US20030027135A1 - Method for rapid detection and identification of bioagents - Google Patents

Method for rapid detection and identification of bioagents Download PDF

Info

Publication number
US20030027135A1
US20030027135A1 US09/798,007 US79800701A US2003027135A1 US 20030027135 A1 US20030027135 A1 US 20030027135A1 US 79800701 A US79800701 A US 79800701A US 2003027135 A1 US2003027135 A1 US 2003027135A1
Authority
US
United States
Prior art keywords
nucleic acid
base
bioagent
mass
amplification product
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US09/798,007
Inventor
David Ecker
Richard Griffey
Rangarajan Sampath
Steven Hofstadler
John McNeil
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ibis Biosciences Inc
Original Assignee
Isis Pharmaceuticals Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Isis Pharmaceuticals Inc filed Critical Isis Pharmaceuticals Inc
Priority to US09/798,007 priority Critical patent/US20030027135A1/en
Assigned to ISIS PHARMACEUTICALS, INC. reassignment ISIS PHARMACEUTICALS, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ECKER, DAVID J., HOFSTADLER, STEVEN, MCNEIL, JOHN, GRIFFEY, RICHARD, SAMPATH, RANGARAJAN
Priority to JP2002570692A priority patent/JP2005504508A/en
Priority to NZ527857A priority patent/NZ527857A/en
Priority to EP02709785A priority patent/EP1364064B1/en
Priority to EP10179791A priority patent/EP2311992B1/en
Priority to AU2002244250A priority patent/AU2002244250B2/en
Priority to RU2003129269/13A priority patent/RU2003129269A/en
Priority to EP10179789.2A priority patent/EP2333118B1/en
Priority to CA2853831A priority patent/CA2853831C/en
Priority to CN201210348178.6A priority patent/CN102912036B/en
Priority to MX2014010221A priority patent/MX349763B/en
Priority to IL15766102A priority patent/IL157661A0/en
Priority to PCT/US2002/006763 priority patent/WO2002070664A2/en
Priority to ES10179789.2T priority patent/ES2448770T3/en
Priority to ES02709785T priority patent/ES2394731T3/en
Priority to CN028091221A priority patent/CN1505685B/en
Priority to EP10179795.9A priority patent/EP2322649B1/en
Priority to ES10179795.9T priority patent/ES2451003T3/en
Priority to CA2439655A priority patent/CA2439655C/en
Priority to MXPA03007927A priority patent/MXPA03007927A/en
Priority to US10/156,608 priority patent/US7108974B2/en
Priority to US10/319,290 priority patent/US20030175696A1/en
Priority to US10/318,881 priority patent/US20030175695A1/en
Priority to US10/319,342 priority patent/US20030175697A1/en
Priority to US10/326,047 priority patent/US20030190605A1/en
Publication of US20030027135A1 publication Critical patent/US20030027135A1/en
Priority to US10/430,253 priority patent/US20040110169A1/en
Priority to US10/435,307 priority patent/US20040202997A1/en
Priority to IL157661A priority patent/IL157661A/en
Priority to ZA200306810A priority patent/ZA200306810B/en
Priority to US10/660,122 priority patent/US7781162B2/en
Priority to US10/660,997 priority patent/US7226739B2/en
Priority to US10/660,996 priority patent/US7255992B2/en
Priority to US10/660,998 priority patent/US7666588B2/en
Priority to US10/728,486 priority patent/US7718354B2/en
Priority to AU2003298030A priority patent/AU2003298030B2/en
Priority to US11/233,630 priority patent/US8017322B2/en
Priority to US11/331,987 priority patent/US8017358B2/en
Priority to US11/331,978 priority patent/US7741036B2/en
Priority to US11/682,259 priority patent/US8563250B2/en
Assigned to IBIS BIOSCIENCES, INC. reassignment IBIS BIOSCIENCES, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: ISIS PHARMACEUTICALS, INC.
Priority to US11/869,449 priority patent/US20080311558A1/en
Priority to US11/929,707 priority patent/US8017743B2/en
Priority to US11/929,910 priority patent/US8214154B2/en
Priority to US11/930,017 priority patent/US8265878B2/en
Priority to US11/929,930 priority patent/US8802372B2/en
Priority to US11/930,741 priority patent/US8815513B2/en
Priority to US12/326,800 priority patent/US8268565B2/en
Priority to US12/605,628 priority patent/US20100145626A1/en
Priority to JP2009245976A priority patent/JP5121803B2/en
Priority to AU2010200893A priority patent/AU2010200893B2/en
Priority to US13/174,254 priority patent/US9416424B2/en
Priority to US14/058,723 priority patent/US20150225780A1/en
Priority to US14/456,806 priority patent/US9752184B2/en
Priority to IL238855A priority patent/IL238855A0/en
Priority to US15/237,261 priority patent/US20170044630A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Definitions

  • the present invention relates to methods for rapid detection and identification of bioagents from environmental, clinical or other samples.
  • the methods provide for detection and characterization of a unique base composition signature (BCS) from any bioagent, including bacteria and viruses.
  • BCS base composition signature
  • the unique BCS is used to rapidly identify the bioagent.
  • PCR polymerase chain reaction
  • detection and data analysis convert the hybridization event into an analytical result.
  • Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated.
  • high-resolution MS alone fails to perform against unknown or bioengineered agents, or in environments where there is a high background level of bioagents (“cluttered” background).
  • Low-resolution MS can fail to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample.
  • 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 detect a particular organism.
  • Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments.
  • Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 10:377-382, 1996).
  • the use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec. 13:1201-1204, 1999).
  • MALDI-TOF matrix-assisted laser desorption ionization-time of flight
  • U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism.
  • U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods.
  • WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry.
  • WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided.
  • PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS.
  • U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes.
  • WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry.
  • Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation.
  • Methods of sequencing a nucleic acid via mass spectrometry methods are also described.
  • WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry.
  • U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing.
  • One embodiment of the present invention is a method of identifying an unknown bioagent comprising (a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; (b) amplifying the variable nucleic acid sequence to produce an amplification product; (c) determining the molecular mass of the amplification product; and (d) comparing the molecular mass to one or more molecular masses of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies the unknown bioagent.
  • the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved.
  • the amplifying step comprises polymerase chain reaction.
  • the amplifying step comprises ligase chain reaction or strand displacement amplification.
  • the bioagent is a bacterium, virus, cell or spore.
  • the nucleic acid is ribosomal RNA.
  • the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.
  • the amplification product is ionized prior to molecular mass determination.
  • the method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers.
  • the method may further comprise the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification products obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d).
  • the one or more molecular mass is contained in a database of molecular masses.
  • the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
  • the molecular mass is determined by mass spectrometry.
  • the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole.
  • the method may further comprise performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.
  • the oligonucleotide primer comprises a base analog or substitute base at positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared to the native base.
  • the primer comprises a universal base at position 3 of each triplet within the primer.
  • the base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp.
  • the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy- ⁇ -D-ribofuranosyl)-imidazole-4-carboxamide.
  • Another embodiment of the present invention is a method of identifying an unknown bioagent comprising (a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; (b) amplifying the variable nucleic acid sequence to produce an amplification product; (c) determining the base composition of the amplification product; and (d) comparing the base composition to one or more base compositions of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies the unknown bioagent.
  • the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved.
  • the amplifying step comprises polymerase chain reaction.
  • the amplifying step comprises ligase chain reaction or strand displacement amplification.
  • the bioagent is a bacterium, virus, cell or spore.
  • the nucleic acid is ribosomal RNA.
  • the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.
  • the amplification product is ionized prior to molecular mass determination.
  • the method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers.
  • the method may further comprise the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more base composition signatures of amplification products obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d).
  • the one or more base compositions is contained in a database of base compositions.
  • the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
  • the molecular mass is determined by mass spectrometry.
  • the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole.
  • the method may further comprise performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.
  • the oligonucleotide primer comprises a base analog or substitute base at positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared to the native base.
  • the primer comprises a universal base at position 3 of each triplet within the primer.
  • the base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp.
