase)	Campylobacter	15791399
		jejuni
UNCA_NC002163	uncA (ATP synthetase alpha chain)	Campylobacter	15791399
		jejuni
AGR-III_NC003923	agr-III (accessory gene regulator-III)	Staphylococcus	21281729
		aureus
ARCC_NC003923	arcC (carbamate kinase)	Staphylococcus	21281729
		aureus
AROE_NC003923	aroE (shikimate 5-dehydrogenase	Staphylococcus	21281729
		aureus
BSA-A_NC003923	bsa-a (glutathione peroxidase)	Staphylococcus	21281729
		aureus
BSA-B_NC003923	bsa-b (epidermin biosynthesis protein	Staphylococcus	21281729
	EpiB)	aureus
GLPF_NC003923	glpF (glycerol transporter)	Staphylococcus	21281729
		aureus
GMK_NC003923	gmk (guanylate kinase)	Staphylococcus	21281729
		aureus
MECI-R_NC003923	mecR1 (truncated methicillin	Staphylococcus	21281729
	resistance protein)	aureus
PTA_NC003923	pta (phosphate acetyltransferase)	Staphylococcus	21281729
		aureus
PVLUK_NC003923	pvluk (Panton-Valentine leukocidin	Staphylococcus	21281729
	chain F precursor)	aureus
SA442_NC003923	sa442 gene	Staphylococcus	21281729
		aureus
SEA_NC003923	sea (staphylococcal enterotoxin A	Staphylococcus	21281729
	precursor)	aureus
SEC_NC003923	sec4 (enterotoxin type C precursor)	Staphylococcus	21281729
		aureus
TPI_NC003923	tpi (triosephosphate isomerase)	Staphylococcus	21281729
		aureus
YQI_NC003923	yqi (acetyl-CoA C-acetyltransferase	Staphylococcus	21281729
	homologue)	aureus
GALE_AF513299	galE (galactose epimerase)	Francisella	23506418
		tularensis
VVHA_NC004460	vVhA (cytotoxin, cytolysin precursor)	Vibrio vulnificus	27366463
TDH_NC004605	tdh (thermostable direct hemolysin A)	Vibrio	28899855
		parahaemolyticus
AGR-II_NC002745	agr-II (accessory gene regulator-II)	Staphylococcus	29165615
		aureus
PARC_NC003997	parC (topoisomerase IV)	Bacillus anthracis	30260195
GYRA_AY291534	gyrA (DNA gyrase subunit A)	Bacillus anthracis	31323274
AGR-I_AJ617706	agr-I (accessory gene regulator-I)	Staphylococcus	46019543
		aureus
AGR-IV_AJ617711	agr-IV (accessory gene regulator-III)	Staphylococcus	46019563
		aureus
BLAZ_NC002952	blaZ (beta lactamase III)	Staphylococcus	49482253
		aureus
ERMA_NC002952	ermA (rRNA methyltransferase A)	Staphylococcus	49482253
		aureus
ERMB_Y13600	ermB (rRNA methyltransferase B)	Staphylococcus	49482253
		aureus
SEA-SEE_NC002952	sea (staphylococcal enterotoxin A	Staphylococcus	49482253
	precursor)	aureus
SEA-SEE_NC002952	sea (staphylococcal enterotoxin A	Staphylococcus	49482253
	precursor)	aureus
SEE_NC002952	sea (staphylococcal enterotoxin A	Staphylococcus	49482253
	precursor)	aureus
SEH_NC002953	seh (staphylococcal enterotoxin H)	Staphylococcus	49484912
		aureus
ERMC_NC005908	ermC (rRNA methyltransferase C)	Staphylococcus	49489772
		aureus
MUTS_AY698802	mutS (DNA mismatch repair protein)	Shigella boydii	52698233
NUC_NC002758	nuc (staphylococcal nuclease)	Staphylococcus	57634611
		aureus
SEB_NC002758	seb (enterotoxin type B precursor)	Staphylococcus	57634611
		aureus
SEG_NC002758	seg (staphylococcal enterotoxin G)	Staphylococcus	57634611
		aureus
SEI_NC002758	sei (staphylococcal enterotoxin I)	Staphylococcus	57634611
		aureus
TSST_NC002758	tsst (toxic shock syndrome toxin-1)	Staphylococcus	57634611
		aureus
TUFB_NC002758	tufB (Elongation factor Tu)	Staphylococcus	57634611
		aureus

Note:
artificial reference sequences represent concatenations of partial gene extractions from the indicated reference gi number. Partial sequences were used to create the concatenated sequence because complete gene sequences were not necessary for primer design.

Example 2

Sample Preparation and PCR

Genomic DNA was prepared from samples using the DNeasy Tissue Kit (Qiagen, Valencia, Calif.) according to the manufacturer's protocols.
All PCR reactions were assembled in 50 μL reaction volumes in a 96-well microtiter plate format using a Packard MPII liquid handling robotic platform and M. J. Dyad thermocyclers (MJ research, Waltham, Mass.) or Eppendorf Mastercycler thermocyclers (Eppendorf, Westbury, N.Y.). The PCR reaction mixture consisted of 4 units of Amplitaq Gold, 1× buffer II (Applied Biosystems, Foster City, Calif.), 1.5 mM MgCl₂, 0.4 M betaine, 800 μM dNTP mixture and 250 nM of each primer. The following typical PCR conditions were used: 95° C. for 10 min followed by 8 cycles of 95° C. for 30 seconds, 48° C. for 30 seconds, and 72° C. 30 seconds with the 48° C. annealing temperature increasing 0.9° C. with each of the eight cycles. The PCR was then continued for 37 additional cycles of 95° C. for 15 seconds, 56° C. for 20 seconds, and 72° C. 20 seconds.

Example 3

Purification of PCR Products for Mass Spectrometry with Ion Exchange Resin-Magnetic Beads

For solution capture of nucleic acids with ion exchange resin linked to magnetic beads, 25 μl of a 2.5 mg/mL suspension of BioClone amine terminated superparamagnetic beads were added to 25 to 50 μl of a PCR (or RT-PCR) reaction containing approximately 10 μM of a typical PCR amplification product. The above suspension was mixed for approximately 5 minutes by vortexing or pipetting, after which the liquid was removed after using a magnetic separator. The beads containing bound PCR amplification product were then washed three times with 50 mM ammonium bicarbonate/50% MeOH or 100 mM ammonium bicarbonate/50% MeOH, followed by three more washes with 50% MeOH. The bound PCR amplicon was eluted with a solution of 25 mM piperidine, 25 mM imidazole, 35% MeOH which included peptide calibration standards.

Example 4

Mass Spectrometry and Base Composition Analysis

The ESI-FTICR mass spectrometer is based on a Bruker Daltonics (Billerica, Mass.) Apex II 70e electrospray ionization Fourier transform ion cyclotron resonance mass spectrometer that employs an actively shielded 7 Tesla superconducting magnet. The active shielding constrains the majority of the fringing magnetic field from the superconducting magnet to a relatively small volume. Thus, components that might be adversely affected by stray magnetic fields, such as CRT monitors, robotic components, and other electronics, can operate in close proximity to the FTICR spectrometer. All aspects of pulse sequence control and data acquisition were performed on a 600 MHz Pentium II data station running Bruker's Xmass software under Windows NT 4.0 operating system. Sample aliquots, typically 15 μl, were extracted directly from 96-well microtiter plates using a CTC HTS PAL autosampler (LEAP Technologies, Carrboro, N.C.) triggered by the FTICR data station. Samples were injected directly into a 10 μl sample loop integrated with a fluidics handling system that supplies the 100 μl/hr flow rate to the ESI source. Ions were formed via electrospray ionization in a modified Analytica (Branford, Conn.) source employing an off axis, grounded electrospray probe positioned approximately 1.5 cm from the metalized terminus of a glass desolvation capillary. The atmospheric pressure end of the glass capillary was biased at 6000 V relative to the ESI needle during data acquisition. A counter-current flow of dry N₂was employed to assist in the desolvation process. Ions were accumulated in an external ion reservoir comprised of an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode, prior to injection into the trapped ion cell where they were mass analyzed. Ionization duty cycles greater than 99% were achieved by simultaneously accumulating ions in the external ion reservoir during ion detection. Each detection event consisted of 1M data points digitized over 2.3 s. To improve the signal-to-noise ratio (S/N), 32 scans were co-added for a total data acquisition time of 74 s.
The ESI-TOF mass spectrometer is based on a Bruker Daltonics MicroTOF™. Ions from the ESI source undergo orthogonal ion extraction and are focused in a reflectron prior to detection. The TOF and FTICR are equipped with the same automated sample handling and fluidics described above. Ions are formed in the standard MicroTOF™ ESI source that is equipped with the same off-axis sprayer and glass capillary as the FTICR ESI source. Consequently, source conditions were the same as those described above. External ion accumulation was also employed to improve ionization duty cycle during data acquisition. Each detection event on the TOF was comprised of 75,000 data points digitized over 75 μs.
The sample delivery scheme allows sample aliquots to be rapidly injected into the electrospray source at high flow rate and subsequently be electrosprayed at a much lower flow rate for improved ESI sensitivity. Prior to injecting a sample, a bolus of buffer was injected at a high flow rate to rinse the transfer line and spray needle to avoid sample contamination/carryover. Following the rinse step, the autosampler injected the next sample and the flow rate was switched to low flow. Following a brief equilibration delay, data acquisition commenced. As spectra were co-added, the autosampler continued rinsing the syringe and picking up buffer to rinse the injector and sample transfer line. In general, two syringe rinses and one injector rinse were required to minimize sample carryover. During a routine screening protocol a new sample mixture was injected every 106 seconds. More recently a fast wash station for the syringe needle has been implemented which, when combined with shorter acquisition times, facilitates the acquisition of mass spectra at a rate of just under one spectrum/minute.
Raw mass spectra were post-calibrated with an internal mass standard and deconvoluted to monoisotopic molecular masses. Unambiguous base compositions were derived from the exact mass measurements of the complementary single-stranded oligonucleotides. Quantitative results are obtained by comparing the peak heights with an internal PCR calibration standard present in every PCR well at 500 molecules per well. Calibration methods are commonly owned and disclosed in U.S. Provisional Patent Application Ser. No. 60/545,425 which is incorporated herein by reference in entirety.

Example 5

De Novo Determination of Base Composition of Amplification Products Using Molecular Mass Modified Deoxynucleotide Triphosphates

Because the molecular masses of the four natural nucleobases have a relatively narrow molecular mass range (A=313.058, G=329.052, C=289.046, T=304.046—See Table 4), a persistent source of ambiguity in assignment of base composition can occur as follows: two nucleic acid strands having different base composition may have a difference of about 1 Da when the base composition difference between the two strands is G
A (−15.994) combined with C
T (+15.000). For example, one 99-mer nucleic acid strand having a base composition of A₂₇G₃₀C₂₁T₂, has a theoretical molecular mass of 30779.058 while another 99-mer nucleic acid strand having a base composition of A₂₆G₃₁C₂₂T₂₀has a theoretical molecular mass of 30780.052. A 1 Da difference in molecular mass may be within the experimental error of a molecular mass measurement and thus, the relatively narrow molecular mass range of the four natural nucleobases imposes an uncertainty factor.
The present invention provides for a means for removing this theoretical 1 Da uncertainty factor through amplification of a nucleic acid with one mass-tagged nucleobase and three natural nucleobases. The term “nucleobase” as used herein is synonymous with other terms in use in the art including “nucleotide,” “deoxynucleotide,” “nucleotide residue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” or deoxynucleotide triphosphate (dNTP).

Addition of significant mass to one of the 4 nucleobases (dNTPs) in an amplification reaction, or in the primers themselves, will result in a significant difference in mass of the resulting amplification product (significantly greater than 1 Da) arising from ambiguities arising from the G A combined with C

T event (Table 4). Thus, the same the G A (−15.994) event combined with 5-Iodo-C

T (−110.900) event would result in a molecular mass difference of 126.894. If the molecular mass of the base composition A₂₇G₃₀5-Iodo-C₂₁T₂₁(33422.958) is compared with A₂₆G₃₁5-Iodo-C₂₂T₂₀, (33549.852) the theoretical molecular mass difference is +126.894. The experimental error of a molecular mass measurement is not significant with regard to this molecular mass difference. Furthermore, the only base composition consistent with a measured molecular mass of the 99-mer nucleic acid is A₂₇G₃₀5-Iodo-C₂₁T₂₁. In contrast, the analogous amplification without the mass tag has 18 possible base compositions.

TABLE 4


Molecular Masses of Natural Nucleobases and the
Mass-Modified Nucleobase 5-Iodo-C and
Mass Differences Resulting from Transitions

Nucleobase	Molecular Mass	Transition	Molecular Mass

A	313.058	A-->T	−9.012
A	313.058	A-->C	−24.012
A	313.058	A-->5-Iodo-C	101.888
A	313.058	A-->G	15.994
T	304.046	T-->A	9.012
T	304.046	T-->C	−15.000
T	304.046	T-->5-Iodo-C	110.900
T	304.046	T-->G	25.006
C	289.046	C-->A	24.012
C	289.046	C-->T	15.000
C	289.046	C-->G	40.006
5-Iodo-C	414.946	5-Iodo-C-->A	−101.888
5-Iodo-C	414.946	5-Iodo-C-->T	−110.900
5-Iodo-C	414.946	5-Iodo-C-->G	−85.894
G	329.052	G-->A	−15.994
G	329.052	G-->T	−25.006
G	329.052	G-->C	−40.006
G	329.052	G-->5-Iodo-C	85.894

Mass spectra of bioagent-identifying amplicons were analyzed independently using a maximum-likelihood processor, such as is widely used in radar signal processing. This processor, referred to as GenX, first makes maximum likelihood estimates of the input to the mass spectrometer for each primer by running matched filters for each base composition aggregate on the input data. This includes the GenX response to a calibrant for each primer.
The algorithm emphasizes performance predictions culminating in probability-of-detection versus probability-of-false-alarm plots for conditions involving complex backgrounds of naturally occurring organisms and environmental contaminants. Matched filters consist of a priori expectations of signal values given the set of primers used for each of the bioagents. A genomic sequence database is used to define the mass base count matched filters. The database contains the sequences of known bacterial bioagents and includes threat organisms as well as benign background organisms. The latter is used to estimate and subtract the spectral signature produced by the background organisms. A maximum likelihood detection of known background organisms is implemented using matched filters and a running-sum estimate of the noise covariance. Background signal strengths are estimated and used along with the matched filters to form signatures which are then subtracted. The maximum likelihood process is applied to this “cleaned up” data in a similar manner employing matched filters for the organisms and a running-sum estimate of the noise-covariance for the cleaned up data.
The amplitudes of all base compositions of bioagent-identifying amplicons for each primer are calibrated and a final maximum likelihood amplitude estimate per organism is made based upon the multiple single primer estimates. Models of all system noise are factored into this two-stage maximum likelihood calculation. The processor reports the number of molecules of each base composition contained in the spectra. The quantity of amplification product corresponding to the appropriate primer set is reported as well as the quantities of primers remaining upon completion of the amplification reaction.
Base count blurring can be carried out as follows. “Electronic PCR” can be conducted on nucleotide sequences of the desired bioagents to obtain the different expected base counts that could be obtained for each primer pair. See for example, ncbi.nlm.nih.gov/sutils/e-pcr/; Schuler, Genome Res. 7:541-50, 1997. In one illustrative embodiment, one or more spreadsheets, such as Microsoft Excel workbooks contain a plurality of worksheets. First in this example, there is a worksheet with a name similar to the workbook name; this worksheet contains the raw electronic PCR data. Second, there is a worksheet named “filtered bioagents base count” that contains bioagent name and base count; there is a separate record for each strain after removing sequences that are not identified with a genus and species and removing all sequences for bioagents with less than 10 strains. Third, there is a worksheet, “Sheet1” that contains the frequency of substitutions, insertions, or deletions for this primer pair. This data is generated by first creating a pivot table from the data in the “filtered bioagents base count” worksheet and then executing an Excel VBA macro. The macro creates a table of differences in base counts for bioagents of the same species, but different strains. One of ordinary skill in the art may understand additional pathways for obtaining similar table differences without undo experimentation.
Application of an exemplary script, involves the user defining a threshold that specifies the fraction of the strains that are represented by the reference set of base counts for each bioagent. The reference set of base counts for each bioagent may contain as many different base counts as are needed to meet or exceed the threshold. The set of reference base counts is defined by taking the most abundant strain's base type composition and adding it to the reference set and then the next most abundant strain's base type composition is added until the threshold is met or exceeded. The current set of data was obtained using a threshold of 55%, which was obtained empirically.
For each base count not included in the reference base count set for that bioagent, the script then proceeds to determine the manner in which the current base count differs from each of the base counts in the reference set. This difference may be represented as a combination of substitutions, Si=Xi, and insertions, Ii=Yi, or deletions, Di=Zi. If there is more than one reference base count, then the reported difference is chosen using rules that aim to minimize the number of changes and, in instances with the same number of changes, minimize the number of insertions or deletions. Therefore, the primary rule is to identify the difference with the minimum sum (Xi+Yi) or (Xi+Zi), e.g., one insertion rather than two substitutions. If there are two or more differences with the minimum sum, then the one that will be reported is the one that contains the most substitutions.
Differences between a base count and a reference composition are categorized as one, two, or more substitutions, one, two, or more insertions, one, two, or more deletions, and combinations of substitutions and insertions or deletions. The different classes of nucleobase changes and their probabilities of occurrence have been delineated in U.S. Patent Application Publication No. 2004209260 (U.S. application Ser. No. 10/418,514) which is incorporated herein by reference in entirety.

Example 6

Use of Broad Range Survey and Division Wide Primer Pairs for Identification of Bacteria in an Epidemic Surveillance Investigation

This investigation employed a set of 16 primer pairs which is herein designated the “surveillance primer set” and comprises broad range survey primer pairs, division wide primer pairs and a single Bacillus clade primer pair. The surveillance primer set is shown in Table 5 and consists of primer pairs originally listed in Table 2. This surveillance set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row. Primer pair 449 (non-T modified) has been modified twice. Its predecessors are primer pairs 70 and 357, displayed below in the same row. Primer pair 360 has also been modified twice and its predecessors are primer pairs 17 and 118.

TABLE 5


Bacterial Primer Pairs of the Surveillance Primer Set

		Forward		Reverse
Primer		Primer		Primer
Pair		(SEQ ID		(SEQ ID
No.	Forward Primer Name	NO:)	Reverse Primer Name	NO:)	Target Gene

346	16S_EC_713_732_TMOD_F	202	16S_EC_789_809_TMOD_R	1110	16S rRNA
10	16S_EC_713_732_F	21	16S_EC_789_809	798	16S rRNA
347	16S_EC_785_806_TMOD_F	560	16S_EC_880_897_TMOD_R	1278	16S rRNA
11	16S_EC_785_806_F	118	16S_EC_880_897_R	830	16S rRNA
348	16S_EC_960_981_TMOD_F	706	16S_EC_1054_1073_TMOD_R	895	16S rRNA
14	16S_EC_960_981_F	672	16S_EC_1054_1073_R	735	16S rRNA
349	23S_EC_1826_1843_TMOD_F	401	23S_EC_1906_1924_TMOD_R	1156	23S rRNA
16	23S_EC_1826_1843_F	80	23S_EC_1906_1924_R	805	23S rRNA
352	INFB_EC_1365_1393_TMOD_F	687	INFB_EC_1439_1467_TMOD_R	1411	infB
34	INFB_EC_1365_1393_F	524	INFB_EC_1439_1467_R	1248	infB
354	RPOC_EC_2218_2241_TMOD_F	405	RPOC_EC_2313_2337_TMOD_R	1072	rpoC
52	RPOC_EC_2218_2241_F	81	RPOC_EC_2313_2337_R	790	rpoC
355	SSPE_BA_115_137_TMOD_F	255	SSPE_BA_197_222_TMOD_R	1402	sspE
58	SSPE_BA_115_137_F	45	SSPE_BA_197_222_R	1201	sspE
356	RPLB_EC_650_679_TMOD_F	232	RPLB_EC_739_762_TMOD_R	592	rplB
66	RPLB_EC_650_679_F	98	RPLB_EC_739_762_R	999	rplB
358	VALS_EC_1105_1124_TMOD_F	385	VALS_EC_1195_1218_TMOD_R	1093	valS
71	VALS_EC_1105_1124_F	77	VALS_EC_1195_1218_R	795	valS
359	RPOB_EC_1845_1866_TMOD_F	659	RPOB_EC_1909_1929_TMOD_R	1250	rpoB
72	RPOB_EC_1845_1866_F	233	RPOB_EC_1909_1929_R	825	rpoB
360	23S_EC_2646_2667_TMOD_F	409	23S_EC_2745_2765_TMOD_R	1434	23S rRNA
118	23S_EC_2646_2667_F	84	23S_EC_2745_2765_R	1389	23S rRNA
17	23S_EC_2645_2669_F	408	23S_EC_2744_2761_R	1252	23S rRNA
361	16S_EC_1090_1111_2_TMOD_F	697	16S_EC_1175_1196_TMOD_R	1398	16S rRNA
3	16S_EC_1090_1111_2_F	651	16S_EC_1175_1196_R	1159	16S rRNA
362	RPOB_EC_3799_3821_TMOD_F	581	RPOB_EC_3862_3888_TMOD_R	1325	rpoB
289	RPOB_EC_3799_3821_F	124	RPOB_EC_3862_3888_R	840	rpoB
363	RPOC_EC_2146_2174_TMOD_F	284	RPOC_EC_2227_2245_TMOD_R	898	rpoC
290	RPOC_EC_2146_2174_F	52	RPOC_EC_2227_2245_R	736	rpoC
367	TUFB_EC_957_979_TMOD_F	308	TUFB_EC_1034_1058_TMOD_R	1276	tufB
293	TUFB_EC_957_979_F	55	TUFB_EC_1034_1058_R	829	tufB
449	RPLB_EC_690_710_F	309	RPLB_EC_737_758_R	1336	rplB
357	RPLB_EC_688_710_TMOD_F	296	RPLB_EC_736_757_TMOD_R	1337	rplB
67	RPLB_EC_688_710_F	54	RPLB_EC_736_757_R	842	rplB

The 16 primer pairs of the surveillance set are used to produce bioagent identifying amplicons whose base compositions are sufficiently different amongst all known bacteria at the species level to identify, at a reasonable confidence level, any given bacterium at the species level. As shown in Tables 6A-E, common respiratory bacterial pathogens can be distinguished by the base compositions of bioagent identifying amplicons obtained using the 16 primer pairs of the surveillance set. In some cases, triangulation identification improves the confidence level for species assignment. For example, nucleic acid from Streptococcus pyogenes can be amplified by nine of the sixteen surveillance primer pairs and Streptococcus pneumoniae can be amplified by ten of the sixteen surveillance primer pairs. The base compositions of the biogent identifying amplicons are identical for only one of the analogous bioagent identifying amplicons and differ in all of the remaining analogous bioagent identifying amplicons by up to four bases per bioagent identifying amplicon. The resolving power of the surveillance set was confirmed by determination of base compositions for 120 isolates of respiratory pathogens representing 70 different bacterial species and the results indicated that natural variations (usually only one or two base substitutions per bioagent identifying amplicon) amongst multiple isolates of the same species did not prevent correct identification of major pathogenic organisms at the species level.