  • the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy- ⁇ -D-ribofuranosyl)-imidazole-4-carboxamide.
  • the present invention also provides a method for detecting a single nucleotide polymorphism in an individual, comprising the steps of (a) isolating nucleic acid from the individual; (b) contacting the nucleic acid with oligonucleotide primers which hybridize to regions of the nucleic acid which flank a region comprising the potential polymorphism; (c) amplifying the region to produce an amplification product; (d) determining the molecular mass of the amplification product; and (e) comparing the molecular mass to the molecular mass of the region in an individual known to have the polymorphism, wherein if the molecular masses are the same then the individual has the polymorphism.
  • the primers hybridize to highly conserved sequences.
  • the polymorphism is associated with a disease.
  • the polymorphism is a blood group antigen.
  • the amplifying step is polymerase chain reaction.
  • the amplification step is ligase chain reaction or strand displacement amplification.
  • the amplification product is ionized prior to mass determination.
  • the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
  • the molecular mass is determined by mass spectrometry.
  • the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole.
  • FT-ICR-MS Fourier transform ion cyclotron resonance mass spectrometry
  • ion trap ion trap
  • quadrupole quadrupole
  • magnetic sector magnetic sector
  • TOF time of flight
  • Q-TOF Q-TOF or triple quadrupole.
  • FIGS. 1 A- 1 I are consensus diagrams that show examples of conserved regions from 16S rRNA (FIGS. 1 A- 1 B), 23S rRNA (3′-half, FIGS. 1 C- 1 D; 5′-half, FIGS. 1 E-F), 23S rRNA Domain I (FIG. 1G), 23S rRNA Domain IV (FIG. 1H) and 16S rRNA Domain III (FIG. 11) which are suitable for use in the present invention.
  • Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
  • Bases in capital letters are greater than 95% conserved; bases in lower case letters are 90-95% conserved, filled circles are 80-90% conserved; and open circles are less than 80% conserved.
  • the label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
  • FIG. 2 shows a typical primer amplified region from the 16S rRNA Domain III shown in FIG. 1C.
  • FIG. 3 is a schematic diagram showing conserved regions in RNase P. Bases in capital letters are greater than 90% conserved; bases in lower case letters are 80-90% conserved; filled circles designate bases which are 70-80% conserved; and open circles designate bases that are less than 70% conserved.
  • FIG. 4 is a schematic diagram of base composition signature determination using nucleotide analog “tags” to determine base composition signatures.
  • FIG. 5 shows the deconvoluted mass spectra of a Bacillus anthracis region with and without the mass tag phosphorothioate A (A*).
  • the two spectra differ in that the measured molecular weight of the mass tag-containing sequence is greater than the unmodified sequence.
  • FIG. 6 shows base composition signature (BCS) spectra from PCR products from Staphylococcus aureus ( S. aureus 16S — 1337F) and Bacillus anthracus ( B. anthr. 16S — 1337F), amplified using the same primers.
  • the two strands differ by only two (AT ⁇ CG) substitutions and are clearly distinguished on the basis of their BCS.
  • FIG. 7 shows that a single difference between two sequences (A 14 in B. anthracis vs. A 15 in B. cereus ) can be easily detected using ESI-TOF mass spectrometry.
  • FIG. 8 is an ESI-TOF of Bacillus anthracis spore coat protein sspE 56mer plus calibrant. The signals unambiguously identify B. anthracis versus other Bacillus species.
  • FIG. 9 is an ESI-TOF of a B. anthracis synthetic 16S — 1228 duplex (reverse and forward strands). The technique easily distinguishes between the forward and reverse strands.
  • FIG. 10 is an ESI-FTICR-MS of a synthetic B. anthracis 16S — 1337 46 base pair duplex.
  • FIG. 11 is an ESI-TOF-MS of a 56mer oligonucleotide (3 scans) from the B. anthracis saspB gene with an internal mass standard.
  • the internal mass standards are designated by asterisks.
  • FIG. 12 is an ESI-TOF-MS of an internal standard with 5 mM TBA-TFA buffer showing that charge stripping with tributylammonium trifluoroacetate reduces the most abundant charge state from [M-8H + ] 8 ⁇ to [M-3H + ] 3 ⁇ .
  • the present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with PCR-based BCS technology using “intelligent primers” which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent.
  • the high-resolution MS technique is used to determine the molecular mass and base composition signature (BCS) of the amplified sequence region.
  • This unique “base composition signature” (BCS) is then input to a maximum-likelihood detection algorithm for matching against a database of base composition signatures in the same amplified region.
  • the present method combines PCR-based amplification technology (which provides specificity) and a molecular mass detection mode (which provides speed and does not require nucleic acid sequencing of the amplified target sequence) for bioagent detection and identification.
  • the present method allows extremely rapid and accurate detection and identification of bioagents compared to existing methods. Furthermore, this rapid detection and identification is possible even when sample material is impure. Thus, the method is useful in a wide variety of fields, including, but not limited to, environmental testing (e.g., detection and discrimination of pathogenic vs. non-pathogenic bacteria in water or other samples), germ warfare (allowing immediate identification of the bioagent and appropriate treatment), pharmacogenetic analysis and medical diagnosis (including cancer diagnosis based on mutations and polymorphisms; drug resistance and susceptibility testing; screening for and/or diagnosis of genetic diseases and conditions; and diagnosis of infectious diseases and conditions).
  • the method leverages ongoing biomedical research in virulence, pathogenicity, drug resistance and genome sequencing into a method which provides greatly improved sensitivity, specificity and reliability compared to existing methods, with lower rates of false positives.
  • the present method can be used to detect and classify any biological agent, including bacteria, viruses, fungi and toxins.
  • the information obtained is used to determine practical information needed for countermeasures, including toxin genes, pathogenicity islands and antibiotic resistance genes.
  • the methods can be used to identify natural or deliberate engineering events including chromosome fragment swapping, molecular breeding (gene shuffling) and emerging infectious diseases.
  • Bacteria have a common set of absolutely required genes. About 250 genes are present in all bacterial species ( Proc. Natl. Acad. Sci. U.S.A. 93:10268, 1996; Science 270:397, 1995), including tiny genomes like Mycoplasma, Ureaplasma and Rickettsia. These genes encode proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like.
  • Operons can also be targeted using the present method.
  • One example of an operon is the bfp operon from enteropathogenic E. coli.
  • Multiple core chromosomal genes can be used to classify bacteria at a genus or genus species level to determine if an organism has threat potential. The method can also be used to detect pathogenicity markers (plasmid or chromosomal) and antibiotic resistance genes to confirm the threat potential of an organism and to direct countermeasures.
  • a theoretically ideal bioagent detector would identify, quantify, and report the complete nucleic acid sequence of every bioagent that reached the sensor.
  • the complete sequence of the nucleic acid component of a pathogen would provide all relevant information about the threat, including its identity and the presence of drug-resistance or pathogenicity markers. This ideal has not yet been achieved.
  • the present invention provides a straightforward strategy for obtaining information with the same practical value using base composition signatures (BCS). While the base composition of a gene fragment is not as information-rich as the sequence itself, there is no need to analyze the complete sequence of the gene if the short analyte sequence fragment is properly chosen.
  • BCS base composition signatures
  • a database of reference sequences can be prepared in which each sequence is indexed to a unique base composition signature, so that the presence of the sequence can be inferred with accuracy from the presence of the signature.
  • base composition signatures are that they can be quantitatively measured in a massively parallel fashion using multiplex PCR (PCR in which two or more primer pairs amplify target sequences simultaneously) and mass spectrometry. These multiple primer amplified regions uniquely identify most threat and ubiquitous background bacteria and viruses. In addition, cluster-specific primer pairs distinguish important local clusters (e.g., anthracis group).
  • a “bioagent” is any organism, living or dead, or a nucleic acid derived from such an organism.
  • bioagents include but are not limited to cells (including but not limited to human clinical samples, bacterial cells and other pathogens) viruses, toxin genes and 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.
  • a base composition signatures is the exact base composition from selected fragments of nucleic acid sequences that uniquely identifies the target gene and source organism. BCS can be thought of as unique indexes of specific genes.