Bacillus anthracis is a well known biological warfare agent which has emerged in domestic terrorism in recent years. Since it was envisioned to produce bioagent identifying amplicons for identification of Bacillus anthracis, additional drill-down analysis primers were designed to target genes present on virulence plasmids of Bacillus anthracis so that additional confidence could be reached in positive identification of this pathogenic organism. Three drill-down analysis primers were designed and are listed in Tables 2 and 6. In Table 6, the drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

TABLE 6


Drill-Down Primer Pairs for Confirmation of Identification of Bacillus anthracis

350	CAPC_BA_274_303_TMOD_F	476	CAPC_BA_349_376_TMOD_R	1314	capC
24	CAPC_BA_274_303_F	109	CAPC_BA_349_376_R	837	capC
351	CYA_BA_1353_1379_TMOD_F	355	CYA_BA_1448_1467_TMOD_R	1423	cyA
30	CYA_BA_1353_1379_F	64	CYA_BA_1448_1467_R	1342	cyA
353	LEF_BA_756_781_TMOD_F	220	LEF_BA_843_872_TMOD_R	1394	lef
37	LEF_BA_756_781_F	26	LEF_BA_843_872_R	1135	lef

Phylogenetic coverage of bacterial space of the sixteen surveillance primers of Table 5 and the three Bacillus anthracis drill-down primers of Table 6 is shown in FIG. 3 which lists common pathogenic bacteria. FIG. 3 is not meant to be comprehensive in illustrating all species identified by the primers. Only pathogenic bacteria are listed as representative examples of the bacterial species that can be identified by the primers and methods of the present invention. Nucleic acid of groups of bacteria enclosed within the polygons of FIG. 3 can be amplified to obtain bioagent identifying amplicons using the primer pair numbers listed in the upper right hand corner of each polygon. Primer coverage for polygons within polygons is additive. As an illustrative example, bioagent identifying amplicons can be obtained for Chlamydia trachomatis by amplification with, for example, primer pairs 346-349, 360 and 361, but not with any of the remaining primers of the surveillance primer set. On the other hand, bioagent identifying amplicons can be obtained from nucleic acid originating from Bacillus anthracis (located within 5 successive polygons) using, for example, any of the following primer pairs: 346-349, 360, 361 (base polygon), 356, 449 (second polygon), 352 (third polygon), 355 (fourth polygon), 350, 351 and 353 (fifth polygon). Multiple coverage of a given organism with multiple primers provides for increased confidence level in identification of the organism as a result of enabling broad triangulation identification.

In Tables 7A-E, base compositions of respiratory pathogens for primer target regions are shown. Two entries in a cell, represent variation in ribosomal DNA operons. The most predominant base composition is shown first and the minor (frequently a single operon) is indicated by an asterisk (*). Entries with NO DATA mean that the primer would not be expected to prime this species due to mismatches between the primer and target region, as determined by theoretical PCR.

TABLE 7A


Base Compositions of Common Respiratory Pathogens
for Bioagent Identifying Amplicons Corresponding to
Primer Pair Nos: 346, 347 and 348

		Primer 346	Primer 347	Primer 348
Organism	Strain	[A G C T]	[A G C T]	[A G C T]

Klebsiella	MGH78578	[29 32 25 13]	[23 38 28 26]	[26 32 28 30]
pneumoniae		[29 31 25 13]*	[23 37 28 26]*	[26 31 28 30]*
Yersinia pestis	CO-92 Biovar	[29 32 25 13]	[22 39 28 26]	[29 30 28 29]
	Orientalis			[30 30 27 29]*
Yersinia pestis	KIM5 P12 (Biovar	[29 32 25 13]	[22 39 28 26]	[29 30 28 29]
	Mediaevalis)
Yersinia pestis	91001	[29 32 25 13]	[22 39 28 26]	[29 30 28 29]
				[30 30 27 29]*
Haemophilus	KW20	[28 31 23 17]	[24 37 25 27]	[29 30 28 29]
influenzae
Pseudomonas	PAO1	[30 31 23 15]	[26 36 29 24]	[26 32 29 29]
aeruginosa			[27 36 29 23]*
Pseudomonas	Pf0-1	[30 31 23 15]	[26 35 29 25]	[28 31 28 29]
fluorescens
Pseudomonas	KT2440	[30 31 23 15]	[28 33 27 27]	[27 32 29 28]
putida
Legionella	Philadelphia-1	[30 30 24 15]	[33 33 23 27]	[29 28 28 31]
pneumophila
Francisella	schu 4	[32 29 22 16]	[28 38 26 26]	[25 32 28 31]
tularensis
Bordetella	Tohama I	[30 29 24 16]	[23 37 30 24]	[30 32 30 26]
pertussis
Burkholderia	J2315	[29 29 27 14]	[27 32 26 29]	[27 36 31 24]
cepacia				[20 42 35 19]*
Burkholderia	K96243	[29 29 27 14]	[27 32 26 29]	[27 36 31 24]
pseudomallei
Neisseria	FA 1090, ATCC	[29 28 24 18]	[27 34 26 28]	[24 36 29 27]
gonorrhoeae	700825
Neisseria	MC58 (serogroup B)	[29 28 26 16]	[27 34 27 27]	[25 35 30 26]
meningitidis
Neisseria	serogroup C, FAM18	[29 28 26 16]	[27 34 27 27]	[25 35 30 26]
meningitidis
Neisseria	Z2491 (serogroup A)	[29 28 26 16]	[27 34 27 27]	[25 35 30 26]
meningitidis
Chlamydophila	TW-183	[31 27 22 19]	NO DATA	[32 27 27 29]
pneumoniae
Chlamydophila	AR39	[31 27 22 19]	NO DATA	[32 27 27 29]
pneumoniae
Chlamydophila	CWL029	[31 27 22 19]	NO DATA	[32 27 27 29]
pneumoniae
Chlamydophila	J138	[31 27 22 19]	NO DATA	[32 27 27 29]
pneumoniae
Corynebacterium	NCTC13129	[29 34 21 15]	[22 38 31 25]	[22 33 25 34]
diphtheriae
Mycobacterium	k10	[27 36 21 15]	[22 37 30 28]	[21 36 27 30]
avium
Mycobacterium	104	[27 36 21 15]	[22 37 30 28]	[21 36 27 30]
avium
Mycobacterium	CSU#93	[27 36 21 15]	[22 37 30 28]	[21 36 27 30]
tuberculosis
Mycobacterium	CDC 1551	[27 36 21 15]	[22 37 30 28]	[21 36 27 30]
tuberculosis
Mycobacterium	H37Rv (lab strain)	[27 36 21 15]	[22 37 30 28]	[21 36 27 30]
tuberculosis
Mycoplasma	M129	[31 29 19 20]	NO DATA	NO DATA
pneumoniae
Staphylococcus	MRSA252	[27 30 21 21]	[25 35 30 26]	[30 29 30 29]
aureus				[29 31 30 29]*
Staphylococcus	MSSA476	[27 30 21 21]	[25 35 30 26]	[30 29 30 29]
aureus				[30 29 29 30]*
Staphylococcus	COL	[27 30 21 21]	[25 35 30 26]	[30 29 30 29]
aureus				[30 29 29 30]*
Staphylococcus	Mu50	[27 30 21 21]	[25 35 30 26]	[30 29 30 29]
aureus				[30 29 29 30]*
Staphylococcus	MW2	[27 30 21 21]	[25 35 30 26]	[30 29 30 29]
aureus				[30 29 29 30]*
Staphylococcus	N315	[27 30 21 21]	[25 35 30 26]	[30 29 30 29]
aureus				[30 29 29 30]*
Staphylococcus	NCTC 8325	[27 30 21 21]	[25 35 30 26]	[30 29 30 29]
aureus			[25 35 31 26]*	[30 29 29 30]
Streptococcus	NEM316	[26 32 23 18]	[24 36 31 25]	[25 32 29 30]
agalactiae			[24 36 30 26]*
Streptococcus	NC_002955	[26 32 23 18]	[23 37 31 25]	[29 30 25 32]
equi
Streptococcus	MGAS8232	[26 32 23 18]	[24 37 30 25]	[25 31 29 31]
pyogenes
Streptococcus	MGAS315	[26 32 23 18]	[24 37 30 25]	[25 31 29 31]
pyogenes
Streptococcus	SSI-1	[26 32 23 18]	[24 37 30 25]	[25 31 29 31]
pyogenes
Streptococcus	MGAS10394	[26 32 23 18]	[24 37 30 25]	[25 31 29 31]
pyogenes
Streptococcus	Manfredo (M5)	[26 32 23 18]	[24 37 30 25]	[25 31 29 31]
pyogenes
Streptococcus	SF370 (M1)	[26 32 23 18]	[24 37 30 25]	[25 31 29 31]
pyogenes
Streptococcus	670	[26 32 23 18]	[25 35 28 28]	[25 32 29 30]
pneumoniae
Streptococcus	R6	[26 32 23 18]	[25 35 28 28]	[25 32 29 30]
pneumoniae
Streptococcus	TIGR4	[26 32 23 18]	[25 35 28 28]	[25 32 30 29]
pneumoniae
Streptococcus	NCTC7868	[25 33 23 18]	[24 36 31 25]	[25 31 29 31]
gordonii
Streptococcus	NCTC 12261	[26 32 23 18]	[25 35 30 26]	[25 32 29 30]
mitis				[24 31 35 29]*
Streptococcus	UA159	[24 32 24 19]	[25 37 30 24]	[28 31 26 31]
mutans

TABLE 7B


Base Compositions of Common Respiratory Pathogens for Bioagent Identifying
Amplicons Corresponding to Primer Pair Nos: 349, 360, and 356

		Primer 349	Primer 360	Primer 356
Organism	Strain	[A G C T]	[A G C T]	[A G C T]

Klebsiella	MGH78578	[25 31 25 22]	[33 37 25 27]	NO DATA
pneumoniae
Yersinia pestis	CO-92 Biovar	[25 31 27 20]	[34 35 25 28]	NO DATA
	Orientalis	[25 32 26 20]*
Yersinia pestis	KIM5 P12 (Biovar	[25 31 27 20]	[34 35 25 28]	NO DATA
	Mediaevalis)	[25 32 26 20]*
Yersinia pestis	91001	[25 31 27 20]	[34 35 25 28]	NO DATA
Haemophilus	KW20	[28 28 25 20]	[32 38 25 27]	NO DATA
influenzae
Pseudomonas	PAO1	[24 31 26 20]	[31 36 27 27]	NO DATA
aeruginosa			[31 36 27 28]*
Pseudomonas	Pf0-1	NO DATA	[30 37 27 28]	NO DATA
fluorescens			[30 37 27 28]
Pseudomonas	KT2440	[24 31 26 20]	[30 37 27 28]	NO DATA
putida
Legionella	Philadelphia-1	[23 30 25 23]	[30 39 29 24]	NO DATA
pneumophila
Francisella	schu 4	[26 31 25 19]	[32 36 27 27]	NO DATA
tularensis
Bordetella	Tohama I	[21 29 24 18]	[33 36 26 27]	NO DATA
pertussis
Burkholderia	J2315	[23 27 22 20]	[31 37 28 26]	NO DATA
cepacia
Burkholderia	K96243	[23 27 22 20]	[31 37 28 26]	NO DATA
pseudomallei
Neisseria	FA 1090, ATCC 700825	[24 27 24 17]	[34 37 25 26]	NO DATA
gonorrhoeae
Neisseria	MC58 (serogroup B)	[25 27 22 18]	[34 37 25 26]	NO DATA
meningitidis
Neisseria	serogroup C, FAM18	[25 26 23 18]	[34 37 25 26]	NO DATA
meningitidis
Neisseria	Z2491 (serogroup A)	[25 26 23 18]	[34 37 25 26]	NO DATA
meningitidis
Chlamydophila	TW-183	[30 28 27 18]	NO DATA	NO DATA
pneumoniae
Chlamydophila	AR39	[30 28 27 18]	NO DATA	NO DATA
pneumoniae
Chlamydophila	CWL029	[30 28 27 18]	NO DATA	NO DATA
pneumoniae
Chlamydophila	J138	[30 28 27 18]	NO DATA	NO DATA
pneumoniae
Corynebacterium	NCTC13129	NO DATA	[29 40 28 25]	NO DATA
diphtheriae
Mycobacterium	k10	NO DATA	[33 35 32 22]	NO DATA
avium
Mycobacterium	104	NO DATA	[33 35 32 22]	NO DATA
avium
Mycobacterium	CSU#93	NO DATA	[30 36 34 22]	NO DATA
tuberculosis
Mycobacterium	CDC 1551	NO DATA	[30 36 34 22]	NO DATA
tuberculosis
Mycobacterium	H37Rv (lab strain)	NO DATA	[30 36 34 22]	NO DATA
tuberculosis
Mycoplasma	M129	[28 30 24 19]	[34 31 29 28]	NO DATA
pneumoniae
Staphylococcus	MRSA252	[26 30 25 20]	[31 38 24 29]	[33 30 31 27]
aureus
Staphylococcus	MSSA476	[26 30 25 20]	[31 38 24 29]	[33 30 31 27]
aureus
Staphylococcus	COL	[26 30 25 20]	[31 38 24 29]	[33 30 31 27]
aureus
Staphylococcus	Mu50	[26 30 25 20]	[31 38 24 29]	[33 30 31 27]
aureus
Staphylococcus	MW2	[26 30 25 20]	[31 38 24 29]	[33 30 31 27]
aureus
Staphylococcus	N315	[26 30 25 20]	[31 38 24 29]	[33 30 31 27]
aureus
Staphylococcus	NCTC 8325	[26 30 25 20]	[31 38 24 29]	[33 30 31 27]
aureus
Streptococcus	NEM316	[28 31 22 20]	[33 37 24 28]	[37 30 28 26]
agalactiae
Streptococcus	NC_002955	[28 31 23 19]	[33 38 24 27]	[37 31 28 25]
equi
Streptococcus	MGAS8232	[28 31 23 19]	[33 37 24 28]	[38 31 29 23]
pyogenes
Streptococcus	MGAS315	[28 31 23 19]	[33 37 24 28]	[38 31 29 23]
pyogenes
Streptococcus	SSI-1	[28 31 23 19]	[33 37 24 28]	[38 31 29 23]
pyogenes
Streptococcus	MGAS10394	[28 31 23 19]	[33 37 24 28]	[38 31 29 23]
pyogenes
Streptococcus	Manfredo (M5)	[28 31 23 19]	[33 37 24 28]	[38 31 29 23]
pyogenes
Streptococcus	SF370 (M1)	[28 31 23 19]	[33 37 24 28]	[38 31 29 23]
pyogenes		[28 31 22 20]*
Streptococcus	670	[28 31 22 20]	[34 36 24 28]	[37 30 29 25]
pneumoniae
Streptococcus	R6	[28 31 22 20]	[34 36 24 28]	[37 30 29 25]
pneumoniae
Streptococcus	TIGR4	[28 31 22 20]	[34 36 24 28]	[37 30 29 25]
pneumoniae
Streptococcus	NCTC7868	[28 32 23 20]	[34 36 24 28]	[36 31 29 25]
gordonii
Streptococcus	NCTC 12261	[28 31 22 20]	[34 36 24 28]	[37 30 29 25]
mitis		[29 30 22 20]*
Streptococcus	UA159	[26 32 23 22]	[34 37 24 27]	NO DATA
mutans

TABLE 7C


Base Compositions of Common Respiratory Pathogens for Bioagent Identifying
Amplicons Corresponding to Primer Pair Nos: 449, 354, and 352

		Primer 449	Primer 354	Primer 352
Organism	Strain	[A G C T]	[A G C T]	[A G C T]

Klebsiella	MGH78578	NO DATA	[27 33 36 26]	NO DATA
pneumoniae
Yersinia pestis	CO-92 Biovar	NO DATA	[29 31 33 29]	[32 28 20 25]
	Orientalis
Yersinia pestis	KIM5 P12 (Biovar	NO DATA	[29 31 33 29]	[32 28 20 25]
	Mediaevalis)
Yersinia pestis	91001	NO DATA	[29 31 33 29]	NO DATA
Haemophilus	KW20	NO DATA	[30 29 31 32]	NO DATA
influenzae
Pseudomonas	PAO1	NO DATA	[26 33 39 24]	NO DATA
aeruginosa
Pseudomonas	Pf0-1	NO DATA	[26 33 34 29]	NO DATA
fluorescens
Pseudomonas	KT2440	NO DATA	[25 34 36 27]	NO DATA
putida
Legionella	Philadelphia-1	NO DATA	NO DATA	NO DATA
pneumophila
Francisella	schu 4	NO DATA	[33 32 25 32]	NO DATA
tularensis
Bordetella	Tohama I	NO DATA	[26 33 39 24]	NO DATA
pertussis
Burkholderia	J2315	NO DATA	[25 37 33 27]	NO DATA
cepacia
Burkholderia	K96243	NO DATA	[25 37 34 26]	NO DATA
pseudomallei
Neisseria	FA 1090, ATCC 700825	[17 23 22 10]	[29 31 32 30]	NO DATA
gonorrhoeae
Neisseria	MC58 (serogroup B)	NO DATA	[29 30 32 31]	NO DATA
meningitidis
Neisseria	serogroup C, FAM18	NO DATA	[29 30 32 31]	NO DATA
meningitidis
Neisseria	Z2491 (serogroup A)	NO DATA	[29 30 32 31]	NO DATA
meningitidis
Chlamydophila	TW-183	NO DATA	NO DATA	NO DATA
pneumoniae
Chlamydophila	AR39	NO DATA	NO DATA	NO DATA
pneumoniae
Chlamydophila	CWL029	NO DATA	NO DATA	NO DATA
pneumoniae
Chlamydophila	J138	NO DATA	NO DATA	NO DATA
pneumoniae
Corynebacterium	NCTC13129	NO DATA	NO DATA	NO DATA
diphtheriae
Mycobacterium	k10	NO DATA	NO DATA	NO DATA
avium
Mycobacterium	104	NO DATA	NO DATA	NO DATA
avium
Mycobacterium	CSU#93	NO DATA	NO DATA	NO DATA
tuberculosis
Mycobacterium	CDC 1551	NO DATA	NO DATA	NO DATA
tuberculosis
Mycobacterium	H37Rv (lab strain)	NO DATA	NO DATA	NO DATA
tuberculosis
Mycoplasma	M129	NO DATA	NO DATA	NO DATA
pneumoniae
Staphylococcus	MRSA252	[17 20 21 17]	[30 27 30 35]	[36 24 19 26]
aureus
Staphylococcus	MSSA476	[17 20 21 17]	[30 27 30 35]	[36 24 19 26]
aureus
Staphylococcus	COL	[17 20 21 17]	[30 27 30 35]	[35 24 19 27]
aureus
Staphylococcus	Mu50	[17 20 21 17]	[30 27 30 35]	[36 24 19 26]
aureus
Staphylococcus	MW2	[17 20 21 17]	[30 27 30 35]	[36 24 19 26]
aureus
Staphylococcus	N315	[17 20 21 17]	[30 27 30 35]	[36 24 19 26]
aureus
Staphylococcus	NCTC 8325	[17 20 21 17]	[30 27 30 35]	[35 24 19 27]
aureus
Streptococcus	NEM316	[22 20 19 14]	[26 31 27 38]	[29 26 22 28]
agalactiae
Streptococcus	NC_002955	[22 21 19 13]	NO DATA	NO DATA
equi
Streptococcus	MGAS8232	[23 21 19 12]	[24 32 30 36]	NO DATA
pyogenes
Streptococcus	MGAS315	[23 21 19 12]	[24 32 30 36]	NO DATA
pyogenes
Streptococcus	SSI-1	[23 21 19 12]	[24 32 30 36]	NO DATA
pyogenes
Streptococcus	MGAS10394	[23 21 19 12]	[24 32 30 36]	NO DATA
pyogenes
Streptococcus	Manfredo (M5)	[23 21 19 12]	[24 32 30 36]	NO DATA
pyogenes
Streptococcus	SF370 (M1)	[23 21 19 12]	[24 32 30 36]	NO DATA
pyogenes
Streptococcus	670	[22 20 19 14]	[25 33 29 35]	[30 29 21 25]
pneumoniae
Streptococcus	R6	[22 20 19 14]	[25 33 29 35]	[30 29 21 25]
pneumoniae
Streptococcus	TIGR4	[22 20 19 14]	[25 33 29 35]	[30 29 21 25]
pneumoniae
Streptococcus	NCTC7868	[21 21 19 14]	NO DATA	[29 26 22 28]
gordonii
Streptococcus	NCTC 12261	[22 20 19 14]	[26 30 32 34]	NO DATA
mitis
Streptococcus	UA159	NO DATA	NO DATA	NO DATA
mutans