  • intelligent primers are primers which bind to sequence regions which flank an intervening variable region.
  • these sequence regions which flank the variable region are highly conserved among different species of bioagent.
  • the sequence regions may be highly conserved among all Bacillus species.
  • highly conserve it is meant that the sequence regions exhibit between about 80-100%, more preferably between about 90-100% and most preferably between about 95-100% identity.
  • Examples of intelligent primers which amplify regions of the 16S and 23S rRNA are shown in FIGS. 1 A- 1 I.
  • a typical primer amplified region in 16S rRNA is shown in FIG. 2.
  • the arrows represent primers which bind to highly conserved regions which flank a variable region in 16S rRNA domain III.
  • the amplified region is the stem-loop structure under 1100-1188
  • One main advantage of the detection methods of the present invention is that the primers need not be specific for a particular bacterial species, or even genus, such as Bacillus or Streptomyces. Instead, the primers recognize highly conserved regions across hundreds of bacterial species including, but not limited to, the species described herein. Thus, the same primer pair can be used to identify any desired bacterium because it will bind to the conserved regions which flank a variable region specific to a single species, or common to several bacterial species, allowing nucleic acid amplification of the intervening sequence and determination of its molecular weight and base composition.
  • primers used in the present method bind to one or more of these regions or portions thereof.
  • the present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with nucleic acid amplification-based BCS technology using “intelligent primers” which hybridize to conserved regions and which bracket variable regions that uniquely identify the bioagent(s).
  • a non-PCR biomass detection mode preferably high-resolution MS
  • nucleic acid amplification-based BCS technology using “intelligent primers” which hybridize to conserved regions and which bracket variable regions that uniquely identify the bioagent(s).
  • PCR ligase chain reaction
  • SDA strand displacement amplification
  • the high-resolution MS technique allows separation of bioagent spectral lines from background spectral lines in highly cluttered environments.
  • the resolved spectral lines are then translated to BCS which are input to a maximum-likelihood detection algorithm matched against spectra for one or more known BCS.
  • the bioagent BCS spectrum is matched against one or more databases of BCS from vast numbers of bioagents.
  • base composition signatures are quantitatively measured in a massively parallel fashion using the polymerase chain reaction (PCR), preferably multiplex PCR, and mass spectrometric (MS) methods. Sufficient quantities of nucleic acids must be present for detection of bioagents by MS. A wide variety of techniques for preparing large amounts of purified nucleic acids or fragments thereof are well known to those of skill in the art.
  • PCR requires one or more pairs of oligonucleotide primers which bind to regions which flank the target sequence(s) to be amplified. These primers prime synthesis of a different strand of DNA, with synthesis occurring in the direction of one primer towards the other primer.
  • the primers, DNA to be amplified, a thermostable DNA polymerase (e.g. Tag polymerase), the four deoxynucleotide triphosphates, and a buffer are combined to initiate DNA synthesis.
  • the solution is denatured by heating, then cooled to allow annealing of newly added primer, followed by another round of DNA synthesis. This process is typically repeated for about 30 cycles, resulting in amplification of the target sequence.
  • the “intelligent primers” define the target sequence region to be amplified and analyzed.
  • the target sequence is a ribosomal RNA (rRNA) gene sequence.
  • rRNA ribosomal RNA
  • rRNA genes Like many genes involved in core life functions, rRNA genes contain sequences that are extraordinarily conserved across bacterial domains interspersed with regions of high variability that are more specific to each species. The variable regions can be utilized to build a database of base composition signatures.
  • the strategy involves creating a structure-based alignment of sequences of the small (16S) and the large (23S) subunits of the rRNA genes. For example, there are currently over 13,000 sequences in the ribosomal RNA database that has been created and maintained by Robin Gutell, University of Texas at Austin, and is publicly available on the Institute for Cellular and Molecular Biology web page (http://www.rna.icmb.utexas.edu/). There is also a publicly available rRNA database created and maintained by the University of Antwerp, Belgium at http://www.rrna.uia.ac.be.
  • regions that are useful as base composition signatures are: a) between about 80 and 100%, preferably >about 95% identity among species of the particular bioagent of interest, of upstream and downstream nucleotide sequences which serve as sequence amplification primer sites; b) an intervening variable region which exhibits no greater than about 5% identity among species; and c) a separation of between about 30 and 1000 nucleotides, preferably no more than about 50-250 nucleotides, and more preferably no more than about 60-100 nucleotides, between the conserved regions.
  • flanking rRNA primer sequences serve as good “universal” primer binding sites to amplify the region of interest for most, if not all, bacterial species.
  • the intervening region between the sets of primers varies in length and/or composition, and thus provides a unique base composition signature.
  • 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 base”.
  • 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 bases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides 14:1001-1003, 1995), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides 14:1053-1056, 1995) or the purine analog 1-(2-deoxy- ⁇ -D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res. 24:3302-3306, 1996).
  • the oligonucleotide primers are designed such that the first and second positions of each triplet are occupied by nucleotide analogs which bind with greater affinity than the unmodified nucleotide.
  • these analogs are 2,6-diaminopurine which binds to thymine, propyne T which binds to adenine and propyne C and phenoxazines, including G-clamp, which binds to G.
  • Propynes are described in U.S. Pat. Nos. 5,645,985, 5,830.653 and 5,484,908, the entire contents of which are incorporated herein by reference.
  • Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, the entire contents of which are incorporated herein by reference.
  • G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, the entire contents of which are incorporated herein by reference.
  • Bacterial biological warfare agents capable of being detected by the present methods include Bacillus anthracis (anthrax), Yersinia pestis (pneumonic plague), Franciscella tularensis (tularemia), Brucella suis, Brucella abortus, Brucella melitensis (undulant fever), Burkholderia mallei (glanders), Burkholderia pseudomalleii (melioidosis), Salmonella typhi (typhoid fever), Rickettsia typhii (epidemic typhus), Rickettsia prowasekii (endemic typhus) and Coxiella burnetii (Q fever), Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas
  • target regions suitable for use in the present invention for detection of bacteria include 5S rRNA and RNase P (FIG. 3).
  • Biological warfare fungus biowarfare agents include coccidioides immitis (Coccidioidomycosis).
  • Biological warfare toxin genes capable of being detected by the methods of the present invention include botulism, T-2 mycotoxins, ricin, staph enterotoxin B, shigatoxin, abrin, aflatoxin, Clostridium perfringens epsilon toxin, conotoxins, diacetoxyscirpenol, tetrodotoxin and saxitoxin.
  • RNA viruses positive-strand and negative-strand
  • Every RNA virus is a family of related viruses (quasispecies). These viruses mutate rapidly and the potential for engineered strains (natural or deliberate) is very high.
  • RNA viruses cluster into families that have conserved RNA structural domains on the viral genome (e.g., virion components, accessory proteins) and conserved housekeeping genes that encode core viral proteins including, for single strand positive strand RNA viruses, RNA-dependent RNA polymerase, double stranded RNA helicase, chymotrypsin-like and papain-like proteases and methyltransferases.
  • Examples of ( ⁇ )-strand RNA viruses include arenaviruses (e.g., sabia virus, lassa fever, Machupo, Argentine hemorrhagic fever, flexal virus), bunyaviruses (e.g., hantavirus, nairovirus, phlebovirus, hantaan virus, Congo-crimean hemorrhagic fever, rift valley fever), and mononegavirales (e.g., filovirus, paramyxovirus, ebola virus, Marburg, equine morbillivirus).
  • arenaviruses e.g., sabia virus, lassa fever, Machupo, Argentine hemorrhagic fever, flexal virus
  • bunyaviruses e.g., hantavirus, nairovirus, phlebovirus, hantaan virus, Congo-crimean hemorrhagic fever, rift valley fever
  • (+)-strand RNA viruses include picornaviruses (e.g., coxsackievirus, echovirus, human coxsackievirus A, human echovirus, human enterovirus, human poliovirus, hepatitis A virus, human parechovirus, human rhinovirus), astroviruses (e.g., human astrovirus), calciviruses (e.g., chiba virus, chitta virus, human calcivirus, norwalk virus), nidovirales (e.g., human coronavirus, human torovirus), flaviviruses (e.g., dengue virus 1-4, Japanese encephalitis virus, Kyanasur forest disease virus, Murray Valley encephalitis virus, Rocio virus, St.
  • picornaviruses e.g., coxsackievirus, echovirus, human coxsackievirus A, human echovirus, human enterovirus, human poliovirus, hepatitis A virus, human pare
  • the hepatitis C virus has a 5′-untranslated region of 340 nucleotides, an open reading frame encoding 9 proteins having 3010 amino acids and a 3′-untranslated region of 240 nucleotides.
  • the 5′-UTR and 3′-UTR are 99% conserved in hepatitis C viruses.
  • the target gene is an RNA-dependent RNA polymerase or a helicase encoded by (+)-strand RNA viruses, or RNA polymerase from a ( ⁇ )-strand RNA virus.
  • (+)-strand RNA viruses are double stranded RNA and replicate by RNA-directed RNA synthesis using RNA-dependent RNA polymerase and the positive strand as a template. Helicase unwinds the RNA duplex to allow replication of the single stranded RNA.
  • viruses include viruses from the family picornaviridae (e.g., poliovirus, coxsackievirus, echovirus), togaviridae (e.g., alphavirus, flavivirus, rubivirus), arenaviridae (e.g., lymphocytic choriomeningitis virus, lassa fever virus), cononaviridae (e.g., human respiratory virus) and Hepatitis A virus.
  • picornaviridae e.g., poliovirus, coxsackievirus, echovirus
  • togaviridae e.g., alphavirus, flavivirus, rubivirus
  • arenaviridae e.g., lymphocytic choriomeningitis virus, lassa fever virus
  • cononaviridae e.g., human respiratory virus
  • Hepatitis A virus e.g., human respiratory virus
  • the detection scheme for the PCR products generated from the bioagent(s) incorporates three features. First, the technique simultaneously detects and differentiates multiple (generally about 6-10) PCR products. Second, the technique provides a BCS that uniquely identifies the bioagent from the possible primer sites. Finally, the detection technique is rapid, allowing multiple PCR reactions to be run in parallel.
  • the method can be used to detect the presence of antibiotic resistance and/or toxin genes in a bacterial species.
  • Bacillus anthracis comprising a tetracycline resistance plasmid and plasmids encoding one or both anthracis toxins (px01 and/or px02) can be detected by using antibiotic resistance primer sets and toxin gene primer sets. If the B. anthracis is positive for tetracycline resistance, then a different antibiotic, for example quinalone, is used.
  • Mass spectrometry (MS)-based detection of PCR products provides all of these features with additional advantages.
  • MS is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product with a unique base composition 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.
  • Intact molecular ions can be 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).
  • ES electrospray ionization
  • MALDI matrix-assisted laser desorption ionization
  • FAB fast atom bombardment
  • MALDI of nucleic acids along with examples of matrices for use in MALDI of nucleic acids, are described in WO 98/54751 (Genetrace, Inc.).
  • Electrospray ionization mass spectrometry 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), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and triple quadrupole.
  • FT-ICR-MS Fourier transform ion cyclotron resonance mass spectrometry
  • ion trap ion trap
  • quadrupole quadrupole
  • magnetic sector magnetic sector
  • TOF time of flight
  • Q-TOF Q-TOF
  • triple quadrupole triple quadrupole
  • the mass spectrometric techniques which can be used in the present invention include, but are not limited to, tandem mass spectrometry, infrared multiphoton dissociation and pyrolytic gas chromatography mass spectrometry (PGC-MS).
  • the bioagent detection system operates continually in bioagent detection mode using pyrolytic GC-MS without PCR for rapid detection of increases in biomass (for example, increases in fecal contamination of drinking water or of germ warfare agents).
  • a continuous sample stream flows directly into the PGC-MS combustion chamber.
  • a PCR process is automatically initiated.
  • Bioagent presence produces elevated levels of large molecular fragments from 100-7,000 Da which are observed in the PGC-MS spectrum.
  • the observed mass spectrum is compared to a threshold level and when levels of biomass are determined to exceed a predetermined threshold, the bioagent classification process described hereinabove(combining PCR and MS, preferably FT-ICR MS) is initiated.
  • alarms or other processes are also initiated by this detected biomass level.
  • tandem mass spectrometry (MS n ) techniques may provide more definitive information pertaining to molecular identity or sequence.
  • Tandem MS involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry. The first stage is used to select an ion or component of a sample from which further structural information is to be obtained. The selected ion is then fragmented using, e.g., blackbody irradiation, infrared multiphoton dissociation, or collisional activation. For example, ions generated by electrospray ionization (ESI) can be fragmented using IR multiphoton dissociation.
  • ESI electrospray ionization
  • This activation leads to dissociation of glycosidic bonds and the phosphate backbone, producing two series of fragment ions, called the w-series (having an intact 3′ terminus and a 5′ phosphate following internal cleavage) and the a-Base series(having an intact 5′ terminus and a 3′ furan).
  • the second stage of mass analysis is then used to detect and measure the mass of these resulting fragments of product ions.
  • Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular sequence of a sample.
  • a nucleotide analog or “tag” is incorporated during amplification (e.g., a 5-(trifluoromethyl) deoxythymidine triphosphate) which has a different molecular weight than the unmodified base so as to improve distinction of masses.
  • tags are described in, for example, PCT WO97/33000. This further limits the number of possible base compositions consistent with any mass.
  • 5-(trifluoromethyl)deoxythymidine triphosphate can be used in place of dTTP in a separate nucleic acid amplification reaction.
  • Measurement of the mass shift between a conventional amplification product and the tagged product is used to quantitate the number of thymidine nucleotides in each of the single strands. Because the strands are complementary, the number of adenosine nucleotides in each strand is also determined.
  • the number of G and C residues in each strand is determined using, for example, the cytidine analog 5-methylcytosine (5-meC) or propyne C.
  • the mass tag phosphorothioate A (A*) was used to distinguish a Bacillus anthracis cluster.
  • the B. anthracis (A 14 G 9 C 14 T 9 ) had an average MW of 14072.26, and the B. anthracis (A 1 A* 13 G 9 C 14 T 9 ) had an average molecular weight of 14281.11 and the phosphorothioate A had an average molecular weight of +16.06 as determined by ESI-TOF MS.
  • the deconvoluted spectra are shown in FIG. 5.
  • Signals from the mass spectrometer may be input to a maximum-likelihood detection and classification algorithm such as is widely used in radar signal processing.
  • the detection processing uses matched filtering of BCS observed in mass-basecount space and allows for detection and subtraction of signatures from known, harmless organisms, and for detection of unknown bioagent threats. Comparison of newly observed bioagents to known bioagents is also possible, for estimation of threat level, by comparing their BCS to those of known organisms and to known forms of pathogenicity enhancement, such as insertion of antibiotic resistance genes or toxin genes.
  • Processing may end with a Bayesian classifier using log likelihood ratios developed from the observed signals and average background levels.
  • the program 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 e.g. GenBank
  • GenBank is used to define the mass basecount matched filters.
  • the database contains known threat agents and benign background organisms. The latter is used to estimate and subtract the 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.
  • a strategy to “triangulate” each organism by measuring signals from multiple core genes is used to reduce false negative and false positive signals, and enable reconstruction of the origin or hybrid or otherwise engineered bioagents.
  • alignments are created from nucleic acid sequence databases. The alignments are then analyzed for regions of conservation and variation, and potential primer binding sites flanking variable regions are identified.
  • amplification target regions for signature analysis are selected which distinguishes organisms based on specific genomic differences (i.e., base composition). For example, detection of signatures for the three part toxin genes typical of B. anthracis (Bowen, J. E. and C. P. Quinn, 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.
  • the present method can also be used to detect single nucleotide polymorphisms (SNPs), or multiple nucleotide polymorphisms, rapidly and accurately.
  • SNP single nucleotide polymorphisms
  • a SNP is defined as a single base pair site in the genome that is different from one individual to another. The difference can be expressed either as a deletion, an insertion or a substitution, and is frequently linked to a disease state. Because they occur every 100-1000 base pairs, SNPs are the most frequently bound type of genetic marker in the human genome.