TABLE 7D


Base Compositions of Common Respiratory Pathogens for Bioagent Identifying
Amplicons Corresponding to Primer Pair Nos: 355, 358, and 359

		Primer 355	Primer 358	Primer 359
Organism	Strain	[A G C T]	[A G C T]	[A G C T]

Klebsiella	MGH78578	NO DATA	[24 39 33 20]	[25 21 24 17]
pneumoniae
Yersinia pestis	CO-92 Biovar	NO DATA	[26 34 35 21]	[23 23 19 22]
	Orientalis
Yersinia pestis	KIM5 P12 (Biovar	NO DATA	[26 34 35 21]	[23 23 19 22]
	Mediaevalis)
Yersinia pestis	91001	NO DATA	[26 34 35 21]	[23 23 19 22]
Haemophilus	KW20	NO DATA	NO DATA	NO DATA
influenzae
Pseudomonas	PAO1	NO DATA	NO DATA	NO DATA
aeruginosa
Pseudomonas	Pf0-1	NO DATA	NO DATA	NO DATA
fluorescens
Pseudomonas	KT2440	NO DATA	[21 37 37 21]	NO DATA
putida
Legionella	Philadelphia-1	NO DATA	NO DATA	NO DATA
pneumophila
Francisella	schu 4	NO DATA	NO DATA	NO DATA
tularensis
Bordetella	Tohama I	NO DATA	NO DATA	NO DATA
pertussis
Burkholderia	J2315	NO DATA	NO DATA	NO DATA
cepacia
Burkholderia	K96243	NO DATA	NO DATA	NO DATA
pseudomallei
Neisseria	FA 1090, ATCC 700825	NO DATA	NO DATA	NO DATA
gonorrhoeae
Neisseria	MC58 (serogroup B)	NO DATA	NO DATA	NO DATA
meningitidis
Neisseria	serogroup C, FAM18	NO DATA	NO DATA	NO DATA
meningitidis
Neisseria	Z2491 (serogroup A)	NO DATA	NO DATA	NO DATA
meningitidis
Chlamydophila	TW-183	NO DATA	NO DATA	NO DATA
pneumoniae
Chlamydophila	AR39	NO DATA	NO DATA	NO DATA
pneumoniae
Chlamydophila	CWL029	NO DATA	NO DATA	NO DATA
pneumoniae
Chlamydophila	J138	NO DATA	NO DATA	NO DATA
pneumoniae
Corynebacterium	NCTC13129	NO DATA	NO DATA	NO DATA
diphtheriae
Mycobacterium	k10	NO DATA	NO DATA	NO DATA
avium
Mycobacterium	104	NO DATA	NO DATA	NO DATA
avium
Mycobacterium	CSU#93	NO DATA	NO DATA	NO DATA
tuberculosis
Mycobacterium	CDC 1551	NO DATA	NO DATA	NO DATA
tuberculosis
Mycobacterium	H37Rv (lab strain)	NO DATA	NO DATA	NO DATA
tuberculosis
Mycoplasma	M129	NO DATA	NO DATA	NO DATA
pneumoniae
Staphylococcus	MRSA252	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	MSSA476	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	COL	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	Mu50	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	MW2	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	N315	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	NCTC 8325	NO DATA	NO DATA	NO DATA
aureus
Streptococcus	NEM316	NO DATA	NO DATA	NO DATA
agalactiae
Streptococcus	NC_002955	NO DATA	NO DATA	NO DATA
equi
Streptococcus	MGAS8232	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	MGAS315	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	SSI-1	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	MGAS10394	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	Manfredo (M5)	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	SF370 (M1)	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	670	NO DATA	NO DATA	NO DATA
pneumoniae
Streptococcus	R6	NO DATA	NO DATA	NO DATA
pneumoniae
Streptococcus	TIGR4	NO DATA	NO DATA	NO DATA
pneumoniae
Streptococcus	NCTC7868	NO DATA	NO DATA	NO DATA
gordonii
Streptococcus	NCTC 12261	NO DATA	NO DATA	NO DATA
mitis
Streptococcus	UA159	NO DATA	NO DATA	NO DATA
mutans

TABLE 7E


Base Compositions of Common Respiratory Pathogens for Bioagent Identifying
Amplicons Corresponding to Primer Pair Nos: 362, 363, and 367

		Primer 362	Primer 363	Primer 367
Organism	Strain	[A G C T]	[A G C T]	[A G C T]

Klebsiella	MGH78578	[21 33 22 16]	[16 34 26 26]	NO DATA
pneumoniae
Yersinia pestis	CO-92 Biovar	[20 34 18 20]	NO DATA	NO DATA
	Orientalis
Yersinia pestis	KIM5 P12 (Biovar	[20 34 18 20]	NO DATA	NO DATA
	Mediaevalis)
Yersinia pestis	91001	[20 34 18 20]	NO DATA	NO DATA
Haemophilus	KW20	NO DATA	NO DATA	NO DATA
influenzae
Pseudomonas	PAO1	[19 35 21 17]	[16 36 28 22]	NO DATA
aeruginosa
Pseudomonas	Pf0-1	NO DATA	[18 35 26 23]	NO DATA
fluorescens
Pseudomonas	KT2440	NO DATA	[16 35 28 23]	NO DATA
putida
Legionella	Philadelphia-1	NO DATA	NO DATA	NO DATA
pneumophila
Francisella	schu 4	NO DATA	NO DATA	NO DATA
tularensis
Bordetella	Tohama I	[20 31 24 17]	[15 34 32 21]	[26 25 34 19]
pertussis
Burkholderia	J2315	[20 33 21 18]	[15 36 26 25]	[25 27 32 20]
cepacia
Burkholderia	K96243	[19 34 19 20]	[15 37 28 22]	[25 27 32 20]
pseudomallei
Neisseria	FA 1090, ATCC 700825	NO DATA	NO DATA	NO DATA
gonorrhoeae
Neisseria	MC58 (serogroup B)	NO DATA	NO DATA	NO DATA
meningitidis
Neisseria	serogroup C, FAM18	NO DATA	NO DATA	NO DATA
meningitidis
Neisseria	Z2491 (serogroup A)	NO DATA	NO DATA	NO DATA
meningitidis
Chlamydophila	TW-183	NO DATA	NO DATA	NO DATA
pneumoniae
Chlamydophila	AR39	NO DATA	NO DATA	NO DATA
pneumoniae
Chlamydophila	CWL029	NO DATA	NO DATA	NO DATA
pneumoniae
Chlamydophila	J138	NO DATA	NO DATA	NO DATA
pneumoniae
Corynebacterium	NCTC13129	NO DATA	NO DATA	NO DATA
diphtheriae
Mycobacterium	k10	[19 34 23 16]	NO DATA	[24 26 35 19]
avium
Mycobacterium	104	[19 34 23 16]	NO DATA	[24 26 35 19]
avium
Mycobacterium	CSU#93	[19 31 25 17]	NO DATA	[25 25 34 20]
tuberculosis
Mycobacterium	CDC 1551	[19 31 24 18]	NO DATA	[25 25 34 20]
tuberculosis
Mycobacterium	H37Rv (lab strain)	[19 31 24 18]	NO DATA	[25 25 34 20]
tuberculosis
Mycoplasma	M129	NO DATA	NO DATA	NO DATA
pneumoniae
Staphylococcus	MRSA252	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	MSSA476	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	COL	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	Mu50	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	MW2	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	N315	NO DATA	NO DATA	NO DATA
aureus
Staphylococcus	NCTC 8325	NO DATA	NO DATA	NO DATA
aureus
Streptococcus	NEM316	NO DATA	NO DATA	NO DATA
agalactiae
Streptococcus	NC_002955	NO DATA	NO DATA	NO DATA
equi
Streptococcus	MGAS8232	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	MGAS315	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	SSI-1	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	MGAS10394	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	Manfredo (M5)	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	SF370 (M1)	NO DATA	NO DATA	NO DATA
pyogenes
Streptococcus	670	NO DATA	NO DATA	NO DATA
pneumoniae
Streptococcus	R6	[20 30 19 23]	NO DATA	NO DATA
pneumoniae
Streptococcus	TIGR4	[20 30 19 23]	NO DATA	NO DATA
pneumoniae
Streptococcus	NCTC7868	NO DATA	NO DATA	NO DATA
gordonii
Streptococcus	NCTC 12261	NO DATA	NO DATA	NO DATA
mitis
Streptococcus	UA159	NO DATA	NO DATA	NO DATA
mutans

Four sets of throat samples from military recruits at different military facilities taken at different time points were analyzed using the primers of the present invention. The first set was collected at a military training center from Nov. 1 to Dec. 20, 2002 during one of the most severe outbreaks of pneumonia associated with group A Streptococcus in the United States since 1968. During this outbreak, fifty-one throat swabs were taken from both healthy and hospitalized recruits and plated on blood agar for selection of putative group A Streptococcus colonies. A second set of 15 original patient specimens was taken during the height of this group A Streptococcus-associated respiratory disease outbreak. The third set were historical samples, including twenty-seven isolates of group A Streptococcus, from disease outbreaks at this and other military training facilities during previous years. The fourth set of samples was collected from five geographically separated military facilities in the continental U.S. in the winter immediately following the severe November/December 2002 outbreak.
Pure colonies isolated from group A Streptococcus-selective media from all four collection periods were analyzed with the surveillance primer set. All samples showed base compositions that precisely matched the four completely sequenced strains of Streptococcus pyogenes. Shown in FIG. 4 is a 3D diagram of base composition (axes A, G and C) of bioagent identifying amplicons obtained with primer pair number 14 (a precursor of primer pair number 348 which targets 16S rRNA). The diagram indicates that the experimentally determined base compositions of the clinical samples closely match the base compositions expected for Streptococcus pyogenes and are distinct from the expected base compositions of other organisms.
In addition to the identification of Streptococcus pyogenes, other potentially pathogenic organisms were identified concurrently. Mass spectral analysis of a sample whose nucleic acid was amplified by primer pair number 349 (SEQ ID NOs: 401:1156) exhibited signals of bioagent identifying amplicons with molecular masses that were found to correspond to analogous base compositions of bioagent identifying amplicons of Streptococcus pyogenes (A27 G32 C24 T18), Neisseria meningitidis (A25 G27 C22 T18), and Haemophilus influenzae (A28 G28 C25 T20) (see FIG. 5 and Table 7B). These organisms were present in a ratio of 4:5:20 as determined by comparison of peak heights with peak height of an internal PCR calibration standard as described in commonly owned U.S. Patent Application Ser. No. 60/545,425 which is incorporated herein by reference in its entirety.
Since certain division-wide primers that target housekeeping genes are designed to provide coverage of specific divisions of bacteria to increase the confidence level for identification of bacterial species, they are not expected to yield bioagent identifying amplicons for organisms outside of the specific divisions. For example, primer pair number 356 (SEQ ID NOs: 449:1380) primarily amplifies the nucleic acid of members of the classes Bacilli and Clostridia and is not expected to amplify proteobacteria such as Neisseria meningitidis and Haemophilus influenzae. As expected, analysis of the mass spectrum of amplification products obtained with primer pair number 356 does not indicate the presence of Neisseria meningitidis and Haemophilus influenzae but does indicate the presence of Streptococcus pyogenes (FIGS. 3 and 6, Table 7B). Thus, these primers or types of primers can confirm the absence of particular bioagents from a sample.
The 15 throat swabs from military recruits were found to contain a relatively small set of microbes in high abundance. The most common were Haemophilus influenza, Neisseria meningitides, and Streptococcus pyogenes. Staphylococcus epidermidis, Moraxella cattarhalis, Corynebacterium pseudodiphtheriticum, and Staphylococcus aureus were present in fewer samples. An equal number of samples from healthy volunteers from three different geographic locations, were identically analyzed. Results indicated that the healthy volunteers have bacterial flora dominated by multiple, commensal non-beta-hemolytic Streptococcal species, including the viridans group streptococci (S. parasangunis, S. vestibularis, S. mitis, S. oralis and S. pneumoniae; data not shown), and none of the organisms found in the military recruits were found in the healthy controls at concentrations detectable by mass spectrometry. Thus, the military recruits in the midst of a respiratory disease outbreak had a dramatically different microbial population than that experienced by the general population in the absence of epidemic disease.

Example 7

Triangulation Genotyping Analysis for Determination of emm-Type of Streptococcus pyogenes in Epidemic Surveillance

As a continuation of the epidemic surveillance investigation of Example 6, determination of sub-species characteristics (genotyping) of Streptococcus pyogenes, was carried out based on a strategy that generates strain-specific signatures according to the rationale of Multi-Locus Sequence Typing (MLST). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced (Enright et al. Infection and Immunity, 2001, 69, 2416-2427). In classic MLST analysis, internal fragments of several housekeeping genes are amplified and sequenced. In the present investigation, bioagent identifying amplicons from housekeeping genes were produced using drill-down primers and analyzed by mass spectrometry. Since mass spectral analysis results in molecular mass, from which base composition can be determined, the challenge was to determine whether resolution of emm classification of strains of Streptococcus pyogenes could be determined.

For the purpose of development of a triangulation genotyping assay, an alignment was constructed of concatenated alleles of seven MLST housekeeping genes (glucose kinase (gki), glutamine transporter protein (gtr), glutamate racemase (murI), DNA mismatch repair protein (mutS), xanthine phosphoribosyl transferase (xpt), and acetyl-CoA acetyl transferase (yqiL)) from each of the 212 previously emm-typed strains of Streptococcus pyogenes. From this alignment, the number and location of primer pairs that would maximize strain identification via base composition was determined. As a result, 6 primer pairs were chosen as standard drill-down primers for determination of emm-type of Streptococcus pyogenes. These six primer pairs are displayed in Table 8. This drill-down set comprises primers with T modifications (note TMOD designation in primer names) which constitutes a functional improvement with regard to prevention of non-templated adenylation (vide supra) relative to originally selected primers which are displayed below in the same row.

TABLE 8


Triangulation Genotyping Analysis Primer Pairs for Group A Streptococcus Drill-Down

		Forward
		Primer
Primer		(SEQ ID		Reverse Primer	Target
Pair No.	Forward Primer Name	NO:)	Reverse Primer Name	(SEQ ID NO:)	Gene

442	SP101_SPET11_358_387_TMOD_F	588	SP101_SPET11_448_473_TMOD_R	998	gki
80	SP101_SPET11_358_387_F	126	SP101_SPET11_448_473_TMOD_R	766	gki
443	SP101_SPET11_600_629_TMOD_F	348	SP101_SPET11_686_714_TMOD_R	1018	gtr
81	SP101_SPET11_600_629_F	62	SP101_SPET11_686_714_R	772	gtr
426	SP101_SPET11_1314_1336_TMOD_F	363	SP101_SPET11_1403_1431_TMOD_R	849	murI
86	SP101_SPET11_1314_1336_F	68	SP101_SPET11_1403_1431_R	711	murI
430	SP101_SPET11_1807_1835_TMOD_F	235	SP101_SPET11_1901_1927_TMOD_R	1439	mutS
90	SP101_SPET11_1807_1835_F	33	SP101_SPET11_1901_1927_R	1412	mutS
438	SP101_SPET11_3075_3103_TMOD_F	473	SP101_SPET11_3168_3196_TMOD_R	875	xpt
96	SP101_SPET11_3075_3103_F	108	SP101_SPET11_3168_3196_R	715	xpt
441	SP101_SPET11_3511_3535_TMOD_F	531	SP101_SPET11_3605_3629_TMOD_R	1294	yqiL
98	SP101_SPET11_3511_3535_F	116	SP101_SPET11_3605_3629_R	832	yqiL

The primers of Table 8 were used to produce bioagent identifying amplicons from nucleic acid present in the clinical samples. The bioagent identifying amplicons which were subsequently analyzed by mass spectrometry and base compositions corresponding to the molecular masses were calculated.

Of the 51 samples taken during the peak of the November/December 2002 epidemic (Table 9A-C rows 1-3), all except three samples were found to represent emm3, a Group A Streptococcus genotype previously associated with high respiratory virulence. The three outliers were from samples obtained from healthy individuals and probably represent non-epidemic strains. Archived samples (Tables 9A-C rows 5-13) from historical collections showed a greater heterogeneity of base compositions and emm types as would be expected from different epidemics occurring at different places and dates. The results of the mass spectrometry analysis and emm gene sequencing were found to be concordant for the epidemic and historical samples.

TABLE 9A


Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus
samples from Six Military Installations Obtained with Primer Pair Nos. 426 and 430

	emm-type by				murI	mutS
# of	Mass	emm-Gene	Location		(Primer Pair	(Primer Pair
Instances	Spectrometry	Sequencing	(sample)	Year	No. 426)	No. 430)

48	3	3	MCRD San	2002	A39 G25 C20 T34	A38 G27 C23 T33
2	6	6	Diego		A40 G24 C20 T34	A38 G27 C23 T33
1	28	28	(Cultured)		A39 G25 C20 T34	A38 G27 C23 T33
15	3	ND			A39 G25 C20 T34	A38 G27 C23 T33
6	3	3	NHRC San	2003	A39 G25 C20 T34	A38 G27 C23 T33
3	5, 58	5	Diego-		A40 G24 C20 T34	A38 G27 C23 T33
6	6	6	Archive		A40 G24 C20 T34	A38 G27 C23 T33
1	11	11	(Cultured)		A39 G25 C20 T34	A38 G27 C23 T33
3	12	12			A40 G24 C20 T34	A38 G26 C24 T33
1	22	22			A39 G25 C20 T34	A38 G27 C23 T33
3	25, 75	75			A39 G25 C20 T34	A38 G27 C23 T33
4	44/61, 82, 9	44/61			A40 G24 C20 T34	A38 G26 C24 T33
2	53, 91	91			A39 G25 C20 T34	A38 G27 C23 T33
1	2	2	Ft.	2003	A39 G25 C20 T34	A38 G27 C24 T32
2	3	3	Leonard		A39 G25 C20 T34	A38 G27 C23 T33
1	4	4	Wood		A39 G25 C20 T34	A38 G27 C23 T33
1	6	6	(Cultured)		A40 G24 C20 T34	A38 G27 C23 T33
11	25 or 75	75			A39 G25 C20 T34	A38 G27 C23 T33
1	25, 75, 33,	75			A39 G25 C20 T34	A38 G27 C23 T33
	34, 4, 52, 84
1	44/61 or 82	44/61			A40 G24 C20 T34	A38 G26 C24 T33
	or 9
2	5 or 58	5			A40 G24 C20 T34	A38 G27 C23 T33
3	1	1	Ft. Sill	2003	A40 G24 C20 T34	A38 G27 C23 T33
2	3	3	(Cultured)		A39 G25 C20 T34	A38 G27 C23 T33
1	4	4			A39 G25 C20 T34	A38 G27 C23 T33
1	28	28			A39 G25 C20 T34	A38 G27 C23 T33
1	3	3	Ft.	2003	A39 G25 C20 T34	A38 G27 C23 T33
1	4	4	Benning		A39 G25 C20 T34	A38 G27 C23 T33
3	6	6	(Cultured)		A40 G24 C20 T34	A38 G27 C23 T33
1	11	11			A39 G25 C20 T34	A38 G27 C23 T33
1	13	94**			A40 G24 C20 T34	A38 G27 C23 T33
1	44/61 or 82	82			A40 G24 C20 T34	A38 G26 C24 T33
	or 9
1	5 or 58	58			A40 G24 C20 T34	A38 G27 C23 T33
1	78 or 89	89			A39 G25 C20 T34	A38 G27 C23 T33
2	5 or 58	ND	Lackland	2003	A40 G24 C20 T34	A38 G27 C23 T33
1	2		AFB		A39 G25 C20 T34	A38 G27 C24 T32
1	81 or 90		(Throat		A40 G24 C20 T34	A38 G27 C23 T33
1	78		Swabs)		A38 G26 C20 T34	A38 G27 C23 T33
3***	No detection				No detection	No detection
7	3	ND	MCRD San	2002	A39 G25 C20 T34	A38 G27 C23 T33
1	3	ND	Diego		No detection	A38 G27 C23 T33
1	3	ND	(Throat		No detection	No detection
1	3	ND	Swabs)		No detection	No detection
2	3	ND			No detection	A38 G27 C23 T33
3	No detection	ND			No detection	No detection