  • sickle cell anemia results from an A-T transition, which encodes a valine rather than a glutamic acid residue.
  • Oligonucleotide primers may be designed such that they bind to sequences which flank a SNP site, followed by nucleotide amplification and mass determination of the amplified product. Because the molecular masses of the resulting product from an individual who does not have sickle cell anemia is different from that of the product from an individual who has the disease, the method can be used to distinguish the two individuals. Thus, the method can be used to detect any known SNP in an individual and thus diagnose or determine increased susceptibility to a disease or condition.
  • blood is drawn from an individual and peripheral blood mononuclear cells (PBMC) are isolated and simultaneously tested, preferably in a high-throughput screening method, for one or more SNPs using appropriate primers based on the known sequences which flank the SNP region.
  • PBMC peripheral blood mononuclear cells
  • the National Center for Biotechnology Information maintains a publicly available database of SNPs (http://www.ncbi.nlm.nih.gov/SNP/).
  • the method of the present invention can also be used for blood typing.
  • the gene encoding A, B or O blood type can differ by four single nucleotide polymorphisms. If the gene contains the sequence CGTGGTGACCCTT, antigen A results. If the gene contains the sequence CGTCGTCACCGCTA antigen B results. If the gene contains the sequence CGTGGT-ACCCCTT, blood group O results (“ ⁇ ” indicates a deletion). These sequences can be distinguished by designing a single primer pair which flanks these regions, followed by amplification and mass determination.
  • nucleic acid is isolated from the organisms and amplified by PCR using standard methods prior to BCS determination by mass spectrometry.
  • Nucleic acid is isolated, for example, by detergent lysis of bacterial cells, centrifugation and ethanol precipitation. Nucleic acid isolation methods are described in, for example, Current Protocols in Molecular Biology (Ausubel et al.) and Molecular Cloning; A Laboratory Manual (Sambrook et al.).
  • the nucleic acid is then amplified using standard methodology, such as PCR, with primers which bind to conserved regions of the nucleic acid which contain an intervening variable sequence as described below.
  • the FTICR instrument is based on a 7 tesla actively shielded superconducting magnet and modified Bruker Daltonics Apex II 70e ion optics and vacuum chamber.
  • the spectrometer is interfaced to a LEAP PAL autosampler and a custom fluidics control system for high throughput screening applications. Samples are analyzed directly from 96-well or 384-well microtiter plates at a rate of about 1 sample/minute.
  • the Bruker data-acquisition platform is supplemented with a lab-built ancillary NT datastation which controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf-excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for sophisticated tandem MS experiments.
  • a lab-built ancillary NT datastation which controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf-excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for sophisticated tandem MS experiments.
  • SWIFT stored waveform inverse Fourier transform
  • analyte solutions are delivered at 150 nL/minute to a 30 mm i.d. fused-silica ESI emitter mounted on a 3-D micromanipulator.
  • the ESI ion optics consist of a heated metal capillary, an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode.
  • the 6.2 cm rf-only hexapole is comprised of 1 mm diameter rods and is operated at a voltage of 380 Vpp at a frequency of 5 MHz.
  • a lab-built electromechanical shutter can be employed to prevent the electrospray plume from entering the inlet capillary unless triggered to the “open” position via a TTL pulse from the data station.
  • a stable electrospray plume is maintained between the ESI emitter and the face of the shutter.
  • the back face of the shutter arm contains an elastomeric seal which can be positioned to form a vacuum seal with the inlet capillary. When the seal is removed, a 1 mm gap between the shutter blade and the capillary inlet allows constant pressure in the external ion reservoir regardless of whether the shutter is in the open or closed position.
  • a “time slice” of ions is allowed to enter the inlet capillary and is subsequently accumulated in the external ion reservoir.
  • the rapid response time of the ion shutter ( ⁇ 25 ms) provides reproducible, user defined intervals during which ions can be injected into and accumulated in the external ion reservoir.
  • a 25 watt CW CO 2 laser operating at 10.6 ⁇ m has been interfaced to the spectrometer to enable infrared multiphoton dissociation (IRMPD) for oligonucleotide sequencing and other tandem MS applications.
  • An aluminum optical bench is positioned approximately 1.5 m from the actively shielded superconducting magnet such that the laser beam is aligned with the central axis of the magnet.
  • the unfocused 3 mm laser beam is aligned to traverse directly through the 3.5 mm holes in the trapping electrodes of the FTICR trapped ion cell and longitudinally traverse the hexapole region of the external ion guide finally impinging on the skimmer cone.
  • This scheme allows IRMPD to be conducted in an m/z selective manner in the trapped ion cell (e.g. following a SWIFT isolation of the species of interest), or in a broadband mode in the high pressure region of the external ion reservoir where collisions with neutral molecules stabilize IRMPD-generated metastable fragment ions resulting in increased fragment ion yield and sequence coverage.
  • Table 1 shows a small cross section of a database of calculated molecular masses for over 9 primer sets and approximately 30 organisms.
  • the primer sets were derived from rRNA alignment. Examples of regions from rRNA consensus alignments are shown in FIGS. 1 A- 1 C. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed.
  • the primer pairs are >95% conserved in the bacterial sequence database (currently over 10,000 organisms).
  • the intervening regions are variable in length and/or composition, thus providing the base composition “signature” (BCS) for each organism.
  • Primer pairs were chosen so the total length of the amplified region is less than about 80-90 nucleotides.
  • the label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
  • FIG. 6 shows the use of ESI-FT-ICR MS for measurement of exact mass.
  • the spectra from 46mer PCR products originating at position 1337 of the 16S rRNA from S. aureus (upper) and B. anthracis (lower) are shown. These data are from the region of the spectrum containing signals from the [M-8H+] 8 ⁇ charge states of the respective 5′-3′ strands.
  • the two strands differ by two (AT ⁇ CG) substitutions, and have measured masses of 14206.396 and 14208.373 ⁇ 0.010 Da, respectively.
  • the possible base compositions derived from the masses of the forward and reverse strands for the B. anthracis products are listed in Table 3. TABLE 3 Possible base composition for B.
  • the pathogen Vibrio cholera can be distinguished from Vibrio parahemolyticus with ⁇ M>600 Da using one of three 16S primer sets shown in Table 2 (16S — 971, 16S — 1228 or 16S — 1294) as shown in Table 4.
  • the two mycoplasma species in the list ( M. genitalium and M. pneumoniae ) can also be distinguished from each other, as can the three mycobacteriae. While the direct mass measurements of amplified products can identify and distinguish a large number of organisms, measurement of the base composition signature provides dramatically enhanced the base composition signature provides dramatically enhanced resolving power for closely related organisms.
  • compositional analysis or fragmentation patterns are used to resolve the differences.
  • the single base difference between the two organisms yields different fragmentation patterns, and despite the presence of the ambiguous/unidentified base N at position 20 in B. anthracis, the two organisms can be identified.
  • Tables 4a-b show examples of primer pairs from Table 1 which distinguish pathogens from background.
  • Table 4 shows the expected molecular weight and base composition of region 16S — 1100-1188 in Mycobacterium avium and Streptomyces sp. TABLE 5 Organism Molecular Base Region name Length weight comp. 16S_1100-1188 Mycobacte- 82 25624.1728 A 16 G 32 C 18 T 16 rium avium 16S_1100-1188 Strepto- 96 29904.871 A 17 G 38 C 27 T 14 myces sp.
  • Table 5 shows base composition (single strand) results for 16S — 1100-1188 primer amplification reactions different species of bacteria. Species which are repeated in the table (e.g., Clostridium botulinum ) are different strains which have different base compositions in the 16S — 1100-1188 region.