TABLE 9B


Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus
samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441

	emm-type by				xpt	yqiL
# of	Mass	emm-Gene	Location		(Primer Pair	(Primer Pair
Instances	Spectrometry	Sequencing	(sample)	Year	No. 438)	No. 441)

48	3	3	MCRD San	2002	A30 G36 C20 T36	A40 G29 C19 T31
2	6	6	Diego		A30 G36 C20 T36	A40 G29 C19 T31
1	28	28	(Cultured)		A30 G36 C20 T36	A41 G28 C18 T32
15	3	ND			A30 G36 C20 T36	A40 G29 C19 T31
6	3	3	NHRC San	2003	A30 G36 C20 T36	A40 G29 C19 T31
3	5, 58	5	Diego-		A30 G36 C20 T36	A40 G29 C19 T31
6	6	6	Archive		A30 G36 C20 T36	A40 G29 C19 T31
1	11	11	(Cultured)		A30 G36 C20 T36	A40 G29 C19 T31
3	12	12			A30 G36 C19 T37	A40 G29 C19 T31
1	22	22			A30 G36 C20 T36	A40 G29 C19 T31
3	25, 75	75			A30 G36 C20 T36	A40 G29 C19 T31
4	44/61, 82, 9	44/61			A30 G36 C20 T36	A41 G28 C19 T31
2	53, 91	91			A30 G36 C19 T37	A40 G29 C19 T31
1	2	2	Ft.	2003	A30 G36 C20 T36	A40 G29 C19 T31
2	3	3	Leonard		A30 G36 C20 T36	A40 G29 C19 T31
1	4	4	Wood		A30 G36 C19 T37	A41 G28 C19 T31
1	6	6	(Cultured)		A30 G36 C20 T36	A40 G29 C19 T31
11	25 or 75	75			A30 G36 C20 T36	A40 G29 C19 T31
1	25, 75, 33,	75			A30 G36 C19 T37	A40 G29 C19 T31
	34, 4, 52, 84
1	44/61 or 82	44/61			A30 G36 C20 T36	A41 G28 C19 T31
	or 9
2	5 or 58	5			A30 G36 C20 T36	A40 G29 C19 T31
3	1	1	Ft. Sill	2003	A30 G36 C19 T37	A40 G29 C19 T31
2	3	3	(Cultured)		A30 G36 C20 T36	A40 G29 C19 T31
1	4	4			A30 G36 C19 T37	A41 G28 C19 T31
1	28	28			A30 G36 C20 T36	A41 G28 C18 T32
1	3	3	Ft.	2003	A30 G36 C20 T36	A40 G29 C19 T31
1	4	4	Benning		A30 G36 C19 T37	A41 G28 C19 T31
3	6	6	(Cultured)		A30 G36 C20 T36	A40 G29 C19 T31
1	11	11			A30 G36 C20 T36	A40 G29 C19 T31
1	13	94**			A30 G36 C20 T36	A41 G28 C19 T31
1	44/61 or 82	82			A30 G36 C20 T36	A41 G28 C19 T31
	or 9
1	5 or 58	58			A30 G36 C20 T36	A40 G29 C19 T31
1	78 or 89	89			A30 G36 C20 T36	A41 G28 C19 T31
2	5 or 58	ND	Lackland	2003	A30 G36 C20 T36	A40 G29 C19 T31
1	2		AFB		A30 G36 C20 T36	A40 G29 C19 T31
1	81 or 90		(Throat		A30 G36 C20 T36	A40 G29 C19 T31
1	78		Swabs)		A30 G36 C20 T36	A41 G28 C19 T31
3***	No detection				No detection	No detection
7	3	ND	MCRD San	2002	A30 G36 C20 T36	A40 G29 C19 T31
1	3	ND	Diego		A30 G36 C20 T36	A40 G29 C19 T31
1	3	ND	(Throat		A30 G36 C20 T36	No detection
1	3	ND	Swabs)		No detection	A40 G29 C19 T31
2	3	ND			A30 G36 C20 T36	A40 G29 C19 T31
3	No detection	ND			No detection	No detection

TABLE 9C


Base Composition Analysis of Bioagent Identifying Amplicons of Group A Streptococcus
samples from Six Military Installations Obtained with Primer Pair Nos. 438 and 441

	emm-type by				gki	gtr
# of	Mass	emm-Gene	Location		(Primer Pair	((Primer Pair
Instances	Spectrometry	Sequencing	(sample)	Year	No. 442)	No. 443)

48	3	3	MCRD San	2002	A32 G35 C17 T32	A39 G28 C16 T32
2	6	6	Diego		A31 G35 C17 T33	A39 G28 C15 T33
1	28	28	(Cultured)		A30 G36 C17 T33	A39 G28 C16 T32
15	3	ND			A32 G35 C17 T32	A39 G28 C16 T32
6	3	3	NHRC San	2003	A32 G35 C17 T32	A39 G28 C16 T32
3	5, 58	5	Diego-		A30 G36 C20 T30	A39 G28 C15 T33
6	6	6	Archive		A31 G35 C17 T33	A39 G28 C15 T33
1	11	11	(Cultured)		A30 G36 C20 T30	A39 G28 C16 T32
3	12	12			A31 G35 C17 T33	A39 G28 C15 T33
1	22	22			A31 G35 C17 T33	A38 G29 C15 T33
3	25, 75	75			A30 G36 C17 T33	A39 G28 C15 T33
4	44/61, 82, 9	44/61			A30 G36 C18 T32	A39 G28 C15 T33
2	53, 91	91			A32 G35 C17 T32	A39 G28 C16 T32
1	2	2	Ft.	2003	A30 G36 C17 T33	A39 G28 C15 T33
2	3	3	Leonard		A32 G35 C17 T32	A39 G28 C16 T32
1	4	4	Wood		A31 G35 C17 T33	A39 G28 C15 T33
1	6	6	(Cultured)		A31 G35 C17 T33	A39 G28 C15 T33
11	25 or 75	75			A30 G36 C17 T33	A39 G28 C15 T33
1	25, 75, 33,	75			A30 G36 C17 T33	A39 G28 C15 T33
	34, 4, 52, 84
1	44/61 or 82	44/61			A30 G36 C18 T32	A39 G28 C15 T33
	or 9
2	5 or 58	5			A30 G36 C20 T30	A39 G28 C15 T33
3	1	1	Ft. Sill	2003	A30 G36 C18 T32	A39 G28 C15 T33
2	3	3	(Cultured)		A32 G35 C17 T32	A39 G28 C16 T32
1	4	4			A31 G35 C17 T33	A39 G28 C15 T33
1	28	28			A30 G36 C17 T33	A39 G28 C16 T32
1	3	3	Ft.	2003	A32 G35 C17 T32	A39 G28 C16 T32
1	4	4	Benning		A31 G35 C17 T33	A39 G28 C15 T33
3	6	6	(Cultured)		A31 G35 C17 T33	A39 G28 C15 T33
1	11	11			A30 G36 C20 T30	A39 G28 C16 T32
1	13	94**			A30 G36 C19 T31	A39 G28 C15 T33
1	44/61 or 82	82			A30 G36 C18 T32	A39 G28 C15 T33
	or 9
1	5 or 58	58			A30 G36 C20 T30	A39 G28 C15 T33
1	78 or 89	89			A30 G36 C18 T32	A39 G28 C15 T33
2	5 or 58	ND	Lackland	2003	A30 G36 C20 T30	A39 G28 C15 T33
1	2		AFB		A30 G36 C17 T33	A39 G28 C15 T33
1	81 or 90		(Throat		A30 G36 C17 T33	A39 G28 C15 T33
1	78		Swabs)		A30 G36 C18 T32	A39 G28 C15 T33
3***	No detection				No detection	No detection
7	3	ND	MCRD San	2002	A32 G35 C17 T32	A39 G28 C16 T32
1	3	ND	Diego		No detection	No detection
1	3	ND	(Throat		A32 G35 C17 T32	A39 G28 C16 T32
1	3	ND	Swabs)		A32 G35 C17 T32	No detection
2	3	ND			A32 G35 C17 T32	No detection
3	No detection	ND			No detection	No detection

Example 8

Design of Calibrant Polynucleotides Based on Bioagent Identifying Amplicons for Identification of Species of Bacteria (Bacterial Bioagent Identifying Amplicons)

This example describes the design of 19 calibrant polynucleotides based on bacterial bioagent identifying amplicons corresponding to the primers of the broad surveillance set (Table 5) and the Bacillus anthracis drill-down set (Table 6).
Calibration sequences were designed to simulate bacterial bioagent identifying amplicons produced by the T modified primer pairs shown in Tables 5 and 6 (primer names have the designation “TMOD”). The calibration sequences were chosen as a representative member of the section of bacterial genome from specific bacterial species which would be amplified by a given primer pair. The model bacterial species upon which the calibration sequences are based are also shown in Table 10. For example, the calibration sequence chosen to correspond to an amplicon produced by primer pair no. 361 is SEQ ID NO: 1445. In Table 10, the forward (_F) or reverse (_R) primer name indicates the coordinates of an extraction representing a gene of a standard reference bacterial genome to which the primer hybridizes e.g.: the forward primer name 16S_EC_—713_—732_TMOD_F indicates that the forward primer hybridizes to residues 713-732 of the gene encoding 16S ribosomal RNA in an E. coli reference sequence (in this case, the reference sequence is an extraction consisting of residues 4033120-4034661 of the genomic sequence of E. coli K12 (GenBank gi number 16127994). Additional gene coordinate reference information is shown in Table 11. The designation “TMOD” in the primer names indicates that the 5′ end of the primer has been modified with a non-matched template T residue which prevents the PCR polymerase from adding non-templated adenosine residues to the 5′ end of the amplification product, an occurrence which may result in miscalculation of base composition from molecular mass data (vide supra).

The 19 calibration sequences described in Tables 10 and 11 were combined into a single calibration polynucleotide sequence (SEQ ID NO: 1464—which is herein designated a “combination calibration polynucleotide”) which was then cloned into a pCR®-Blunt vector (Invitrogen, Carlsbad, Calif.). This combination calibration polynucleotide can be used in conjunction with the primers of Tables 5 or 6 as an internal standard to produce calibration amplicons for use in determination of the quantity of any bacterial bioagent. Thus, for example, when the combination calibration polynucleotide vector is present in an amplification reaction mixture, a calibration amplicon based on primer pair 346 (16S rRNA) will be produced in an amplification reaction with primer pair 346 and a calibration amplicon based on primer pair 363 (rpoC) will be produced with primer pair 363. Coordinates of each of the 19 calibration sequences within the calibration polynucleotide (SEQ ID NO: 1464) are indicated in Table 11.

TABLE 10


Bacterial Primer Pairs for Production of Bacterial Bioagent Identifying
Amplicons and Corresponding Representative Calibration Sequences

						Calibra-
		Forward		Reverse	Calibration	tion
		Primer		Primer	Sequence	Sequence
Primer		(SEQ ID		(SEQ ID	Model	(SEQ ID
Pair No.	Forward Primer Name	NO:)	Reverse Primer Name	NO:)	Species	NO:)

361	16S_EC_1090_1111_2_TMOD_F	697	16S_EC_1175_1196_TMOD_R	1398	Bacillus	1445
					anthracis
346	16S_EC_713_732_TMOD_F	202	16S_EC_789_809_TMOD_R	1110	Bacillus	1446
					anthracis
347	16S_EC_785_806_TMOD_F	560	16S_EC_880_897_TMOD_R	1278	Bacillus	1447
					anthracis
348	16S_EC_960_981_TMOD_F	706	16S_EC_1054_1073_TMOD_R	895	Bacillus	1448
					anthracis
349	23S_EC_1826_1843_TMOD_F	401	23S_EC_1906_1924_TMOD_R	1156	Bacillus	1449
					anthracis
360	23S_EC_2646_2667_TMOD_F	409	23S_EC_2745_2765_TMOD_R	1434	Bacillus	1450
					anthracis
350	CAPC_BA_274_303_TMOD_F	476	CAPC_BA_349_376_TMOD_R	1314	Bacillus	1451
					anthracis
351	CYA_BA_1353_1379_TMOD_F	355	CYA_BA_1448_1467_TMOD_R	1423	Bacillus	1452
					anthracis
352	INFB_EC_1365_1393_TMOD_F	687	INFB_EC_1439_1467_TMOD_R	1411	Bacillus	1453
					anthracis
353	LEF_BA_756_781_TMOD_F	220	LEF_BA_843_872_TMOD_R	1394	Bacillus	1454
					anthracis
356	RPLB_EC_650_679_TMOD_F	449	RPLB_EC_739_762_TMOD_R	1380	Clostridium	1455
					botulinum
449	RPLB_EC_690_710_F	309	RPLB_EC_737_758_R	1336	Clostridium	1456
					botulinum
359	RPOB_EC_1845_1866_TMOD_F	659	RPOB_EC_1909_1929_TMOD_R	1250	Yersinia	1457
					Pestis
362	RPOB_EC_3799_3821_TMOD_F	581	RPOB_EC_3862_3888_TMOD_R	1325	Burkholderia	1458
					mallei
363	RPOC_EC_2146_2174_TMOD_F	284	RPOC_EC_2227_2245_TMOD_R	898	Burkholderia	1459
					mallei
354	RPOC_EC_2218_2241_TMOD_F	405	RPOC_EC_2313_2337_TMOD_R	1072	Bacillus	1460
					anthracis
355	SSPE_BA_115_137_TMOD_F	255	SSPE_BA_197_222_TMOD_R	1402	Bacillus	1461
					anthracis
367	TUFB_EC_957_979_TMOD_F	308	TUFB_EC_1034_1058_TMOD_R	1276	Burkholderia	1462
					mallei
358	VALS_EC_1105_1124_TMOD_F	385	VALS_EC_1195_1218_TMOD_R	1093	Yersinia	1463
					Pestis

TABLE 11


Primer Pair Gene Coordinate References and Calibration Polynucleotide Sequence
Coordinates within the Combination Calibration Polynucleotide

				Coordinates of
	Gene Extraction			Calibration Sequence in
Bacterial	Coordinates	Reference GenBank GI		Combination Calibration
Gene and	of Genomic or Plasmid	No. of Genomic (G) or	Primer	Polynucleotide (SEQ ID
Species	Sequence	Plasmid (P) Sequence	Pair No.	NO: 1464)

16S E. coli	4033120 . . . 4034661	16127994 (G)	346	16 . . . 109
16S E. coli	4033120 . . . 4034661	16127994 (G)	347	83 . . . 190
16S E. coli	4033120 . . . 4034661	16127994 (G)	348	246 . . . 353
16S E. coli	4033120 . . . 4034661	16127994 (G)	361	368 . . . 469
23S E. coli	4166220 . . . 4169123	16127994 (G)	349	743 . . . 837
23S E. coli	4166220 . . . 4169123	16127994 (G)	360	865 . . . 981
rpoB E. coli.	4178823 . . . 4182851	16127994 (G)	359	1591 . . . 1672
	(complement strand)
rpoB E. coli	4178823 . . . 4182851	16127994 (G)	362	2081 . . . 2167
	(complement strand)
rpoC E. coli	4182928 . . . 4187151	16127994 (G)	354	1810 . . . 1926
rpoC E. coli	4182928 . . . 4187151	16127994 (G)	363	2183 . . . 2279
infB E. coli	3313655 . . . 3310983	16127994 (G)	352	1692 . . . 1791
	(complement strand)
tufB E. coli	4173523 . . . 4174707	16127994 (G)	367	2400 . . . 2498
rplB E. coli	3449001 . . . 3448180	16127994 (G)	356	1945 . . . 2060
rplB E. coli	3449001 . . . 3448180	16127994 (G)	449	1986 . . . 2055
valS E. coli	4481405 . . . 4478550	16127994 (G)	358	1462 . . . 1572
	(complement strand)
capC	56074 . . . 55628 (complement	6470151 (P)	350	2517 . . . 2616
B. anthracis	strand)
cya	156626 . . . 154288	4894216 (P)	351	1338 . . . 1449
B. anthracis	(complement strand)
lef	127442 . . . 129921	4894216 (P)	353	1121 . . . 1234
B. anthracis
sspE	226496 . . . 226783	30253828 (G)	355	1007-1104
B. anthracis

Example 9

Use of a Calibration Polynucleotide for Determining the Quantity of Bacillus Anthracis in a Sample Containing a Mixture of Microbes

The process described in this example is shown in FIG. 2. The capC gene is a gene involved in capsule synthesis which resides on the pX02 plasmid of Bacillus anthracis. Primer pair number 350 (see Tables 10 and 11) was designed to identify Bacillus anthracis via production of a bacterial bioagent identifying amplicon. Known quantities of the combination calibration polynucleotide vector described in Example 8 were added to amplification mixtures containing bacterial bioagent nucleic acid from a mixture of microbes which included the Ames strain of Bacillus anthracis. Upon amplification of the bacterial bioagent nucleic acid and the combination calibration polynucleotide vector with primer pair no. 350, bacterial bioagent identifying amplicons and calibration amplicons were obtained and characterized by mass spectrometry. A mass spectrum measured for the amplification reaction is shown in FIG. 7. The molecular masses of the bioagent identifying amplicons provided the means for identification of the bioagent from which they were obtained (Ames strain of Bacillus anthracis) and the molecular masses of the calibration amplicons provided the means for their identification as well. The relationship between the abundance (peak height) of the calibration amplicon signals and the bacterial bioagent identifying amplicon signals provides the means of calculation of the copies of the pX02 plasmid of the Ames strain of Bacillus anthracis. Methods of calculating quantities of molecules based on internal calibration procedures are well known to those of ordinary skill in the art.
Averaging the results of 10 repetitions of the experiment described above, enabled a calculation that indicated that the quantity of Ames strain of Bacillus anthracis present in the sample corresponds to approximately 10 copies of pX02 plasmid.

Example 10

Triangulation Genotyping Analysis of Campylobacter Species

A series of triangulation genotyping analysis primers were designed as described in Example 1 with the objective of identification of different strains of Campylobacter jejuni. The primers are listed in Table 12 with the designation “CJST_CJ.” Housekeeping genes to which the primers hybridize and produce bioagent identifying amplicons include: tkt (transketolase), glyA (serine hydroxymethyltransferase), gltA (citrate synthase), aspA (aspartate ammonia lyase), glnA (glutamine synthase), pgm (phosphoglycerate mutase), and uncA (ATP synthetase alpha chain).

TABLE 12


Campylobacter Genotyping Primer Pairs

Primer
Pair		Forward Primer		Reverse Primer
No.	Forward Primer Name	(SEQ ID NO:)	Reverse Primer Name	(SEQ ID NO:)	Target Gene

1053	CJST_CJ_1080_1110_F	681	CJST_CJ_1166_1198_R	1022	gltA
1047	CJST_CJ_584_616_F	315	CJST_CJ_663_692_R	1379	glnA
1048	CJST_CJ_360_394_F	346	CJST_CJ_442_476_R	955	aspA
1049	CJST_CJ_2636_2668_F	504	CJST_CJ_2753_2777_R	1409	tkt
1054	CJST_CJ_2060_2090_F	323	CJST_CJ_2148_2174_R	1068	pgm
1064	CJST_CJ_1680_1713_F	479	CJST_CJ_1795_1822_R	938	glyA

The primers were used to amplify nucleic acid from 50 food product samples provided by the USDA, 25 of which contained Campylobacter jejuni and 25 of which contained Campylobacter coli. Primers used in this study were developed primarily for the discrimination of Campylobacter jejuni clonal complexes and for distinguishing Campylobacter jejuni from Campylobacter coli. Finer discrimination between Campylobacter coli types is also possible by using specific primers targeted to loci where closely-related Campylobacter coli isolates demonstrate polymorphisms between strains. The conclusions of the comparison of base composition analysis with sequence analysis are shown in Tables 13A-C.

TABLE 13A


Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-down
MLST Primer Pair Nos: 1048 and 1047

						Base	Base
						Composition of	Composition of
			MLST type or			Bioagent	Bioagent
			Clonal	MLST Type		Identifying	Identifying
			Complex by	or Clonal		Amplicon	Amplicon
			Base	Complex by		Obtained with	Obtained with
		Isolate	Composition	Sequence		Primer Pair No:	Primer Pair
Group	Species	origin	analysis	analysis	Strain	1048 (aspA)	No: 1047 (glnA)

J-1	C. jejuni	Goose	ST 690/	ST 991	RM3673	A30 G25 C16 T46	A47 G21 C16 T25
			692/707/991
J-2	C. jejuni	Human	Complex	ST 356,	RM4192	A30 G25 C16 T46	A48 G21 C17 T23
			206/48/353	complex
				353
J-3	C. jejuni	Human	Complex	ST 436	RM4194	A30 G25 C15 T47	A48 G21 C18 T22
			354/179
J-4	C. jejuni	Human	Complex 257	ST 257,	RM4197	A30 G25 C16 T46	A48 G21 C18 T22
				complex
				257
J-5	C. jejuni	Human	Complex 52	ST 52,	RM4277	A30 G25 C16 T46	A48 G21 C17 T23
				complex 52
J-6	C. jejuni	Human	Complex 443	ST 51,	RM4275	A30 G25 C15 T47	A48 G21 C17 T23
				complex	RM4279	A30 G25 C15 T47	A48 G21 C17 T23
				443
J-7	C. jejuni	Human	Complex 42	ST 604,	RM1864	A30 G25 C15 T47	A48 G21 C18 T22
				complex 42
J-8	C. jejuni	Human	Complex	ST 362,	RM3193	A30 G25 C15 T47	A48 G21 C18 T22
			42/49/362	complex
				362
J-9	C. jejuni	Human	Complex	ST 147,	RM3203	A30 G25 C15 T47	A47 G21 C18 T23
			45/283	Complex 45
	C. jejuni	Human	Consistent	ST 828	RM4183	A31 G27 C20 T39	A48 G21 C16 T24
C-1	C. coli		with 74	ST 832	RM1169	A31 G27 C20 T39	A48 G21 C16 T24
			closely	ST 1056	RM1857	A31 G27 C20 T39	A48 G21 C16 T24
		Poultry	related	ST 889	RM1166	A31 G27 C20 T39	A48 G21 C16 T24
			sequence	ST 829	RM1182	A31 G27 C20 T39	A48 G21 C16 T24
			types (none	ST 1050	RM1518	A31 G27 C20 T39	A48 G21 C16 T24
			belong to a	ST 1051	RM1521	A31 G27 C20 T39	A48 G21 C16 T24
			clonal	ST 1053	RM1523	A31 G27 C20 T39	A48 G21 C16 T24
			complex)	ST 1055	RM1527	A31 G27 C20 T39	A48 G21 C16 T24
				ST 1017	RM1529	A31 G27 C20 T39	A48 G21 C16 T24
				ST 860	RM1840	A31 G27 C20 T39	A48 G21 C16 T24
				ST 1063	RM2219	A31 G27 C20 T39	A48 G21 C16 T24
				ST 1066	RM2241	A31 G27 C20 T39	A48 G21 C16 T24
				ST 1067	RM2243	A31 G27 C20 T39	A48 G21 C16 T24
				ST 1068	RM2439	A31 G27 C20 T39	A48 G21 C16 T24
		Swine		ST 1016	RM3230	A31 G27 C20 T39	A48 G21 C16 T24
				ST 1069	RM3231	A31 G27 C20 T39	A48 G21 C16 T24
				ST 1061	RM1904	A31 G27 C20 T39	A48 G21 C16 T24
		Unknown		ST 825	RM1534	A31 G27 C20 T39	A48 G21 C16 T24
				ST 901	RM1505	A31 G27 C20 T39	A48 G21 C16 T24
C-2	C. coli	Human	ST 895	ST 895	RM1532	A31 G27 C19 T40	A48 G21 C16 T24
C-3	C. coli	Poultry	Consistent	ST 1064	RM2223	A31 G27 C20 T39	A48 G21 C16 T24
			with 63	ST 1082	RM1178	A31 G27 C20 T39	A48 G21 C16 T24
			closely	ST 1054	RM1525	A31 G27 C20 T39	A48 G21 C16 T24
			related	ST 1049	RM1517	A31 G27 C20 T39	A48 G21 C16 T24
		Marmoset	sequence	ST 891	RM1531	A31 G27 C20 T39	A48 G21 C16 T24
			types (none
			belong to a
			clonal
			complex)

TABLE 13B


Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-
down MLST Primer Pair Nos: 1053 and 1064

						Base	Base
						Composition of	Composition of
			MLST type or			Bioagent	Bioagent
			Clonal	MLST Type		Identifying	Identifying
			Complex by	or Clonal		Amplicon	Amplicon
			Base	Complex by		Obtained with	Obtained with
		Isolate	Composition	Sequence		Primer Pair	Primer Pair
Group	Species	origin	analysis	analysis	Strain	No: 1053 (gltA)	No: 1064 (glyA)

J-1	C. jejuni	Goose	ST 690/	ST 991	RM3673	A24 G25 C23 T47	A40 G29 C29 T45
			692/707/991
J-2	C. jejuni	Human	Complex	ST 356,	RM4192	A24 G25 C23 T47	A40 G29 C29 T45
			206/48/353	complex
				353
J-3	C. jejuni	Human	Complex	ST 436	RM4194	A24 G25 C23 T47	A40 G29 C29 T45
			354/179
J-4	C. jejuni	Human	Complex 257	ST 257,	RM4197	A24 G25 C23 T47	A40 G29 C29 T45
				complex
				257
J-5	C. jejuni	Human	Complex 52	ST 52,	RM4277	A24 G25 C23 T47	A39 G30 C26 T48
				complex 52
J-6	C. jejuni	Human	Complex 443	ST 51,	RM4275	A24 G25 C23 T47	A39 G30 C28 T46
				complex	RM4279	A24 G25 C23 T47	A39 G30 C28 T46
				443
J-7	C. jejuni	Human	Complex 42	ST 604,	RM1864	A24 G25 C23 T47	A39 G30 C26 T48
				complex 42
J-8	C. jejuni	Human	Complex	ST 362,	RM3193	A24 G25 C23 T47	A38 G31 C28 T46
			42/49/362	complex
				362
J-9	C. jejuni	Human	Complex	ST 147,	RM3203	A24 G25 C23 T47	A38 G31 C28 T46
			45/283	Complex 45
	C. jejuni	Human	Consistent	ST 828	RM4183	A23 G24 C26 T46	A39 G30 C27 T47
C-1	C. coli		with 74	ST 832	RM1169	A23 G24 C26 T46	A39 G30 C27 T47
			closely	ST 1056	RM1857	A23 G24 C26 T46	A39 G30 C27 T47
		Poultry	related	ST 889	RM1166	A23 G24 C26 T46	A39 G30 C27 T47
			sequence	ST 829	RM1182	A23 G24 C26 T46	A39 G30 C27 T47
			types (none	ST 1050	RM1518	A23 G24 C26 T46	A39 G30 C27 T47
			belong to a	ST 1051	RM1521	A23 G24 C26 T46	A39 G30 C27 T47
			clonal	ST 1053	RM1523	A23 G24 C26 T46	A39 G30 C27 T47
			complex)	ST 1055	RM1527	A23 G24 C26 T46	A39 G30 C27 T47
				ST 1017	RM1529	A23 G24 C26 T46	A39 G30 C27 T47
				ST 860	RM1840	A23 G24 C26 T46	A39 G30 C27 T47
				ST 1063	RM2219	A23 G24 C26 T46	A39 G30 C27 T47
				ST 1066	RM2241	A23 G24 C26 T46	A39 G30 C27 T47
				ST 1067	RM2243	A23 G24 C26 T46	A39 G30 C27 T47
				ST 1068	RM2439	A23 G24 C26 T46	A39 G30 C27 T47
		Swine		ST 1016	RM3230	A23 G24 C26 T46	A39 G30 C27 T47
				ST 1069	RM3231	A23 G24 C26 T46	NO DATA
				ST 1061	RM1904	A23 G24 C26 T46	A39 G30 C27 T47
		Unknown		ST 825	RM1534	A23 G24 C26 T46	A39 G30 C27 T47
				ST 901	RM1505	A23 G24 C26 T46	A39 G30 C27 T47
C-2	C. coli	Human	ST 895	ST 895	RM1532	A23 G24 C26 T46	A39 G30 C27 T47
C-3	C. coli	Poultry	Consistent	ST 1064	RM2223	A23 G24 C26 T46	A39 G30 C27 T47
			with 63	ST 1082	RM1178	A23 G24 C26 T46	A39 G30 C27 T47
			closely	ST 1054	RM1525	A23 G24 C25 T47	A39 G30 C27 T47
			related	ST 1049	RM1517	A23 G24 C26 T46	A39 G30 C27 T47
		Marmoset	sequence	ST 891	RM1531	A23 G24 C26 T46	A39 G30 C27 T47
			types (none
			belong to a
			clonal
			complex)

TABLE 13C


Results of Base Composition Analysis of 50 Campylobacter Samples with Drill-
down MLST Primer Pair Nos: 1054 and 1049

						Base	Base
						Composition of	Composition of
			MLST type or			Bioagent	Bioagent
			Clonal	MLST Type		Identifying	Identifying
			Complex by	or Clonal		Amplicon	Amplicon
			Base	Complex by		Obtained with	Obtained with
		Isolate	Composition	Sequence		Primer Pair No:	Primer Pair
Group	Species	origin	analysis	analysis	Strain	1054 (pgm)	No: 1049 (tkt)

J-1	C. jejuni	Goose	ST 690/	ST 991	RM3673	A26 G33 C18 T38	A41 G28 C35 T38
			692/707/991
J-2	C. jejuni	Human	Complex	ST 356,	RM4192	A26 G33 C19 T37	A41 G28 C36 T37
			206/48/353	complex
				353
J-3	C. jejuni	Human	Complex	ST 436	RM4194	A27 G32 C19 T37	A42 G28 C36 T36
			354/179
J-4	C. jejuni	Human	Complex 257	ST 257,	RM4197	A27 G32 C19 T37	A41 G29 C35 T37
				complex
				257
J-5	C. jejuni	Human	complex 52	ST 52,	RM4277	A26 G33 C18 T38	A41 G28 C36 T37
				complex 52
J-6	C. jejuni	Human	Complex 443	ST 51,	RM4275	A27 G31 C19 T38	A41 G28 C36 T37
				complex	RM4279	A27 G31 C19 T38	A41 G28 C36 T37
				443
J-7	C. jejuni	Human	Complex 42	ST 604,	RM1864	A27 G32 C19 T37	A42 G28 C35 T37
				complex 42
J-8	C. jejuni	Human	Complex	ST 362,	RM3193	A26 G33 C19 T37	A42 G28 C35 T37
			42/49/362	complex
				362
J-9	C. jejuni	Human	Complex	ST 147,	RM3203	A28 G31 C19 T37	A43 G28 C36 T35
			45/283	Complex 45
	C. jejuni	Human	Consistent	ST 828	RM4183	A27 G30 C19 T39	A46 G28 C32 T36
C-1	C. coli		with 74	ST 832	RM1169	A27 G30 C19 T39	A46 G28 C32 T36
			closely	ST 1056	RM1857	A27 G30 C19 T39	A46 G28 C32 T36
		Poultry	related	ST 889	RM1166	A27 G30 C19 T39	A46 G28 C32 T36
			sequence	ST 829	RM1182	A27 G30 C19 T39	A46 G28 C32 T36
			types (none	ST 1050	RM1518	A27 G30 C19 T39	A46 G28 C32 T36
			belong to a	ST 1051	RM1521	A27 G30 C19 T39	A46 G28 C32 T36
			clonal	ST 1053	RM1523	A27 G30 C19 T39	A46 G28 C32 T36
			complex)	ST 1055	RM1527	A27 G30 C19 T39	A46 G28 C32 T36
				ST 1017	RM1529	A27 G30 C19 T39	A46 G28 C32 T36
				ST 860	RM1840	A27 G30 C19 T39	A46 G28 C32 T36
				ST 1063	RM2219	A27 G30 C19 T39	A46 G28 C32 T36
				ST 1066	RM2241	A27 G30 C19 T39	A46 G28 C32 T36
				ST 1067	RM2243	A27 G30 C19 T39	A46 G28 C32 T36
				ST 1068	RM2439	A27 G30 C19 T39	A46 G28 C32 T36
		Swine		ST 1016	RM3230	A27 G30 C19 T39	A39 G30 C27 T47
				ST 1069	RM3231	A27 G30 C19 T39	A46 G28 C32 T36
				ST 1061	RM1904	A27 G30 C19 T39	A46 G28 C32 T36
		Unknown		ST 825	RM1534	A27 G30 C19 T39	A46 G28 C32 T36
				ST 901	RM1505	A27 G30 C19 T39	A46 G28 C32 T36
C-2	C. coli	Human	ST 895	ST 895	RM1532	A27 G30 C19 T39	A46 G28 C32 T36
C-3	C. coli	Poultry	Consistent	ST 1064	RM2223	A27 G30 C19 T39	A46 G28 C32 T36
			with 63	ST 1082	RM1178	A27 G30 C19 T39	A46 G28 C32 T36
			closely	ST 1054	RM1525	A27 G30 C19 T39	A46 G28 C32 T36
			related	ST 1049	RM1517	A27 G30 C19 T39	A46 G28 C32 T36
		Marmoset	sequence	ST 891	RM1531	A27 G30 C19 T39	A46 G28 C32 T36
			types (none
			belong to a
			clonal
			complex)

The base composition analysis method was successful in identification of 12 different strain groups. Campylobacter jejuni and Campylobacter coli are generally differentiated by all loci. Ten clearly differentiated Campylobacter jejuni isolates and 2 major Campylobacter coli groups were identified even though the primers were designed for strain typing of Campylobacter jejuni. One isolate (RM4183) which was designated as Campylobacter jejuni was found to group with Campylobacter coli and also appears to actually be Campylobacter coli by full MLST sequencing.

Example 11

Identification of Acinetobacter baumannii Using Broad Range Survey and Division-Wide Primers in Epidemiological Surveillance

To test the capability of the broad range survey and division-wide primer sets of Table 5 in identification of Acinetobacter species, 183 clinical samples were obtained from individuals participating in, or in contact with individuals participating in Operation Iraqi Freedom (including US service personnel, US civilian patients at the Walter Reed Army Institute of Research (WRAIR), medical staff, Iraqi civilians and enemy prisoners. In addition, 34 environmental samples were obtained from hospitals in Iraq, Kuwait, Germany, the United States and the USNS Comfort, a hospital ship.
Upon amplification of nucleic acid obtained from the clinical samples, primer pairs 346-349, 360, 361, 354, 362 and 363 (Table 5) all produced bacterial bioagent amplicons which identified Acinetobacter baumannii in 215 of 217 samples. The organism Klebsiella pneumoniae was identified in the remaining two samples. In addition, 14 different strain types (containing single nucleotide polymorphisms relative to a reference strain of Acinetobacter baumannii) were identified and assigned arbitrary numbers from 1 to 14. Strain type 1 was found in 134 of the sample isolates and strains 3 and 7 were found in 46 and 9 of the isolates respectively.
The epidemiology of strain type 7 of Acinetobacter baumannii was investigated. Strain 7 was found in 4 patients and 5 environmental samples (from field hospitals in Iraq and Kuwait). The index patient infected with strain 7 was a pre-war patient who had a traumatic amputation in March of 2003 and was treated at a Kuwaiti hospital. The patient was subsequently transferred to a hospital in Germany and then to WRAIR. Two other patients from Kuwait infected with strain 7 were found to be non-infectious and were not further monitored. The fourth patient was diagnosed with a strain 7 infection in September of 2003 at WRAIR. Since the fourth patient was not related involved in Operation Iraqi Freedom, it was inferred that the fourth patient was the subject of a nosocomial infection acquired at WRAIR as a result of the spread of strain 7 from the index patient.
The epidemiology of strain type 3 of Acinetobacter baumannii was also investigated. Strain type 3 was found in 46 samples, all of which were from patients (US service members, Iraqi civilians and enemy prisoners) who were treated on the USNS Comfort hospital ship and subsequently returned to Iraq or Kuwait. The occurrence of strain type 3 in a single locale may provide evidence that at least some of the infections at that locale were a result of nosocomial infections.
This example thus illustrates an embodiment of the present invention wherein the methods of analysis of bacterial bioagent identifying amplicons provide the means for epidemiological surveillance.

Example 12

Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Acinetobacter baumanii

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, an additional 21 primer pairs were selected based on analysis of housekeeping genes of the genus Acinetobacter. Genes to which the drill-down triangulation genotyping analysis primers hybridize for production of bacterial bioagent identifying amplicons include anthranilate synthase component I (trpE), adenylate kinase (adk), adenine glycosylase (mutY), fulmarate hydratase (fumC), and pyrophosphate phospho-hydratase (ppa). These 21 primer pairs are indicated with reference to sequence listings in Table 14. Primer pair numbers 1151-1154 hybridize to and amplify segments of trpE. Primer pair numbers 1155-1157 hybridize to and amplify segments of adk. Primer pair numbers 1158-1164 hybridize to and amplify segments of mutY. Primer pair numbers 1165-1170 hybridize to and amplify segments of fumC. Primer pair number 1171 hybridizes to and amplifies a segment of ppa. Primer pair numbers: 2846-2848 hybridize to and amplify segments of the parC gene of DNA topoisomerase which include a codon known to confer quinolone drug resistance upon sub-types of Acinetobacter baumannii. Primer pair numbers 2852-2854 hybridize to and amplify segments of the gyrA gene of DNA gyrase which include a codon known to confer quinolone drug resistance upon sub-types of Acinetobacter baumannii. Primer pair numbers 2922 and 2972 are speciating primers which are useful for identifying different species members of the genus Acinetobacter. The primer names given in Table 14A (with the exception of primer pair numbers 2846-2848, 2852-2854) indicate the coordinates to which the primers hybridize to a reference sequence which comprises a concatenation of the genes TrpE, efp (elongation factor p), adk, mutT, fumC, and ppa. For example, the forward primer of primer pair 1151 is named AB_MLST-11-OIF007_—62_—91_F because it hybridizes to the Acinetobacter primer reference sequence of strain type 11 in sample 007 of Operation Iraqi Freedom (OIF) at positions 62 to 91. DNA was sequenced from strain type 11 and from this sequence data and an artificial concatenated sequence of partial gene extractions was assembled for use in design of the triangulation genotyping analysis primers. The stretches of arbitrary residues “N”s in the concatenated sequence were added for the convenience of separation of the partial gene extractions (40N for AB_MLST (SEQ ID NO: 1444)).

The hybridization coordinates of primer pair numbers 2846-2848 are with respect to GenBank Accession number X95819. The hybridization coordinates of primer pair numbers 2852-2854 are with respect to GenBank Accession number AY642140. Sequence residue “I” appearing in the forward and reverse primers of primer pair number 2972 represents inosine.

TABLE 14A


Triangulation Genotyping Analysis Primer Pairs for Identification of Sub-species
characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter

Primer		Forward Primer		Reverse Primer
Pair No.	Forward Primer Name	(SEQ ID NO:)	Reverse Primer Name	(SEQ ID NO:)

1151	AB_MLST-11-OIF007_62_91_F	454	AB_MLST-11-OIF007_169_203_R	1418
1152	AB_MLST-11-OIF007_185_214_F	243	AB_MLST-11-OIF007_291_324_R	969
1153	AB_MLST-11-OIF007_260_289_F	541	AB_MLST-11-OIF007_364_393_R	1400
1154	AB_MLST-11-OIF007_206_239_F	436	AB_MLST-11-OIF007_318_344_R	1036
1155	AB_MLST-11-OIF007_522_552_F	378	AB_MLST-11-OIF007_587_610_R	1392
1156	AB_MLST-11-OIF007_547_571_F	250	AB_MLST-11-OIF007_656_686_R	902
1157	AB_MLST-11-OIF007_601_627_F	256	AB_MLST-11-OIF007_710_736_R	881
1158	AB_MLST-11-OIF007_1202_1225_F	384	AB_MLST-11-OIF007_1266_1296_R	878
1159	AB_MLST-11-OIF007_1202_1225_F	384	AB_MLST-11-OIF007_1299_1316_R	1199
1160	AB_MLST-11-OIF007_1234_1264_F	694	AB_MLST-11-OIF007_1335_1362_R	1215
1161	AB_MLST-11-OIF007_1327_1356_F	225	AB_MLST-11-OIF007_1422_1448_R	1212
1162	AB_MLST-11-OIF007_1345_1369_F	383	AB_MLST-11-OIF007_1470_1494_R	1083
1163	AB_MLST-11-OIF007_1351_1375_F	662	AB_MLST-11-OIF007_1470_1494_R	1083
1164	AB_MLST-11-OIF007_1387_1412_F	422	AB_MLST-11-OIF007_1470_1494_R	1083
1165	AB_MLST-11-OIF007_1542_1569_F	194	AB_MLST-11-OIF007_1656_1680_R	1173
1166	AB_MLST-11-OIF007_1566_1593_F	684	AB_MLST-11-OIF007_1656_1680_R	1173
1167	AB_MLST-11-OIF007_1611_1638_F	375	AB_MLST-11-OIF007_1731_1757_R	890
1168	AB_MLST-11-OIF007_1726_1752_F	182	AB_MLST-11-OIF007_1790_1821_R	1195
1169	AB_MLST-11-OIF007_1792_1826_F	656	AB_MLST-11-OIF007_1876_1909_R	1151
1170	AB_MLST-11-OIF007_1792_1826_F	656	AB_MLST-11-OIF007_1895_1927_R	1224
1171	AB_MLST-11-OIF007_1970_2002_F	618	AB_MLST-11-OIF007_2097_2118_R	1157
2846	PARC_X95819_33_58_F	302	PARC_X95819_121_153_R	852
2847	PARC_X95819_33_58_F	199	PARC_X95819_157_178_R	889
2848	PARC_X95819_33_58_F	596	PARC_X95819_97_128_R	1169
2852	GYRA_AY642140_−1_24_F	150	GYRA_AY642140_71_100_R	1242
2853	GYRA_AY642140_26_54_F	166	GYRA_AY642140_121_146_R	1069
2854	GYRA_AY642140_26_54_F	166	GYRA_AY642140_58_89_R	1168
2922	AB_MLST-11-OIF007_991_1018_F	583	AB_MLST-11-OIF007_1110_1137_R	923
2972	AB_MLST-11-OIF007_1007_1034_F	592	AB_MLST-11-OIF007_1126_1153_R	924

TABLE 14B


Triangulation Genotyping Analysis Primer Pairs for Identification of Sub-species
characteristics (Strain Type) of Members of the Bacterial Genus Acinetobacter

	Forward
Primer	Primer		Reverse
Pair	(SEQ ID		Primer
No.	NO:)	SEQUENCE	(SEQ ID NO:)	SEQUENCE