  • tuberculosis A 20 G 33 C 21 T 16 Nocardia asteroides A 20 G 33 C 21 T 16 Fusobacterium necroforum A 21 G 26 C 22 T 18 Listeria monocytogenes A 21 G 27 C 19 T 19 Clostridium botulinum A 21 G 27 C 19 T 21 Neisseria gonorrhoeae A 21 G 28 C 21 T 18 Bartonella quintana A 21 G 30 C 22 T 16 Enterococcus faecalis A 22 G 27 C 20 T 19 Bacillus megaterium A 22 G 28 C 20 T 18 Bacillus subtilis A 22 G 28 C 21 T 17 Pseudomonas aeruginosa A 22 G 29 C 23 T 15 Legionella pneumophila A 22 G 32 C 20 T 16 Mycoplasma pneumoniae A 23 G 20 C 14 T 16 Clostridium botulinum A 23 G 26 C 20 T 19 Enterococcus faecium A 23 G 26 C 21 T 18 Acinetobacter calco
  • the same organism having different base compositions are different strains. Groups of organisms which are highlighted or in italics have the same base compositions in the amplified region. Some of these organisms can be distinguished using multiple primers. For example, Bacillus anthracis can be distinguished from Bacillus cereus and Bacillus thuringiensis using the primer 16S — 971-1062 (Table 6). Other primer pairs which produce unique base composition signatures are shown in Table 6 (bold). Clusters containing very similar threat and ubiquitous non-threat organisms (e.g. anthracis cluster) are distinguished at high resolution with focused sets of primer pairs.
  • the known biowarfare agents in Table 6 are Bacillus anthracis, Yersinia pestis, Francisella tularensis and Rickettsia prowazekii .
  • B. anthracis has an ambiguous base at position 20.
  • the mass measurement accuracy that can be obtained using an internal mass standard in the ESI-MS study of PCR products is shown in FIG. 8.
  • the mass standard was a 20-mer phosphorothioate oligonucleotide added to a solution containing a 56-mer PCR product from the B. anthracis spore coat protein sspE.
  • the mass of the expected PCR product distinguishes B. anthracis from other species of Bacillus such as B. thuringiensis and B. cereus.
  • ESI-TOF MS spectra were obtained on a synthetic 56-mer oligonucleotide (5 ⁇ M )from the saspB gene of B. anthracis containing an internal mass standard at an ESI of 1.7 ⁇ L/min as a function of sample consumption.
  • the results show that the signal to noise is improved as more scans are summed, and that the standard and the product are visible after only 100 scans.

Abstract

Method for detecting and identifying unknown bioagents, including bacteria, viruses and the like, by a combination of nucleic acid amplification and molecular weight determination using primers which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The result is a “base composition signature” (BCS) which is then matched against a database of base composition signatures, by which the bioagent is identified.

Description

    STATEMENT OF GOVERNMENT SUPPORT
  • [0001] This invention was made with United States Government support under DARPA/SPO contract BAA00-09. The United States Government may have certain rights in the invention.
  • FIELD OF THE INVENTION
  • The present invention relates to methods for rapid detection and identification of bioagents from environmental, clinical or other samples. The methods provide for detection and characterization of a unique base composition signature (BCS) from any bioagent, including bacteria and viruses. The unique BCS is used to rapidly identify the bioagent. [0002]
  • BACKGROUND OF THE INVENTION
  • Rapid and definitive microbial identification is desirable for a variety of industrial, medical, environmental, quality, and research reasons. Traditionally, the microbiology laboratory has functioned to identify the etiologic agents of infectious diseases through direct examination and culture of specimens. Since the mid-1980s, researchers have repeatedly demonstrated the practical utility of molecular biology techniques, many of which form the basis of clinical diagnostic assays. Some of these techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and separation and purification of nucleic acids (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). These procedures, in general, are time-consuming and tedious. Another option is the polymerase chain reaction (PCR) or other amplification procedure which amplifies a specific target DNA sequence based on the flanking primers used. Finally, detection and data analysis convert the hybridization event into an analytical result. [0003]
  • Other techniques for detection of bioagents include high-resolution mass spectrometry (MS), low-resolution MS, fluorescence, radioiodination, DNA chips and antibody techniques. None of these techniques is entirely satisfactory. [0004]
  • Mass spectrometry provides detailed information about the molecules being analyzed, including high mass accuracy. It is also a process that can be easily automated. However, high-resolution MS alone fails to perform against unknown or bioengineered agents, or in environments where there is a high background level of bioagents (“cluttered” background). Low-resolution MS can fail to detect some known agents, if their spectral lines are sufficiently weak or sufficiently close to those from other living organisms in the sample. 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 detect a particular organism. [0005]
  • Antibodies face more severe diversity limitations than arrays. If antibodies are designed against highly conserved targets to increase diversity, the false alarm problem will dominate, again because threat organisms are very similar to benign ones. Antibodies are only capable of detecting known agents in relatively uncluttered environments. [0006]
  • Several groups have described detection of PCR products using high resolution electrospray ionization—Fourier transform—ion cyclotron resonance mass spectrometry (ESI-FT-ICR MS). Accurate measurement of exact mass combined with knowledge of the number of at least one nucleotide allowed calculation of the total base composition for PCR duplex products of approximately 100 base pairs. (Aaserud et al., [0007] J. Am. Soc. Mass Spec. 7:1266-1269, 1996; Muddiman et al., Anal. Chem. 69:1543-1549, 1997; Wunschel et al., Anal. Chem. 70:1203-1207, 1998; Muddiman et al., Rev. Anal. Chem. 17:1-68, 1998). Electrospray ionization-Fourier transform-ion cyclotron resistance (ESI-FT-ICR) MS may be used to determine the mass of double-stranded, 500 base-pair PCR products via the average molecular mass (Hurst et al., Rapid Commun. Mass Spec. 10:377-382, 1996). The use of matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for characterization of PCR products has been described. (Muddiman et al., Rapid Commun. Mass Spec. 13:1201-1204, 1999). However, the degradation of DNAs over about 75 nucleotides observed with MALDI limited the utility of this method.
  • U.S. Pat. No. 5,849,492 describes a method for retrieval of phylogenetically informative DNA sequences which comprise searching for a highly divergent segment of genomic DNA surrounded by two highly conserved segments, designing the universal primers for PCR amplification of the highly divergent region, amplifying the genomic DNA by PCR technique using universal primers, and then sequencing the gene to determine the identity of the organism. [0008]
  • U.S. Pat. No. 5,965,363 discloses methods for screening nucleic acids for polymorphisms by analyzing amplified target nucleic acids using mass spectrometric techniques and to procedures for improving mass resolution and mass accuracy of these methods. [0009]
  • WO 99/14375 describes methods, PCR primers and kits for use in analyzing preselected DNA tandem nucleotide repeat alleles by mass spectrometry. [0010]
  • WO 98/12355 discloses methods of determining the mass of a target nucleic acid by mass spectrometric analysis, by cleaving the target nucleic acid to reduce its length, making the target single-stranded and using MS to determine the mass of the single-stranded shortened target. Also disclosed are methods of preparing a double-stranded target nucleic acid for MS analysis comprising amplification of the target nucleic acid, binding one of the strands to a solid support, releasing the second strand and then releasing the first strand which is then analyzed by MS. Kits for target nucleic acid preparation are also provided. [0011]
  • PCT WO97/33000 discloses methods for detecting mutations in a target nucleic acid by nonrandomly fragmenting the target into a set of single-stranded nonrandom length fragments and determining their masses by MS. [0012]
  • U.S. Pat. No. 5,605,798 describes a fast and highly accurate mass spectrometer-based process for detecting the presence of a particular nucleic acid in a biological sample for diagnostic purposes. [0013]
  • WO 98/21066 describes processes for determining the sequence of a particular target nucleic acid by mass spectrometry. Processes for detecting a target nucleic acid present in a biological sample by PCR amplification and mass spectrometry detection are disclosed, as are methods for detecting a target nucleic acid in a sample by amplifying the target with primers that contain restriction sites and tags, extending and cleaving the amplified nucleic acid, and detecting the presence of extended product, wherein the presence of a DNA fragment of a mass different from wild-type is indicative of a mutation. Methods of sequencing a nucleic acid via mass spectrometry methods are also described. [0014]