1151	454	TGAGATTGCTGAACATTTAATGCTGATTGA	1418	TTGTACATTTGAAACAATATGCATGACATGTGAAT

1152	243	TATTGTTTCAAATGTACAAGGTGAAGTGCG	969	TCACAGGTTCTACTTCATCAATAATTTCCATTGC

1153	541	TGGAACGTTATCAGGTGCCCCAAAAATTCG	1400	TTGCAATCGACATATCCATTTCACCATGCC

1154	436	TGAAGTGCGTGATGATATCGATGCACTTGATGTA	1036	TCCGCCAAAAACTCCCCTTTTCACAGG

1155	378	TCGGTTTAGTAAAAGAACGTATTGCTCAACC	1392	TTCTGCTTGAGGAATAGTGCGTGG

1156	250	TCAACCTGACTGCGTGAATGGTTGT	902	TACGTTCTACGATTTCTTCATCAGGTACATC

1157	256	TCAAGCAGAAGCTTTGGAAGAAGAAGG	881	TACAACGTGATAAACACGACCAGAAGC

1158	384	TCGTGCCCGCAATTTGCATAAAGC	878	TAATGCCGGGTAGTGCAATCCATTCTTCTAG

1159	384	TCGTGCCCGCAATTTGCATAAAGC	1199	TGCACCTGCGGTCGAGCG

1160	694	TTGTAGCACAGCAAGGCAAATTTCCTGAAAC	1215	TGCCATCCATAATCACGCCATACTGACG

1181	225	TAGGTTTACGTCAGTATGGCGTGATTATGG	1212	TGCCAGTTTCCACATTTCACGTTCGTG

1162	383	TCGTGATTATGGATGGCAACGTGAA	1083	TCGCTTGAGTGTAGTCATGATTGCG

1163	662	TTATGGATGGCAACGTGAAACGCGT	1083	TCGCTTGAGTGTAGTCATGATTGCG

1164	422	TCTTTGCCATTGAAGATGACTTAAGC	1083	TCGCTTGAGTGTAGTCATGATTGCG

1165	194	TACTAGCGGTAAGCTTAAACAAGATTGC	1173	TGAGTCGGGTTCACTTTACCTGGCA

1166	684	TTGCCAATGATATTCGTTGGTTAGCAAG	1173	TGAGTCGGGTTCACTTTACCTGGCA

1167	375	TCGGCGAAATCCGTATTCCTGAAAATGA	890	TACCGGAAGCACCAGCGACATTAATAG

1168	182	TACCACTATTAATGTCGCTGGTGCTTC	1195	TGCAACTGAATAGATTGCAGTAAGTTATAAGC

1169	656	TTATAACTTACTGCAATCTATTCAGTTGCTTGGTG	1151	TGAATTATGCAAGAAGTGATCAATTTTCTCACGA

1170	656	TTATAACTTACTGCAATCTATTCAGTTGCTTGGTG	1224	TGCCGTAACTAACATAAGAGAATTATGCAAGAA

1171	618	TGGTTATGTACCAAATACTTTGTCTGAAGATGG	1157	TGACGGCATCGATACCACCGTC

2846	302	TCCAAAAAAATCAGCGCGTACAGTGG	852	TAAAGGATAGCGGTAACTAAATGGCTGAGCCAT

2847	199	TACTTGGTAAATACCACCCACATGGTGA	889	TACCCCAGTTCCCCTGACCTTC

2848	596	TGGTAAATACCACCCACATGGTGAC	1169	TGAGCCATGAGTACCATGGCTTCATAACATGC

2852	150	TAAATGTGCCCGTGTCGTTGGTGAC	1242	TGCTAAAGTCTTGAGCCATACGAACAATGG

2853	166	TAATCGGTAAATATCACCCGCATGGTGAC	1069	TCGATCGAACCGAAGTTACCCTGACC

2854	166	TAATCGGTAAATATCACCCGCATGGTGAC	1168	TGAGCCATACGAACAATGGTTTCATAAACAGC

2922	583	TGGGCGATGCTGCGAAATGGTTAAAAGA	923	TAGTATCACCACGTACACCCGGATCAGT

2972	592	TGGGIGATGCTGCIAAATGGTTAAAAGA	924	TAGTATCACCACGTACICCIGGATCAGT

Analysis of bioagent identifying amplicons obtained using the primers of Table 14B for over 200 samples from Operation Iraqi Freedom resulted in the identification of 50 distinct strain type clusters. The largest cluster, designated strain type 11 (ST11) includes 42 sample isolates, all of which were obtained from US service personnel and Iraqi civilians treated at the 28^thCombat Support Hospital in Baghdad. Several of these individuals were also treated on the hospital ship USNS Comfort. These observations are indicative of significant epidemiological correlation/linkage.
All of the sample isolates were tested against a broad panel of antibiotics to characterize their antibiotic resistance profiles. As an example of a representative result from antibiotic susceptibility testing, ST11 was found to consist of four different clusters of isolates, each with a varying degree of sensitivity/resistance to the various antibiotics tested which included penicillins, extended spectrum penicillins, cephalosporins, carbepenem, protein synthesis inhibitors, nucleic acid synthesis inhibitors, anti-metabolites, and anti-cell membrane antibiotics. Thus, the genotyping power of bacterial bioagent identifying amplicons, particularly drill-down bacterial bioagent identifying amplicons, has the potential to increase the understanding of the transmission of infections in combat casualties, to identify the source of infection in the environment, to track hospital transmission of nosocomial infections, and to rapidly characterize drug-resistance profiles which enable development of effective infection control measures on a time-scale previously not achievable.

Example 13

Triangulation Genotyping Analysis and Codon Analysis of Acinetobacter baumannii Samples from Two Health Care Facilities

In this investigation, 88 clinical samples were obtained from Walter Reed Hospital and 95 clinical samples were obtained from Northwestern Medical Center. All samples from both healthcare facilities were suspected of containing sub-types of Acinetobacter baumannii, at least some of which were expected to be resistant to quinolone drugs. Each of the 183 samples was analyzed by the method of the present invention. DNA was extracted from each of the samples and amplified with eight triangulation genotyping analysis primer pairs represented by primer pair numbers: 1151, 1156, 1158, 1160, 1165, 1167, 1170, and 1171. The DNA was also amplified with speciating primer pair number 2922 and codon analysis primer pair numbers 2846-2848 which interrogate a codon present in the parc gene, and primer pair numbers 2852-2854 which bracket a codon present in the gyrA gene. The parc and gyrA codon mutations are both responsible for causing drug resistance in Acinetobacter baumannii. During evolution of drug resistant strains, the gyrA mutation usually occurs before the parC mutation. Amplification products were measured by ESI-TOF mass spectrometry as indicated in Example 4. The base compositions of the amplification products were calculated from the average molecular masses of the amplification products and are shown in Tables 15-18. The entries in each of the tables are grouped according to strain type number, which is an arbitrary number assigned to Acinetobacter baumannii strains in the order of observance beginning from the triangulation genotyping analysis OIF genotyping study described in Example 12. For example, strain type 11 which appears in samples from the Walter Reed Hospital is the same strain as the strain type 11 mentioned in Example 12. Ibis# refers to the order in which each sample was analyzed. Isolate refers to the original sample isolate numbering system used at the location from which the samples were obtained (either Walter Reed Hospital or Northwestern Medical Center). ST=strain type. ND=not detected. Base compositions highlighted with bold type indicate that the base composition is a unique base composition for the amplification product obtained with the pair of primers indicated.

TABLE 15A


Base Compositions of Amplification Products of 88 A. baumannii Samples Obtained from
Walter Reed Hospital and Amplified with Codon Analysis Primer Pairs Targeting the gyrA Gene

				PP No: 2852	PP No: 2853	PP No: 2854
Species	Ibis#	Isolate	ST	gyrA	gyrA	gyrA

A. baumannii	20	1082	1	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. baumannii	13	854	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	22	1162	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	27	1230	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	31	1367	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	37	1459	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	55	1700	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	64	1777	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	73	1861	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	74	1877	10	ND	A29G28C21T43	A17G13C13T21
A. baumannii	86	1972	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	3	684	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	6	720	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	7	726	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	19	1079	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	21	1123	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	23	1188	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	33	1417	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	34	1431	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	38	1496	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	40	1523	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	42	1640	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	50	1666	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	51	1668	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	52	1695	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	65	1781	11	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	44	1649	12	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	49A	1658.1	12	A25G23C22T31	A29G28C21T43	A17G13C13T21
A. baumannii	49B	1658.2	12	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	56	1707	12	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	80	1893	12	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	5	693	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	8	749	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	10	839	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	14	865	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	16	888	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	29	1326	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	35	1440	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	41	1524	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	46	1652	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	47	1653	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	48	1657	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	57	1709	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	61	1727	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	63	1762	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	67	1806	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	75	1881	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	77	1886	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	1	649	46	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	2	653	46	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	39	1497	16	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	24	1198	15	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	28	1243	15	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	43	1648	15	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	62	1746	15	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	4	689	15	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	68	1822	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	69	1823A	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	70	1823B	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	71	1826	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	72	1860	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	81	1924	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	82	1929	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	85	1966	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	11	841	3	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. baumannii	32	1415	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	45	1651	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	54	1697	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	58	1712	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	60	1725	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	66	1802	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	76	1883	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	78	1891	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	79	1892	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	83	1947	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	84	1964	24	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	53	1696	24	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. baumannii	36	1458	49	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	59	1716	9	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. baumannii	9	805	30	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. baumannii	18	967	39	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. baumannii	30	1322	48	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. baumannii	26	1218	50	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. sp. 13TU	15	875	A1	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. sp. 13TU	17	895	A1	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. sp. 3	12	853	B7	A25G22C22T32	A30G29C22T40	A17G13C14T20
A. johnsonii	25	1202	NEW1	A25G22C22T32	A30G29C22T40	A17G13C14T20
A. sp. 2082	87	2082	NEW2	A25G22C22T32	A31G28C22T40	A17G13C14T20

TABLE 15B


Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter
Reed Hospital and Amplified with Codon Analysis Primer Pairs Targeting the parC Gene

				PP No: 2846	PP No: 2847	PP No: 2848
Species	Ibis#	Isolate	ST	parC	parC	parC

A. baumannii	20	1082	1	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	13	854	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	22	1162	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	27	1230	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	31	1367	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	37	1459	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	55	1700	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	64	1777	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	73	1861	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	74	1877	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	86	1972	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	3	684	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	6	720	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	7	726	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	19	1079	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	21	1123	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	23	1188	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	33	1417	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	34	1431	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	38	1496	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	40	1523	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	42	1640	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	50	1666	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	51	1668	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	52	1695	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	65	1781	11	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	44	1649	12	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	49A	1658.1	12	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	49B	1658.2	12	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	56	1707	12	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	80	1893	12	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	5	693	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	8	749	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	10	839	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	14	865	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	16	888	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	29	1326	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	35	1440	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	41	1524	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	46	1652	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	47	1653	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	48	1657	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	57	1709	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	61	1727	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	63	1762	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	67	1806	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	75	1881	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	77	1886	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	1	649	46	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	2	653	46	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	39	1497	16	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	24	1198	15	A33G26C28T34	A29G29C23T33	A16G14C14T16
A. baumannii	28	1243	15	A33G26C28T34	A29G29C23T33	A16G14C14T16
A. baumannii	43	1648	15	A33G26C28T34	A29G29C23T33	A16G14C14T16
A. baumannii	62	1746	15	A33G26C28T34	A29G29C23T33	A16G14C14T16
A. baumannii	4	689	15	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	68	1822	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	69	1823A	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	70	1823B	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	71	1826	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	72	1860	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	81	1924	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	82	1929	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	85	1966	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	11	841	3	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	32	1415	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	45	1651	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	54	1697	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	58	1712	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	60	1725	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	66	1802	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	76	1883	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	78	1891	24	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	79	1892	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	83	1947	24	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	84	1964	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	53	1696	24	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	36	1458	49	A34G26C29T32	A30G28C24T32	A16G14C15T15
A. baumannii	59	1716	9	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	9	805	30	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	18	967	39	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	30	1322	48	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	26	1218	50	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. sp. 13TU	15	875	A1	A32G26C28T35	A28G28C24T34	A16G14C15T15
A. sp. 13TU	17	895	A1	A32G26C28T35	A28G28C24T34	A16G14C15T15
A. sp. 3	12	853	B7	A29G26C27T39	A26G32C21T35	A16G14C15T15
A. johnsonii	25	1202	NEW1	A32G28C26T35	A29G29C22T34	A16G14C15T15
A. sp. 2082	87	2082	NEW2	A33G27C26T35	A31G28C20T35	A16G14C15T15

TABLE 16A


Base Compositions Determined from A. baumannii DNA Samples Obtained from
Northwestern Medical Center and Amplified with Codon Analysis
Primer Pairs Targeting the gyrA Gene

				PP No: 2852	PP No: 2853	PP No: 2854
Species	Ibis#	Isolate	ST	gyrA	gyrA	gyrA

A. baumannii	54	536	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	87	665	3	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	8	80	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	9	91	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	10	92	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	11	131	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	12	137	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	21	218	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	26	242	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	94	678	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	1	9	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	2	13	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	3	19	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	4	24	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	5	36	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	6	39	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	13	139	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	15	165	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	16	170	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	17	186	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	20	202	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	22	221	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	24	234	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	25	239	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	33	370	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	34	389	10	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	19	201	14	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	27	257	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	29	301	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	31	354	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	36	422	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	37	424	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	38	434	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	39	473	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	40	482	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	44	512	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	45	516	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	47	522	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	48	526	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	50	528	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	52	531	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	53	533	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	56	542	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	59	550	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	62	556	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	64	557	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	70	588	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	73	603	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	74	605	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	75	606	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	77	611	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	79	622	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	83	643	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	85	653	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	89	669	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	93	674	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	23	228	51	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	32	369	52	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	35	393	52	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	30	339	53	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	41	485	53	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	42	493	53	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	43	502	53	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	46	520	53	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	49	527	53	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	51	529	53	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	65	562	53	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	68	579	53	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	57	546	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	58	548	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	60	552	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	61	555	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	63	557	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	66	570	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	67	578	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	69	584	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	71	593	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	72	602	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	76	609	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	78	621	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	80	625	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	81	628	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	82	632	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	84	649	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	86	655	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	88	668	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	90	671	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	91	672	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	92	673	54	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	18	196	55	A25G23C22T31	A29G28C21T43	A17G13C13T21
A. baumannii	55	537	27	A25G23C21T32	A29G28C21T43	A17G13C13T21
A. baumannii	28	263	27	A25G23C22T31	A29G28C22T42	A17G13C14T20
A. sp. 3	14	164	B7	A25G22C22T32	A30G29C22T40	A17G13C14T20
mixture	7	71	—	ND	ND	A17G13C15T19

TABLE 16B


Base Compositions Determined from A. baumannii DNA Samples Obtained from
Northwestern Medical Center and Amplified with Codon Analysis
Primer Pairs Targeting the parC Gene

				PP No: 2846	PP No: 2847	PP No: 2848
Species	Ibis#	Isolate	ST	parC	parC	parC

A. baumannii	54	536	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	87	665	3	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	8	80	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	9	91	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	10	92	10	A33G26C28T34	A29G28C25T32	ND
A. baumannii	11	131	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	12	137	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	21	218	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	26	242	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	94	678	10	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	1	9	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	2	13	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	3	19	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	4	24	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	5	36	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	6	39	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	13	139	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	15	165	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	16	170	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	17	186	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	20	202	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	22	221	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	24	234	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	25	239	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	33	370	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	34	389	10	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	19	201	14	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	27	257	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	29	301	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	31	354	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	36	422	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	37	424	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	38	434	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	39	473	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	40	482	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	44	512	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	45	516	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	47	522	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	48	526	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	50	528	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	52	531	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	53	533	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	56	542	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	59	550	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	62	556	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	64	557	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	70	588	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	73	603	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	74	605	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	75	606	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	77	611	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	79	622	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	83	643	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	85	653	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	89	669	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	93	674	51	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	23	228	51	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	32	369	52	A34G25C28T34	A30G27C25T32	A16G14C14T16
A. baumannii	35	393	52	A34G25C28T34	A30G27C25T32	A16G14C14T16
A. baumannii	30	339	53	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	41	485	53	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	42	493	53	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	43	502	53	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	46	520	53	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	49	527	53	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	51	529	53	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	65	562	53	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	68	579	53	A34G25C29T33	A30G27C26T31	A16G14C15T15
A. baumannii	57	546	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	58	548	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	60	552	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	61	555	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	63	557	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	66	570	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	67	578	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	69	584	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	71	593	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	72	602	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	76	609	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	78	621	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	80	625	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	81	628	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	82	632	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	84	649	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	86	655	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	88	668	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	90	671	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	91	672	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	92	673	54	A33G26C28T34	A29G28C25T32	A16G14C14T16
A. baumannii	18	196	55	A33G27C28T33	A29G28C25T31	A15G14C15T16
A. baumannii	55	537	27	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. baumannii	28	263	27	A33G26C29T33	A29G28C26T31	A16G14C15T15
A. sp. 3	14	164	B7	A35G25C29T32	A30G28C17T39	A16G14C15T15
mixture	7	71	—	ND	ND	A17G14C15T14

TABLE 17A


Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter
Reed Hospital and Amplified with Speciating Primer Pair No. 2922 and Triangulation
Genotyping Analysis Primer Pair Nos. 1151 and 1156

				PP No: 2922	PP No: 1151	PP No: 1156
Species	Ibis#	Isolate	ST	efp	trpE	Adk

A. baumannii	20	1082	1	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	13	854	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	22	1162	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	27	1230	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	31	1367	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	37	1459	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	55	1700	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	64	1777	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	73	1861	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	74	1877	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	86	1972	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	3	684	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	6	720	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	7	726	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	19	1079	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	21	1123	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	23	1188	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	33	1417	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	34	1431	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	38	1496	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	40	1523	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	42	1640	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	50	1666	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	51	1668	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	52	1695	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	65	1781	11	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	44	1649	12	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	49A	1658.1	12	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	49B	1658.2	12	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	56	1707	12	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	80	1893	12	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	5	693	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	8	749	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	10	839	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	14	865	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	16	888	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	29	1326	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	35	1440	14	A44G35C25T43	ND	A44G32C27T37
A. baumannii	41	1524	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	46	1652	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	47	1653	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	48	1657	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	57	1709	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	61	1727	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	63	1762	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	67	1806	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	75	1881	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	77	1886	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	1	649	46	A44G35C25T43	A44G35C22T41	A44G32C26T38
A. baumannii	2	653	46	A44G35C25T43	A44G35C22T41	A44G32C26T38
A. baumannii	39	1497	16	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	24	1198	15	A44G35C25T43	A44G35C22T41	A44G32C26T38
A. baumannii	28	1243	15	A44G35C25T43	A44G35C22T41	A44G32C26T38
A. baumannii	43	1648	15	A44G35C25T43	A44G35C22T41	A44G32C26T38
A. baumannii	62	1746	15	A44G35C25T43	A44G35C22T41	A44G32C26T38
A. baumannii	4	689	15	A44G35C25T43	A44G35C22T41	A44G32C26T38
A. baumannii	68	1822	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	69	1823A	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	70	1823B	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	71	1826	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	72	1860	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	81	1924	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	82	1929	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	85	1966	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	11	841	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	32	1415	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	45	1651	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	54	1697	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	58	1712	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	60	1725	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	66	1802	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	76	1883	24	ND	A43G36C20T43	A44G32C27T37
A. baumannii	78	1891	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	79	1892	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	83	1947	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	84	1964	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	53	1696	24	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	36	1458	49	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	59	1716	9	A44G35C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	9	805	30	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	18	967	39	A45G34C25T43	A44G35C22T41	A44G32C26T38
A. baumannii	30	1322	48	A44G35C25T43	A43G36C20T43	A44G32C27T37
A. baumannii	26	1218	50	A44G35C25T43	A44G35C21T42	A44G32C26T38
A. sp. 13TU	15	875	A1	A47G33C24T43	A46G32C20T44	A44G33C27T36
A. sp. 13TU	17	895	A1	A47G33C24T43	A46G32C20T44	A44G33C27T36
A. sp. 3	12	853	B7	A46G35C24T42	A42G34C20T46	A43G33C24T40
A. johnsonii	25	1202	NEW1	A46G35C23T43	A42G35C21T44	A43G33C23T41
A. sp. 2082	87	2082	NEW2	A46G36C22T43	A42G32C20T48	A42G34C23T41

TABLE 17B


Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter
Reed Hospital and Amplified with Triangulation Genotyping Analysis Primer
Pair Nos. 1158 and 1160 and 1165

				PP No: 1158	PP No: 1160	PP No: 1165
Species	Ibis#	Isolate	ST	mutY	mutY	fumC

A. baumannii	20	1082	1	A27G21C25T22	A32G35C29T33	A40G33C30T36
A. baumannii	13	854	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	22	1162	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	27	1230	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	31	1367	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	37	1459	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	55	1700	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	64	1777	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	73	1861	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	74	1877	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	86	1972	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	3	684	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	6	720	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	7	726	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	19	1079	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	21	1123	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	23	1188	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	33	1417	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	34	1431	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	38	1496	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	40	1523	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	42	1640	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	50	1666	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	51	1668	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	52	1695	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	65	1781	11	A27G21C25T22	A32G34C28T35	A40G33C30T36
A. baumannii	44	1649	12	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	49A	1658.1	12	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	49B	1658.2	12	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	56	1707	12	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	80	1893	12	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	5	693	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	8	749	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	10	839	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	14	865	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	16	888	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	29	1326	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	35	1440	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	41	1524	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	46	1652	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	47	1653	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	48	1657	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	57	1709	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	61	1727	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	63	1762	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	67	1806	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	75	1881	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	77	1886	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	1	649	46	A29G19C26T21	A31G35C29T34	A40G33C29T37
A. baumannii	2	653	46	A29G19C26T21	A31G35C29T34	A40G33C29T37
A. baumannii	39	1497	16	A29G19C26T21	A31G35C29T34	A40G34C29T36
A. baumannii	24	1198	15	A29G19C26T21	A31G35C29T34	A40G33C29T37
A. baumannii	28	1243	15	A29G19C26T21	A31G35C29T34	A40G33C29T37
A. baumannii	43	1648	15	A29G19C26T21	A31G35C29T34	A40G33C29T37
A. baumannii	62	1746	15	A29G19C26T21	A31G35C29T34	A40G33C29T37
A. baumannii	4	689	15	A29G19C26T21	A31G35C29T34	A40G33C29T37
A. baumannii	68	1822	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	69	1823A	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	70	1823B	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	71	1826	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	72	1860	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	81	1924	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	82	1929	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	85	1966	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	11	841	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	32	1415	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	45	1651	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	54	1697	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	58	1712	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	60	1725	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	66	1802	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	76	1883	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	78	1891	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	79	1892	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	83	1947	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	84	1964	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	53	1696	24	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	36	1458	49	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	59	1716	9	A27G21C25T22	A32G35C28T34	A39G33C30T37
A. baumannii	9	805	30	A27G21C25T22	A32G35C28T34	A39G33C30T37
A. baumannii	18	967	39	A27G21C26T21	A32G35C28T34	A39G33C30T37
A. baumannii	30	1322	48	A28G21C24T22	A32G35C29T33	A40G33C30T36
A. baumannii	26	1218	50	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. sp. 13TU	15	875	A1	A27G21C25T22	A30G36C26T37	A41G34C28T36
A. sp. 13TU	17	895	A1	A27G21C25T22	A30G36C26T37	A41G34C28T36
A. sp. 3	12	853	B7	A26G23C23T23	A30G36C27T36	A39G37C26T37
A. johnsonii	25	1202	NEW1	A25G23C24T23	A30G35C30T34	A38G37C26T38
A. sp. 2082	87	2082	NEW2	A26G22C24T23	A31G35C28T35	A42G34C27T36

TABLE 17C


Base Compositions Determined from A. baumannii DNA Samples Obtained from Walter
Reed Hospital and Amplified with Triangulation Genotyping Analysis Primer
Pair Nos. 1167 and 1170 and 1171

				PP No: 1167	PP No: 1170	PP No: 1171
Species	Ibis#	Isolate	ST	fumC	fumC	ppa

A. baumannii	20	1082	1	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	13	854	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	22	1162	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	27	1230	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	31	1367	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	37	1459	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	55	1700	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	64	1777	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	73	1861	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	74	1877	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	86	1972	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	3	684	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	6	720	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	7	726	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	19	1079	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	21	1123	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	23	1188	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	33	1417	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	34	1431	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	38	1496	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	40	1523	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	42	1640	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	50	1666	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	51	1668	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	52	1695	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	65	1781	11	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	44	1649	12	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	49A	1658.1	12	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	49B	1658.2	12	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	56	1707	12	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	80	1893	12	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	5	693	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	8	749	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	10	839	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	14	865	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	16	888	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	29	1326	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	35	1440	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	41	1524	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	46	1652	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	47	1653	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	48	1657	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	57	1709	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	61	1727	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	63	1762	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	67	1806	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	75	1881	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	77	1886	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	1	649	46	A41G35C32T39	A37G28C20T51	A35G37C32T45
A. baumannii	2	653	46	A41G35C32T39	A37G28C20T51	A35G37C32T45
A. baumannii	39	1497	16	A41G35C32T39	A37G28C20T51	A35G37C30T47
A. baumannii	24	1198	15	A41G35C32T39	A37G28C20T51	A35G37C30T47
A. baumannii	28	1243	15	A41G35C32T39	A37G28C20T51	A35G37C30T47
A. baumannii	43	1648	15	A41G35C32T39	A37G28C20T51	A35G37C30T47
A. baumannii	62	1746	15	A41G35C32T39	A37G28C20T51	A35G37C30T47
A. baumannii	4	689	15	A41G35C32T39	A37G28C20T51	A35G37C30T47
A. baumannii	68	1822	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	69	1823A	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	70	1823B	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	71	1826	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	72	1860	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	81	1924	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	82	1929	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	85	1966	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	11	841	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	32	1415	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	45	1651	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	54	1697	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	58	1712	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	60	1725	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	66	1802	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	76	1883	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	78	1891	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	79	1892	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	83	1947	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	84	1964	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	53	1696	24	A40G35C34T38	A39G26C22T49	A35G37C33T44
A. baumannii	36	1458	49	A40G35C34T38	A39G26C22T49	A35G37C30T47
A. baumannii	59	1716	9	A40G35C32T40	A38G27C20T51	A36G35C31T47
A. baumannii	9	805	30	A40G35C32T40	A38G27C21T50	A35G36C29T49
A. baumannii	18	967	39	A40G35C33T39	A38G27C20T51	A35G37C30T47
A. baumannii	30	1322	48	A40G35C35T37	A38G27C21T50	A35G37C30T47
A. baumannii	26	1218	50	A40G35C34T38	A38G27C21T50	A35G37C33T44
A. sp. 13TU	15	875	A1	A41G39C31T36	A37G26C24T49	A34G38C31T46
A. sp. 13TU	17	895	A1	A41G39C31T36	A37G26C24T49	A34G38C31T46
A. sp. 3	12	853	B7	A43G37C30T37	A36G27C24T49	A34G37C31T47
A. johnsonii	25	1202	NEW1	A42G38C31T36	A40G27C19T50	A35G37C32T45
A. sp. 2082	87	2082	NEW2	A43G37C32T35	A37G26C21T52	A35G38C31T45

TABLE 18A


Base Compositions Determined from A. baumannii DNA Samples Obtained from
Northwestern Medical Center and Amplified with Speciating Primer Pair No. 2922
and Triangulation Genotyping Analysis Primer Pair Nos. 1151 and 1156

				PP No: 2922	PP No: 1151	PP No: 1156
Species	Ibis#	Isolate	ST	efp	trpE	adk

A. baumannii	54	536	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	87	665	3	A44G35C24T44	A44G35C22T41	A44G32C26T38
A. baumannii	8	80	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	9	91	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	10	92	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	11	131	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	12	137	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	21	218	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	26	242	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	94	678	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	1	9	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	2	13	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	3	19	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	4	24	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	5	36	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	6	39	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	13	139	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	15	165	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	16	170	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	17	186	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	20	202	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	22	221	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	24	234	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	25	239	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	33	370	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	34	389	10	A45G34C25T43	A44G35C21T42	A44G32C26T38
A. baumannii	19	201	14	A44G35C25T43	A44G35C22T41	A44G32C27T37
A. baumannii	27	257	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	29	301	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	31	354	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	36	422	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	37	424	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	38	434	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	39	473	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	40	482	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	44	512	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	45	516	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	47	522	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	48	526	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	50	528	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	52	531	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	53	533	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	56	542	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	59	550	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	62	556	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	64	557	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	70	588	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	73	603	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	74	605	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	75	606	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	77	611	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	79	622	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	83	643	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	85	653	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	89	669	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	93	674	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	23	228	51	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	32	369	52	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	35	393	52	A44G35C25T43	A43G36C20T43	A44G32C26T38
A. baumannii	30	339	53	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	41	485	53	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	42	493	53	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	43	502	53	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	46	520	53	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	49	527	53	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	51	529	53	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	65	562	53	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	68	579	53	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	57	546	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	58	548	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	60	552	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	61	555	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	63	557	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	66	570	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	67	578	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	69	584	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	71	593	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	72	602	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	76	609	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	78	621	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	80	625	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	81	628	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	82	632	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	84	649	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	86	655	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	88	668	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	90	671	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	91	672	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	92	673	54	A44G35C25T43	A44G35C20T43	A44G32C26T38
A. baumannii	18	196	55	A44G35C25T43	A44G35C20T43	A44G32C27T37
A. baumannii	55	537	27	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. baumannii	28	263	27	A44G35C25T43	A44G35C19T44	A44G32C27T37
A. sp. 3	14	164	B7	A46G35C24T42	A42G34C20T46	A43G33C24T40
mixture	7	71	?	mixture	ND	ND

TABLE 18B


Base Compositions Determined from A. baumannii DNA Samples Obtained
from Northwestern Medical Center and Amplified with Triangulation
Genotyping Analysis Primer Pair Nos. 1158, 1160 and 1165

				PP No: 1158	PP No: 1160	PP No: 1165
Species	Ibis#	Isolate	ST	mutY	mutY	fumC

A. baumannii	54	536	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	87	665	3	A27G20C27T21	A32G35C28T34	A40G33C30T36
A. baumannii	8	80	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	9	91	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	10	92	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	11	131	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	12	137	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	21	218	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	26	242	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	94	678	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	1	9	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	2	13	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	3	19	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	4	24	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	5	36	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	6	39	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	13	139	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	15	165	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	16	170	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	17	186	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	20	202	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	22	221	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	24	234	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	25	239	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	33	370	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	34	389	10	A27G21C26T21	A32G35C28T34	A40G33C30T36
A. baumannii	19	201	14	A27G21C25T22	A31G36C28T34	A40G33C29T37
A. baumannii	27	257	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	29	301	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	31	354	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	36	422	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	37	424	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	38	434	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	39	473	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	40	482	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	44	512	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	45	516	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	47	522	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	48	526	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	50	528	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	52	531	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	53	533	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	56	542	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	59	550	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	62	556	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	64	557	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	70	588	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	73	603	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	74	605	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	75	606	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	77	611	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	79	622	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	83	643	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	85	653	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	89	669	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	93	674	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	23	228	51	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	32	369	52	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	35	393	52	A27G21C25T22	A32G35C28T34	A40G33C29T37
A. baumannii	30	339	53	A28G20C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	41	485	53	A28G20C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	42	493	53	A28G20C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	43	502	53	A28G20C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	46	520	53	A28G20C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	49	527	53	A28G20C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	51	529	53	A28G20C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	65	562	53	A28G20C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	68	579	53	A28G20C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	57	546	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	58	548	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	60	552	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	61	555	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	63	557	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	66	570	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	67	578	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	69	584	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	71	593	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	72	602	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	76	609	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	78	621	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	80	625	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	81	628	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	82	632	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	84	649	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	86	655	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	88	668	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	90	671	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	91	672	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	92	673	54	A27G21C26T21	A32G34C29T34	A40G33C30T36
A. baumannii	18	196	55	A27G21C25T22	A31G36C27T35	A40G33C29T37
A. baumannii	55	537	27	A27G21C25T22	A32G35C28T34	A40G33C30T36
A. baumannii	28	263	27	A27G21C25T22	A32G35C28T34	A40G33C30T36
A. sp. 3	14	164	B7	A26G23C23T23	A30G36C27T36	A39G37C26T37
mixture	7	71	?	ND	ND	ND

TABLE 18C


Base Compositions Determined from A. baumannii DNA Samples Obtained
from Northwestern Medical Center and Amplified with Triangulation
Genotyping Analysis Primer Pair Nos. 1167, 1170 and 1171

				PP No: 1167	PP No: 1170	PP No: 1171
Species	Ibis#	Isolate	ST	fumC	fumC	ppa

A. baumannii	54	536	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	87	665	3	A41G34C35T37	A38G27C20T51	A35G37C31T46
A. baumannii	8	80	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	9	91	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	10	92	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	11	131	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	12	137	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	21	218	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	26	242	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	94	678	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	1	9	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	2	13	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	3	19	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	4	24	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	5	36	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	6	39	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	13	139	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	15	165	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	16	170	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	17	186	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	20	202	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	22	221	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	24	234	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	25	239	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	33	370	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	34	389	10	A41G34C34T38	A38G27C21T50	A35G37C33T44
A. baumannii	19	201	14	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	27	257	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	29	301	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	31	354	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	36	422	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	37	424	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	38	434	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	39	473	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	40	482	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	44	512	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	45	516	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	47	522	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	48	526	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	50	528	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	52	531	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	53	533	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	56	542	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	59	550	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	62	556	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	64	557	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	70	588	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	73	603	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	74	605	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	75	606	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	77	611	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	79	622	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	83	643	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	85	653	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	89	669	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	93	674	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	23	228	51	A40G35C34T38	A38G27C21T50	A35G37C30T47
A. baumannii	32	369	52	A40G35C34T38	A38G27C21T50	A35G37C31T46
A. baumannii	35	393	52	A40G35C34T38	A38G27C21T50	A35G37C31T46
A. baumannii	30	339	53	A40G35C35T37	A38G27C21T50	A35G37C31T46
A. baumannii	41	485	53	A40G35C35T37	A38G27C21T50	A35G37C31T46
A. baumannii	42	493	53	A40G35C35T37	A38G27C21T50	A35G37C31T46
A. baumannii	43	502	53	A40G35C35T37	A38G27C21T50	A35G37C31T46
A. baumannii	46	520	53	A40G35C35T37	A38G27C21T50	A35G37C31T46
A. baumannii	49	527	53	A40G35C35T37	A38G27C21T50	A35G37C31T46
A. baumannii	51	529	53	A40G35C35T37	A38G27C21T50	A35G37C31T46
A. baumannii	65	562	53	A40G35C35T37	A38G27C21T50	A35G37C31T46
A. baumannii	68	579	53	A40G35C35T37	A38G27C21T50	A35G37C31T46
A. baumannii	57	546	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	58	548	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	60	552	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	61	555	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	63	557	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	66	570	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	67	578	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	69	584	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	71	593	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	72	602	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	76	609	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	78	621	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	80	625	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	81	628	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	82	632	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	84	649	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	86	655	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	88	668	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	90	671	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	91	672	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	92	673	54	A40G35C34T38	A39G26C22T49	A35G37C31T46
A. baumannii	18	196	55	A42G34C33T38	A38G27C20T51	A35G37C31T46
A. baumannii	55	537	27	A40G35C33T39	A38G27C20T51	A35G37C33T44
A. baumannii	28	263	27	A40G35C33T39	A38G27C20T51	A35G37C33T44
A. sp. 3	14	164	B7	A43G37C30T37	A36G27C24T49	A34G37C31T47
mixture	7	71	—	ND	ND	ND

Base composition analysis of the samples obtained from Walter Reed hospital indicated that a majority of the strain types identified were the same strain types already characterized by the OIF study of Example 12. This is not surprising since at least some patients from which clinical samples were obtained in OIF were transferred to the Walter Reed Hospital (WRAIR). Examples of these common strain types include: ST10, ST11, ST12, ST14, ST15, ST16 and ST46. A strong correlation was noted between these strain types and the presence of mutations in the gyrA and parC which confer quinolone drug resistance.
In contrast, the results of base composition analysis of samples obtained from Northwestern Medical Center indicate the presence of 4 major strain types: ST10, ST51, ST53 and ST54. All of these strain types have the gyrA quinolone resistance mutation and most also have the parC quinolone resistance mutation, with the exception of ST35. This observation is consistent with the current understanding that the gyrA mutation generally appears before the parC mutation and suggests that the acquisition of these drug resistance mutations is rather recent and that resistant isolates are taking over the wild-type isolates. Another interesting observation was that a single isolate of ST3 (isolate 841) displays a triangulation genotyping analysis pattern similar to other isolates of ST3, but the codon analysis amplification product base compositions indicate that this isolate has not yet undergone the quinolone resistance mutations in gyrA and parC.
The six isolates that represent species other than Acinetobacter baumannii in the samples obtained from the Walter Reed Hospital were each found to not carry the drug resistance mutations.
The results described above involved analysis of 183 samples using the methods and compositions of the present invention. Results were provided to collaborators at the Walter Reed hospital and Northwestern Medical center within a week of obtaining samples. This example highlights the rapid throughout characteristics of the analysis platform and the resolving power of triangulation genotyping analysis and codon analysis for identification of and determination of drug resistance in bacteria.

Example 14: Identification of Drug Resistance Genes and Virulence Factors in Staphylococcus aureus

An eight primer pair panel was designed for identification of drug resistance genes and virulence factors of Staphylococcus aureus and is shown in Table 19. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 19.

TABLE 19


Primer Pairs for Identification of Drug Resistance Genes and
Virulence Factors in Staphylococcus aureus

		Forward		Reverse
Primer		Primer		Primer
Pair		(SEQ ID		(SEQ ID	Target
No.	Forward Primer Name	NO:)	Reverse Primer Name	NO:)	Gene

879	MECA_Y14051_4507_4530_F	288	MECA_Y14051_4555_4581_R	1269	mecA
2056	MECI-R_NC003923-41798-	698	MECI-R_NC003923-41798-	1420	MecI-R
	41609_33_60_F		41609_86_113_R
2081	ERMA_NC002952-55890-	217	ERMA_NC002952-55890-	1167	ermA
	56621_366_395_F		56621_438_465_R
2086	ERMC_NC005908-2004-	399	ERMC_NC005908-2004-	1041	ermC
	2738_85_116_F		2738_173_206_R
2095	PVLUK_NC003923-1529595-	456	PVLUK_NC003923-1529595-	1261	Pv-luk
	1531285_688_713_F		1531285_775_804_R
2249	TUFB_NC002758-615038-	430	TUFB_NC002758-615038-	1321	tufB
	616222_696_725_F		616222_793_820_R
2256	NUC_NC002758-894288-	174	NUC_NC002758-894288-	853	Nuc
	894974_316_345_F		894974_396_421_R
2313	MUPR_X75439_2486_2516_F	172	MUPR_X75439_2548_2574_R	1360	mupR

Primer pair numbers 2256 and 2249 are confirmation primers designed with the aim of high level identification of Staphylococcus aureus. The nuc gene is a Staphylococcus aureus-specific marker gene. The tufB gene is a universal housekeeping gene but the bioagent identifying amplicon defined by primer pair number 2249 provides a unique base composition (A43 G28 C19 T35) which distinguishes Staphylococcus aureus from other members of the genus Staphylococcus.
High level methicillin resistance in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair numbers 879 and 2056. Analyses have indicated that primer pair number 879 is not expected to prime S. sciuri homolog or Enterococcus faecalis/faciem ampicillin-resistant PBP5 homologs.
Macrolide and erythromycin resistance in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair numbers 2081 and 2086.
Resistance to mupriocin in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair number 2313.
Virulence in a given strain of Staphylococcus aureus is indicated by bioagent identifying amplicons defined by primer pair number 2095. This primer pair can simultaneously and identify the pvl (lukS-PV) gene and the lukD gene which encodes a homologous enterotoxin. A bioagent identifying amplicon of the lukD gene has a six nucleobase length difference relative to the lukS-PV gene.

A total of 32 blinded samples of different strains of Staphylococcus aureus were provided by the Center for Disease Control (CDC). Each sample was analyzed by PCR amplification with the eight primer pair panel, followed by purification and measurement of molecular masses of the amplification products by mass spectrometry. Base compositions for the amplification products were calculated. The base compositions provide the information summarized above for each primer pair. The results are shown in Tables 20A and B. One result noted upon un-blinding of the samples is that each of the PVL+ identifications agreed with PVL+ identified in the same samples by standard PCR assays. These results indicate that the panel of eight primer pairs is useful for identification of drug resistance and virulence sub-species characteristics for Staphylococcus aureus. It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.