  • WO 97/37041, WO 99/31278 and U.S. Pat. No. 5,547,835 describe methods of sequencing nucleic acids using mass spectrometry. U.S. Pat. Nos. 5,622,824, 5,872,003 and 5,691,141 describe methods, systems and kits for exonuclease-mediated mass spectrometric sequencing. [0015]
  • Thus, there is a need for a method for bioagent detection and identification which is both specific and rapid, and in which no nucleic acid sequencing is required. The present invention addresses this need. [0016]
  • SUMMARY OF THE INVENTION
  • One embodiment of the present invention is a method of identifying an unknown bioagent comprising (a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; (b) amplifying the variable nucleic acid sequence to produce an amplification product; (c) determining the molecular mass of the amplification product; and (d) comparing the molecular mass to one or more molecular masses of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies the unknown bioagent. In one aspect of this preferred embodiment, the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved. Preferably, the amplifying step comprises polymerase chain reaction. Alternatively, the amplifying step comprises ligase chain reaction or strand displacement amplification. In one aspect of this preferred embodiment, the bioagent is a bacterium, virus, cell or spore. Advantageously, the nucleic acid is ribosomal RNA. In another aspect, the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase. Preferably, the amplification product is ionized prior to molecular mass determination. The method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers. The method may further comprise the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification products obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d). Preferably, the one or more molecular mass is contained in a database of molecular masses. In another aspect of this preferred embodiment, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole. The method may further comprise performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine. In one aspect, the oligonucleotide primer comprises a base analog or substitute base at [0017] positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared to the native base. Preferably, the primer comprises a universal base at position 3 of each triplet within the primer. The base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp. Preferably, the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
  • Another embodiment of the present invention is a method of identifying an unknown bioagent comprising (a) contacting nucleic acid from the bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of the nucleic acid and flank a variable nucleic acid sequence; (b) amplifying the variable nucleic acid sequence to produce an amplification product; (c) determining the base composition of the amplification product; and (d) comparing the base composition to one or more base compositions of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies the unknown bioagent. In one aspect of this preferred embodiment, the sequences to which the at least one pair of oligonucleotide primers hybridize are highly conserved. Preferably, the amplifying step comprises polymerase chain reaction. Alternatively, the amplifying step comprises ligase chain reaction or strand displacement amplification. In one aspect of this preferred embodiment, the bioagent is a bacterium, virus, cell or spore. Advantageously, the nucleic acid is ribosomal RNA. In another aspect, the nucleic acid encodes RNase P or an RNA-dependent RNA polymerase. Preferably, the amplification product is ionized prior to molecular mass determination. The method may further comprise the step of isolating nucleic acid from the bioagent prior to contacting the nucleic acid with the at least one pair of oligonucleotide primers. The method may further comprise the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more base composition signatures of amplification products obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d). Preferably, the one or more base compositions is contained in a database of base compositions. In another aspect of this preferred embodiment, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole. The method may further comprise performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine. In one aspect, the oligonucleotide primer comprises a base analog or substitute base at [0018] positions 1 and 2 of each triplet within the primer, wherein the base analog or substitute base binds with increased affinity to its complement compared to the native base. Preferably, the primer comprises a universal base at position 3 of each triplet within the primer. The base analog or substitute base may be 2,6-diaminopurine, propyne T, propyne G, phenoxazines or G-clamp. Preferably, the universal base is inosine, guanidine, uridine, 5-nitroindole, 3-nitropyrrole, dP or dK, or 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
  • The present invention also provides a method for detecting a single nucleotide polymorphism in an individual, comprising the steps of (a) isolating nucleic acid from the individual; (b) contacting the nucleic acid with oligonucleotide primers which hybridize to regions of the nucleic acid which flank a region comprising the potential polymorphism; (c) amplifying the region to produce an amplification product; (d) determining the molecular mass of the amplification product; and (e) comparing the molecular mass to the molecular mass of the region in an individual known to have the polymorphism, wherein if the molecular masses are the same then the individual has the polymorphism. [0019]
  • In one aspect of this preferred embodiment, the primers hybridize to highly conserved sequences. Preferably, the polymorphism is associated with a disease. Alternatively, the polymorphism is a blood group antigen. In one aspect of the preferred embodiment, the amplifying step is polymerase chain reaction. Alternatively, the amplification step is ligase chain reaction or strand displacement amplification. Preferably, the amplification product is ionized prior to mass determination. In one aspect, the amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment. Advantageously, the molecular mass is determined by mass spectrometry. Preferably, the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF or triple quadrupole.[0020]
  • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. [0021] 1A-1I are consensus diagrams that show examples of conserved regions from 16S rRNA (FIGS. 1A-1B), 23S rRNA (3′-half, FIGS. 1C-1D; 5′-half, FIGS. 1E-F), 23S rRNA Domain I (FIG. 1G), 23S rRNA Domain IV (FIG. 1H) and 16S rRNA Domain III (FIG. 11) which are suitable for use in the present invention. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram. Bases in capital letters are greater than 95% conserved; bases in lower case letters are 90-95% conserved, filled circles are 80-90% conserved; and open circles are less than 80% conserved. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
  • FIG. 2 shows a typical primer amplified region from the 16S rRNA Domain III shown in FIG. 1C. [0022]
  • FIG. 3 is a schematic diagram showing conserved regions in RNase P. Bases in capital letters are greater than 90% conserved; bases in lower case letters are 80-90% conserved; filled circles designate bases which are 70-80% conserved; and open circles designate bases that are less than 70% conserved. [0023]
  • FIG. 4 is a schematic diagram of base composition signature determination using nucleotide analog “tags” to determine base composition signatures. [0024]
  • FIG. 5 shows the deconvoluted mass spectra of a [0025] Bacillus anthracis region with and without the mass tag phosphorothioate A (A*). The two spectra differ in that the measured molecular weight of the mass tag-containing sequence is greater than the unmodified sequence.
  • FIG. 6 shows base composition signature (BCS) spectra from PCR products from [0026] Staphylococcus aureus ( S. aureus 16S1337F) and Bacillus anthracus (B. anthr. 16S1337F), amplified using the same primers. The two strands differ by only two (AT→CG) substitutions and are clearly distinguished on the basis of their BCS.
  • FIG. 7 shows that a single difference between two sequences (A[0027] 14 in B. anthracis vs. A15 in B. cereus) can be easily detected using ESI-TOF mass spectrometry.
  • FIG. 8 is an ESI-TOF of [0028] Bacillus anthracis spore coat protein sspE 56mer plus calibrant. The signals unambiguously identify B. anthracis versus other Bacillus species.
  • FIG. 9 is an ESI-TOF of a [0029] B. anthracis synthetic 16S1228 duplex (reverse and forward strands). The technique easily distinguishes between the forward and reverse strands.
  • FIG. 10 is an ESI-FTICR-MS of a [0030] synthetic B. anthracis 16S1337 46 base pair duplex.
  • FIG. 11 is an ESI-TOF-MS of a 56mer oligonucleotide (3 scans) from the [0031] B. anthracis saspB gene with an internal mass standard. The internal mass standards are designated by asterisks.
  • FIG. 12 is an ESI-TOF-MS of an internal standard with 5 mM TBA-TFA buffer showing that charge stripping with tributylammonium trifluoroacetate reduces the most abundant charge state from [M-8H[0032] +]8− to [M-3H+]3−.