TABLE 20A


Drug Resistance and Virulence Identified in Blinded Samples
of Various Strains of Staphylococcus aureus
with Primer Pair Nos. 2081, 2086, 2095 and 2256

		Primer	Primer	Primer
Sample	Primer Pair No.	Pair No.	Pair No.	Pair No.
Index No.	2081 (ermA)	2086 (ermC)	2095 (pv-luk)	2256 (nuc)

CDC0010	−	−	PVL−/lukD+	+
CDC0015	−	−	PVL+/lukD+	+
CDC0019	−	+	PVL−/lukD+	+
CDC0026	+	−	PVL−/lukD+	+
CDC0030	+	−	PVL−/lukD+	+
CDC004	−	−	PVL+/lukD+	+
CDC0014	−	+	PVL+/lukD+	+
CDC008	−	−	PVL−/lukD+	+
CDC001	+	−	PVL−/lukD+	+
CDC0022	+	−	PVL−/lukD+	+
CDC006	+	−	PVL−/lukD+	+
CDC007	−	−	PVL−/lukD+	+
CDCVRSA1	+	−	PVL−/lukD+	+
CDCVRSA2	+	+	PVL−/lukD+	+
CDC0011	+	−	PVL−/lukD+	+
CDC0012	−	−	PVL+/lukD−	+
CDC0021	+	−	PVL−/lukD+	+
CDC0023	+	−	PVL−/lukD+	+
CDC0025	+	−	PVL−/lukD+	+
CDC005	−	−	PVL−/lukD+	+
CDC0018	+	−	PVL+/lukD−	+
CDC002	−	−	PVL−/lukD+	+
CDC0028	+	−	PVL−/lukD+	+
CDC003	−	−	PVL−/lukD+	+
CDC0013	−	−	PVL+/lukD+	+
CDC0016	−	−	PVL−/lukD+	+
CDC0027	+	−	PVL−/lukD+	+
CDC0029	−	−	PVL+/lukD+	+
CDC0020	−	+	PVL−/lukD+	+
CDC0024	−	−	PVL−/lukD+	+
CDC0031	−	−	PVL−/lukD+	+

TABLE 20B


Drug Resistance and Virulence Identified in Blinded Samples of Various Strains of
Staphylococcus aureus with Primer Pair Nos. 2249, 879, 2056, and 2313

Sample	Primer Pair No. 2249	Primer Pair No.	Primer Pair No.	Primer Pair No.
Index No.	(tufB)	879 (mecA)	2056 (mecI-R)	2313 (mupR)

CDC0010	Staphylococcus aureus	+	+	−
CDC0015	Staphylococcus aureus	−	−	−
CDC0019	Staphylococcus aureus	+	+	−
CDC0026	Staphylococcus aureus	+	+	−
CDC0030	Staphylococcus aureus	+	+	−
CDC004	Staphylococcus aureus	+	+	−
CDC0014	Staphylococcus aureus	+	+	−
CDC008	Staphylococcus aureus	+	+	−
CDC001	Staphylococcus aureus	+	+	−
CDC0022	Staphylococcus aureus	+	+	−
CDC006	Staphylococcus aureus	+	+	+
CDC007	Staphylococcus aureus	+	+	−
CDCVRSA1	Staphylococcus aureus	+	+	−
CDCVRSA2	Staphylococcus aureus	+	+	−
CDC0011	Staphylococcus aureus	−	−	−
CDC0012	Staphylococcus aureus	+	+	−
CDC0021	Staphylococcus aureus	+	+	−
CDC0023	Staphylococcus aureus	+	+	−
CDC0025	Staphylococcus aureus	+	+	−
CDC005	Staphylococcus aureus	+	+	−
CDC0018	Staphylococcus aureus	+	+	−
CDC002	Staphylococcus aureus	+	+	−
CDC0028	Staphylococcus aureus	+	+	−
CDC003	Staphylococcus aureus	+	+	−
CDC0013	Staphylococcus aureus	+	+	−
CDC0016	Staphylococcus aureus	+	+	−
CDC0027	Staphylococcus aureus	+	+	−
CDC0029	Staphylococcus aureus	+	+	−
CDC0020	Staphylococcus aureus	−	−	−
CDC0024	Staphylococcus aureus	+	+	−
CDC0031	Staphylococcus scleiferi	−	−	−

Example 15

Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Staphylococcus aureus

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of eight triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within six different housekeeping genes which are listed in Table 21. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 21.

TABLE 21


Primer Pairs for Triangulation Genotyping Analysis of Staphylococcus aureus

2146	ARCC_NC003923-2725050-	437	ARCC_NC003923-2725050-	1137	arcC
	2724595_131_161_F		2724595_214_245_R
2149	AROE_NC003923-1674726-	530	AROE_NC003923-1674726-	891	aroE
	1674277_30_62_F		1674277_155_181_R
2150	AROE_NC003923-1674726-	474	AROE_NC003923-1674726-	869	aroE
	1674277_204_232_F		1674277_308_335_R
2156	GMK_NC003923-1190906-	268	GMK_NC003923-1190906-	1284	gmk
	1191334_301_329_F		1191334_403_432_R
2157	PTA_NC003923-628885-	418	PTA_NC003923-628885-	1301	pta
	629355_237_263_F		629355_314_345_R
2161	TPI_NC003923-830671-	318	TPI_NC003923-830671-	1300	tpi
	831072_1_34_F		831072_97_129_R
2163	YQI_NC003923-378916-	440	YQI_NC003923-378916-	1076	yqi
	379431_142_167_F		379431_259_284_R
2166	YQI_NC003923-378916-	219	YQI_NC003923-378916-	1013	yqi
	379431_275_300_F		379431_364_396_R

The same samples analyzed for drug resistance and virulence in Example 14 were subjected to triangulation genotyping analysis. The primer pairs of Table 21 were used to produce amplification products by PCR, which were subsequently purified and measured by mass spectrometry. Base compositions were calculated from the molecular masses and are shown in Tables 22A and 22B.

TABLE 22A


Triangulation Genotyping Analysis of Blinded Samples of Various Strains of
Staphylococcus aureus with Primer Pair Nos. 2146, 2149, 2150 and 2156

Sample		Primer Pair No.	Primer Pair No.	Primer Pair No.	Primer Pair No.
Index No.	Strain	2146 (arcC)	2149(aroE)	2150 (aroE)	2156 (gmk)

CDC0010	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0015	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0019	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0026	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0030	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC004	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0014	COL	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC008	????	A44 G24 C18 T29	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC001	Mu50	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC0022	Mu50	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC006	Mu50	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC0011	MRSA252	A45 G24 C18 T28	A58 G24 C19 T51	A41 G36 C12 T43	A51 G29 C21 T31
CDC0012	MRSA252	A45 G24 C18 T28	A58 G24 C19 T51	A41 G36 C12 T43	A51 G29 C21 T31
CDC0021	MRSA252	A45 G24 C18 T28	A58 G24 C19 T51	A41 G36 C12 T43	A51 G29 C21 T31
CDC0023	ST:110	A45 G24 C18 T28	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0025	ST:110	A45 G24 C18 T28	A59 G24 C18 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC005	ST:338	A44 G24 C18 T29	A59 G23 C19 T51	A40 G36 C14 T42	A51 G29 C21 T31
CDC0018	ST:338	A44 G24 C18 T29	A59 G23 C19 T51	A40 G36 C14 T42	A51 G29 C21 T31
CDC002	ST:108	A46 G23 C20 T26	A58 G24 C19 T51	A42 G36 C12 T42	A51 G29 C20 T32
CDC0028	ST:108	A46 G23 C20 T26	A58 G24 C19 T51	A42 G36 C12 T42	A51 G29 C20 T32
CDC003	ST:107	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC0013	ST:12	ND	A59 G24 C18 T51	A40 G36 C13 T43	A51 G29 C21 T31
CDC0016	ST:120	A45 G23 C18 T29	A58 G24 C19 T51	A40 G37 C13 T42	A51 G29 C21 T31
CDC0027	ST:105	A45 G23 C20 T27	A58 G24 C18 T52	A40 G36 C13 T43	A51 G29 C21 T31
CDC0029	MSSA476	A45 G23 C20 T27	A58 G24 C19 T51	A40 G36 C13 T43	A50 G30 C20 T32
CDC0020	ST:15	A44 G23 C21 T27	A59 G23 C18 T52	A40 G36 C13 T43	A50 G30 C20 T32
CDC0024	ST:137	A45 G23 C20 T27	A57 G25 C19 T51	A40 G36 C13 T43	A51 G29 C22 T30
CDC0031	***	No product	No product	No product	No product

TABLE 22B


Triangulation Genotyping Analysis of Blinded Samples of Various Strains of
Staphylococcus aureus with Primer Pair Nos. 2146, 2149, 2150 and 2156

Sample		Primer Pair No.	Primer Pair No.	Primer Pair No.	Primer Pair No.
Index No.	Strain	2157 (pta)	2161 (tpi)	2163 (yqi)	2166 (yqi)

CDC0010	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0015	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0019	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0026	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0030	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC004	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0014	COL	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC008	unknown	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC001	Mu50	A33 G25 C22 T29	A50 G28 C22 T29	A42 G36 C22 T43	A36 G31 C19 T36
CDC0022	Mu50	A33 G25 C22 T29	A50 G28 C22 T29	A42 G36 C22 T43	A36 G31 C19 T36
CDC006	Mu50	A33 G25 C22 T29	A50 G28 C22 T29	A42 G36 C22 T43	A36 G31 C19 T36
CDC0011	MRSA252	A32 G25 C23 T29	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC0012	MRSA252	A32 G25 C23 T29	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC0021	MRSA252	A32 G25 C23 T29	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC0023	ST:110	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0025	ST:110	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC005	ST:338	A32 G25 C24 T28	A51 G27 C21 T30	A42 G36 C22 T43	A37 G30 C18 T37
CDC0018	ST:338	A32 G25 C24 T28	A51 G27 C21 T30	A42 G36 C22 T43	A37 G30 C18 T37
CDC002	ST:108	A33 G25 C23 T28	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC0028	ST:108	A33 G25 C23 T28	A50 G28 C22 T29	A42 G36 C22 T43	A37 G30 C18 T37
CDC003	ST:107	A32 G25 C23 T29	A51 G28 C22 T28	A41 G37 C22 T43	A37 G30 C18 T37
CDC0013	ST:12	A32 G25 C23 T29	A51 G28 C22 T28	A42 G36 C22 T43	A37 G30 C18 T37
CDC0016	ST:120	A32 G25 C24 T28	A50 G28 C21 T30	A42 G36 C22 T43	A37 G30 C18 T37
CDC0027	ST:105	A33 G25 C22 T29	A50 G28 C22 T29	A43 G36 C21 T43	A36 G31 C19 T36
CDC0029	MSSA476	A33 G25 C22 T29	A50 G28 C22 T29	A42 G36 C22 T43	A36 G31 C19 T36
CDC0020	ST:15	A33 G25 C22 T29	A50 G28 C21 T30	A42 G36 C22 T43	A36 G31 C18 T37
CDC0024	ST:137	A33 G25 C22 T29	A51 G28 C22 T28	A42 G36 C22 T43	A37 G30 C18 T37
CDC0031	***	A34 G25 C25 T25	A51 G27 C24 T27	No product	No product

Note:
*** The sample CDC0031 was identified as Staphylococcus scleiferi as indicated in Example 14. Thus, the triangulation genotyping primers designed for Staphylococcus aureus would generally not be expected to prime and produce amplification products of this organism. Tables 22A and 22B indicate that amplification products are obtained for this organism only with primer pair numbers 2157 and 2161.

A total of thirteen different genotypes of Staphylococcus aureus were identified according to the unique combinations of base compositions across the eight different bioagent identifying amplicons obtained with the eight primer pairs. These results indicate that this eight primer pair panel is useful for analysis of unknown or newly emerging strains of Staphylococcus aureus. It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.

Example 16

Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Members of the Bacterial Genus Vibrio

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of eight triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within seven different housekeeping genes which are listed in Table 23. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 23.

TABLE 23


Primer Pairs for Triangulation Genotyping Analysis of
Members of the Bacterial Genus Vibrio

1098	RNASEP_VBC_331_349_F	325	RNASEP_VBC_388_414_R	1163	RNAse P
2000	CTXB_NC002505_46_70_F	278	CTXB_NC002505_132_162_R	1039	ctxB
2001	FUR_NC002505_87_113_F	465	FUR_NC002505_205_228_R	1037	fur
2011	GYRB_NC002505_1161_1190_F	148	GYRB_NC002505_1255_1284_R	1172	gyrB
2012	OMPU_NC002505_85_110_F	190	OMPU_NC002505_154_180_R	1254	ompU
2014	OMPU_NC002505_431_455_F	266	OMPU_NC002505_544_567_R	1094	ompU
2323	CTXA_NC002505-1568114-	508	CTXA_NC002505-1568114-	1297	ctxA
	1567341_122_149_F		1567341_186_214_R
2927	GAPA_NC002505_694_721_F	259	GAPA_NC_002505_29_58_R	1060	gapA

A group of 50 bacterial isolates containing multiple strains of both environmental and clinical isolates of Vibrio cholerae, 9 other Vibrio species, and 3 species of Photobacteria were tested using this panel of primer pairs. Base compositions of amplification products obtained with these 8 primer pairs were used to distinguish amongst various species tested, including sub-species differentiation within Vibrio cholerae isolates. For instance, the non-O1/non-O139 isolates were clearly resolved from the O1 and the O139 isolates, as were several of the environmental isolates of Vibrio cholerae from the clinical isolates.
It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.

Example 17

Selection and Use of Triangulation Genotyping Analysis Primer Pairs for Members of the Bacterial Genus Pseudomonas

To combine the power of high-throughput mass spectrometric analysis of bioagent identifying amplicons with the sub-species characteristic resolving power provided by triangulation genotyping analysis, a panel of twelve triangulation genotyping analysis primer pairs was selected. The primer pairs are designed to produce bioagent identifying amplicons within seven different housekeeping genes which are listed in Table 24. The primer sequences are found in Table 2 and are cross-referenced by the primer pair numbers, primer pair names or SEQ ID NOs listed in Table 24.

TABLE 24


Primer Pairs for Triangulation Genotyping Analysis of
Members of the Bacterial Genus Pseudomonas

2949	ACS_NC002516-970624-	376	ACS_NC002516-970624-	1265	acsA
	971013_299_316_F		971013_364_383_R
2950	ARO_NC002516-26883-	267	ARO_NC002516-26883-	1341	aroE
	27380_4_26_F		27380_111_128_R
2951	ARO_NC002516-26883-	705	ARO_NC002516-26883-	1056	aroE
	27380_356_377_F		27380_459_484_R
2954	GUA_NC002516-4226546-	710	GUA_NC002516-4226546-	1259	guaA
	4226174_155_178_F		4226174_265_287_R
2956	GUA_NC002516-4226546-	374	GUA_NC002516-4226546-	1111	guaA
	4226174_242_263_F		4226174_355_371_R
2957	MUT_NC002516-5551158-	545	MUT_NC002516-5551158-	978	mutL
	5550717_5_26_F		5550717_99_116_R
2959	NUO_NC002516-2984589-	249	NUO_NC002516-2984589-	1095	nuoD
	2984954_8_26_F		2984954_97_117_R
2960	NUO_NC002516-2984589-	195	NUO_NC002516-2984589-	1376	nuoD
	2984954_218_239_F		2984954_301_326_R
2961	PPS_NC002516-1915014-	311	PPS_NC002516-1915014-	1014	pps
	1915383_44_63_F		1915383_140_165_R
2962	PPS_NC002516-1915014-	365	PPS_NC002516-1915014-	1052	pps
	1915383_240_258_F		1915383_341_360_R
2963	TRP_NC002516-671831-	527	TRP_NC002516-671831-	1071	trpE
	672273_24_42_F		672273_131_150_R
2964	TRP_NC002516-671831-	490	TRP_NC002516-671831-	1182	trpE
	672273_261_282_F		672273_362_383_R

It is expected that a kit comprising one or more of the members of this panel will be a useful embodiment of the present invention.
The present invention includes any combination of the various species and subgeneric groupings falling within the generic disclosure. This invention therefore includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
While in accordance with the patent statutes, description of the various embodiments and examples have been provided, the scope of the invention is not to be limited thereto or thereby. Modifications and alterations of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention.
Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims, rather than by the specific examples which have been presented by way of example.
Each reference (including, but not limited to, journal articles, U.S. and non-U.S. patents, patent application publications, international patent application publications, gene bank gi or accession numbers, internet web sites, and the like) cited in the present application is incorporated herein by reference in its entirety.

Claims

1. An oligonucleotide primer pair comprising a forward primer and a reverse primer, each comprising between 13 and 35 linked nucleotides in length, configured to generate an amplicon that is between 45 and 200 linked nucleotides in length, said forward primer configured to hybridize with at least 70% complementarity to a first portion of a region of Genbank gi number: 57634611, and said reverse primer configured to hybridize with at least 70% complementarity to a second portion of said region of Genbank gi number: 57634611, wherein said region of Genbank gi number: 57634611 begins with the 5′ end of SEQ ID NO.: 174, and extends to the 5′ end of SEQ ID NO.: 899.

2. The oligonucleotide primer pair of claim 1, wherein said forward primer comprises at least 70% sequence identity with SEQ ID NO: 174.

3. The oligonucleotide primer pair of claim 2, wherein said forward primer comprises at least 80% sequence identity with SEQ ID NO: 174.

4. The oligonucleotide primer pair of claim 3, wherein said forward primer comprises at least 90% sequence identity with SEQ ID NO: 174.

5. The oligonucleotide primer pair of claim 1, wherein said forward primer is SEQ ID NO: 174.

6. The oligonucleotide primer pair of claim 1, wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO: 853.

7. The oligonucleotide primer pair of claim 6, wherein said reverse primer comprises at least 80% sequence identity with SEQ ID NO: 853.

8. The oligonucleotide primer pair of claim 7, wherein said reverse primer comprises at least 90% sequence identity with SEQ ID NO: 853.

9. The oligonucleotide primer pair of claim 1, wherein said reverse primer is SEQ ID NO: 853.

10. The oligonucleotide primer pair of claim 1, wherein at least one of said forward primer and said reverse primer comprises at least one modified nucleobase.

11. The oligonucleotide primer pair of claim 10, wherein at least one of said at least one modified nucleobase is a mass modified nucleobase.

12. The oligonucleotide primer pair of claim 11, wherein said mass modified nucleobase is 5-Iodo-C.

13. The composition of claim 11, wherein said mass modified nucleobase comprises a molecular mass modifying tag.

14. The oligonucleotide primer pair of claim 10, wherein at least one of said at least one modified nucleobase is a universal nucleobase.

15. The oligonucleotide primer pair of claim 14, wherein said universal nucleobase is inosine.

16. The oligonucleotide primer pair of claim 1, wherein at least one of said forward primer and said reverse primer comprises a non-templated T residue at its 5′ end.

17. A kit for identifying a Staphylococcus aureus bioagent comprising:

i) a first oligonucleotide primer pair comprising a forward primer and a reverse primer, each comprising between 13 and 35 linked nucleotides in length, configured to generate an amplicon that is between 45 and 200 linked nucleotides in length, said forward primer configured to hybridize with at least 70% complementarity to a first portion of a region of Genbank gi number: 57634611, and said reverse primer configured to hybridize with at least 70% complementarity to a second portion of said region of Genbank gi number: 57634611, wherein said region of Genbank gi number: 57634611 begins with the 5′ end of SEQ ID NO: 174 and extends to the 5′ end of SEQ ID NO: 899; and

ii) at least one additional primer pair, wherein the primers of each of said at least one additional primer pair are configured to hybridize to conserved sequence regions within a Staphylococcus aureus gene selected from the group consisting of: mecA, mecRI, ermA, ermC, pvluk, tufB and mupR.

18. The kit of claim 17, wherein each of said at least one additional primer pair comprises SEQ ID NO: 217:SEQ ID NO: 1167, SEQ ID NO: 399:SEQ ID NO:1041, SEQ ID NO: 456:SEQ ID NO: 1261, SEQ ID NO: 430:SEQ ID NO: 1321, SEQ ID NO: 288:SEQ ID NO:1269, SEQ ID NO: 698:SEQ ID NO: 1420, or SEQ ID NO: 205:SEQ ID NO: 876.

19. The kit of claim 17, wherein said first oligonucleotide primer pair and said at least one additional primer pair consists of eight oligonucleotide primer pairs having at least 70% sequence identity with the primer pairs: SEQ ID NO: 217:SEQ ID NO: 1167, SEQ ID NO: 399:SEQ ID NO:1041, SEQ ID NO: 456:SEQ ID NO: 1261, SEQ ID NO: 174:SEQ ID NO: 853, SEQ ID NO: 430:SEQ ID NO: 1321, SEQ ID NO: 288:SEQ ID NO:1269, SEQ ID NO: 698:SEQ ID NO: 1420, and SEQ ID NO: 205:SEQ ID NO: 876.

20. A method for identifying a Staphylococcus aureus bioagent in a sample comprising:

a) amplifying a nucleic acid from said sample using an oligonucleotide primer pair comprising a forward primer and a reverse primer, each comprising between 13 and 35 linked nucleotides in length, said forward primer configured to hybridize with at least 70% complementarity to a first portion of a region of Genbank gi number: 57634611, and said reverse primer configured to hybridize with at least 70% complementarity to a second portion of said region of Genbank gi number: 57634611, wherein said region of Genbank gi number: 57634611 begins with the 5′ end of SEQ ID NO.: 174, and extends to the 5′ end of SEQ ID NO.: 899; wherein said amplifying generates at least one amplification product that comprises between 45 and 200 linked nucleotides; and

b) determining the molecular mass of said at least one amplification product by mass spectrometry.

21. The method of claim 20 further comprising comparing said determined molecular mass to a database comprising a plurality of molecular masses of bioagent identifying amplicons, wherein a match between said determined molecular mass and a molecular mass comprised in said database identifies said Staphylococcus aureus bioagent in said sample.

22. The method of claim 20 further comprising calculating a base composition of said at least one amplification product using said molecular mass.

23. The method of claim 22 further comprising comparing said calculated base composition to a database comprising a plurality of base compositions of bioagent identifying amplicons, wherein a match between said calculated base composition and a base composition comprised in said database identifies said Staphylococcus aureus bioagent in said sample.

24. The method of claim 20, wherein said forward primer comprises at least 70% sequence identity with SEQ ID NO: 174.

25. The method of claim 20, wherein said reverse primer comprises at least 70% sequence identity with SEQ ID NO: 853.

26. The method of claim 20 further comprising repeating said amplifying and determining steps using at least one additional oligonucleotide primer pair wherein the primers of each of said at least one additional primer pair are designed to hybridize to conserved sequence regions within a Staphylococcus aureus gene selected from the group consisting of mecA, mecRI, ermA, ermC, pvluk, tufB, mupR, and nuc.

27. The method of claim 20, wherein said identifying comprises detecting the presence of said Staphylococcus aureus bioagent in said sample.

28. The method of claim 20, wherein said identifying comprises determining either the sensitivity or the resistance of said Staphylococcus aureus bioagent in said sample to one or more antibiotics.

29. The method of claim 20, wherein said identifying comprises identifying a sub-species characteristic, strain, or genotype of said Staphylococcus aureus bioagent in said sample.