  • DETAILED DESCRIPTION OF THE INVENTION
  • The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with PCR-based BCS technology using “intelligent primers” which hybridize to conserved sequence regions of nucleic acids derived from a bioagent and which bracket variable sequence regions that uniquely identify the bioagent. The high-resolution MS technique is used to determine the molecular mass and base composition signature (BCS) of the amplified sequence region. This unique “base composition signature” (BCS) is then input to a maximum-likelihood detection algorithm for matching against a database of base composition signatures in the same amplified region. The present method combines PCR-based amplification technology (which provides specificity) and a molecular mass detection mode (which provides speed and does not require nucleic acid sequencing of the amplified target sequence) for bioagent detection and identification. [0033]
  • The present method allows extremely rapid and accurate detection and identification of bioagents compared to existing methods. Furthermore, this rapid detection and identification is possible even when sample material is impure. Thus, the method is useful in a wide variety of fields, including, but not limited to, environmental testing (e.g., detection and discrimination of pathogenic vs. non-pathogenic bacteria in water or other samples), germ warfare (allowing immediate identification of the bioagent and appropriate treatment), pharmacogenetic analysis and medical diagnosis (including cancer diagnosis based on mutations and polymorphisms; drug resistance and susceptibility testing; screening for and/or diagnosis of genetic diseases and conditions; and diagnosis of infectious diseases and conditions). The method leverages ongoing biomedical research in virulence, pathogenicity, drug resistance and genome sequencing into a method which provides greatly improved sensitivity, specificity and reliability compared to existing methods, with lower rates of false positives. [0034]
  • The present method can be used to detect and classify any biological agent, including bacteria, viruses, fungi and toxins. As one example, where the agent is a biological threat, the information obtained is used to determine practical information needed for countermeasures, including toxin genes, pathogenicity islands and antibiotic resistance genes. In addition, the methods can be used to identify natural or deliberate engineering events including chromosome fragment swapping, molecular breeding (gene shuffling) and emerging infectious diseases. [0035]
  • Bacteria have a common set of absolutely required genes. About 250 genes are present in all bacterial species ([0036] Proc. Natl. Acad. Sci. U.S.A. 93:10268, 1996; Science 270:397, 1995), including tiny genomes like Mycoplasma, Ureaplasma and Rickettsia. These genes encode proteins involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake, secretion and the like. Examples of these proteins are DNA polymerase III beta, elongation factor TU, heat shock protein groEL, RNA polymerase beta, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase and elongation factor G. Operons can also be targeted using the present method. One example of an operon is the bfp operon from enteropathogenic E. coli. Multiple core chromosomal genes can be used to classify bacteria at a genus or genus species level to determine if an organism has threat potential. The method can also be used to detect pathogenicity markers (plasmid or chromosomal) and antibiotic resistance genes to confirm the threat potential of an organism and to direct countermeasures.
  • A theoretically ideal bioagent detector would identify, quantify, and report the complete nucleic acid sequence of every bioagent that reached the sensor. The complete sequence of the nucleic acid component of a pathogen would provide all relevant information about the threat, including its identity and the presence of drug-resistance or pathogenicity markers. This ideal has not yet been achieved. However, the present invention provides a straightforward strategy for obtaining information with the same practical value using base composition signatures (BCS). While the base composition of a gene fragment is not as information-rich as the sequence itself, there is no need to analyze the complete sequence of the gene if the short analyte sequence fragment is properly chosen. A database of reference sequences can be prepared in which each sequence is indexed to a unique base composition signature, so that the presence of the sequence can be inferred with accuracy from the presence of the signature. The advantage of base composition signatures is that they can be quantitatively measured in a massively parallel fashion using multiplex PCR (PCR in which two or more primer pairs amplify target sequences simultaneously) and mass spectrometry. These multiple primer amplified regions uniquely identify most threat and ubiquitous background bacteria and viruses. In addition, cluster-specific primer pairs distinguish important local clusters (e.g., anthracis group). [0037]
  • In the context of this invention, a “bioagent” is any organism, living or dead, or a nucleic acid derived from such an organism. Examples of bioagents include but are not limited to cells (including but not limited to human clinical samples, bacterial cells and other pathogens) viruses, toxin genes and 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. [0038]
  • As used herein, a [0039]
    Figure US20030027135A1-20030206-P00010
    base composition signatures
    Figure US20030027135A1-20030206-P00010
    (BCS) is the exact base composition from selected fragments of nucleic acid sequences that uniquely identifies the target gene and source organism. BCS can be thought of as unique indexes of specific genes.
  • As used herein, [0040]
    Figure US20030027135A1-20030206-P00010
    intelligent primers
    Figure US20030027135A1-20030206-P00010
    are primers which bind to sequence regions which flank an intervening variable region. In a preferred embodiment, these sequence regions which flank the variable region are highly conserved among different species of bioagent. For example, the sequence regions may be highly conserved among all Bacillus species. By the term
    Figure US20030027135A1-20030206-P00010
    highly conserve
    Figure US20030027135A1-20030206-P00010
    , it is meant that the sequence regions exhibit between about 80-100%, more preferably between about 90-100% and most preferably between about 95-100% identity. Examples of intelligent primers which amplify regions of the 16S and 23S rRNA are shown in FIGS. 1A-1I. A typical primer amplified region in 16S rRNA is shown in FIG. 2. The arrows represent primers which bind to highly conserved regions which flank a variable region in 16S rRNA domain III. The amplified region is the stem-loop structure under
    Figure US20030027135A1-20030206-P00010
    1100-1188
    Figure US20030027135A1-20030206-P00010
  • One main advantage of the detection methods of the present invention is that the primers need not be specific for a particular bacterial species, or even genus, such as Bacillus or Streptomyces. Instead, the primers recognize highly conserved regions across hundreds of bacterial species including, but not limited to, the species described herein. Thus, the same primer pair can be used to identify any desired bacterium because it will bind to the conserved regions which flank a variable region specific to a single species, or common to several bacterial species, allowing nucleic acid amplification of the intervening sequence and determination of its molecular weight and base composition. For example, the 16S[0041] 971-1062, 16S1228-1310 and 16S1100-1188 regions are 98-99% conserved in about 900 species of bacteria (16S=16S rRNA, numbers indicate nucleotide position). In one embodiment of the present invention, primers used in the present method bind to one or more of these regions or portions thereof.
  • The present invention provides a combination of a non-PCR biomass detection mode, preferably high-resolution MS, with nucleic acid amplification-based BCS technology using “intelligent primers” which hybridize to conserved regions and which bracket variable regions that uniquely identify the bioagent(s). Although the use of PCR is preferred, other nucleic acid amplification techniques may also be used, including ligase chain reaction (LCR) and strand displacement amplification (SDA). The high-resolution MS technique allows separation of bioagent spectral lines from background spectral lines in highly cluttered environments. The resolved spectral lines are then translated to BCS which are input to a maximum-likelihood detection algorithm matched against spectra for one or more known BCS. Preferably, the bioagent BCS spectrum is matched against one or more databases of BCS from vast numbers of bioagents. Preferably, the matching is done using a maximum-likelihood detection algorithm. [0042]
  • In a preferred embodiment, base composition signatures are quantitatively measured in a massively parallel fashion using the polymerase chain reaction (PCR), preferably multiplex PCR, and mass spectrometric (MS) methods. Sufficient quantities of nucleic acids must be present for detection of bioagents by MS. A wide variety of techniques for preparing large amounts of purified nucleic acids or fragments thereof are well known to those of skill in the art. PCR requires one or more pairs of oligonucleotide primers which bind to regions which flank the target sequence(s) to be amplified. These primers prime synthesis of a different strand of DNA, with synthesis occurring in the direction of one primer towards the other primer. The primers, DNA to be amplified, a thermostable DNA polymerase (e.g. Tag polymerase), the four deoxynucleotide triphosphates, and a buffer are combined to initiate DNA synthesis. The solution is denatured by heating, then cooled to allow annealing of newly added primer, followed by another round of DNA synthesis. This process is typically repeated for about 30 cycles, resulting in amplification of the target sequence. [0043]
  • The “intelligent primers” define the target sequence region to be amplified and analyzed. In one embodiment, the target sequence is a ribosomal RNA (rRNA) gene sequence. With the complete sequences of many of the smallest microbial genomes now available, it is possible to identify a set of genes that defines “minimal life” and identify composition signatures that uniquely identify each gene and organism. Genes that encode core life functions such as DNA replication, transcription, ribosome structure, translation, and transport are distributed broadly in the bacterial genome and are preferred regions for BCS analysis. Ribosomal RNA (rRNA) genes comprise regions that provide useful base composition signatures. Like many genes involved in core life functions, rRNA genes contain sequences that are extraordinarily conserved across bacterial domains interspersed with regions of high variability that are more specific to each species. The variable regions can be utilized to build a database of base composition signatures. The strategy involves creating a structure-based alignment of sequences of the small (16S) and the large (23S) subunits of the rRNA genes. For example, there are currently over 13,000 sequences in the ribosomal RNA database that has been created and maintained by Robin Gutell, University of Texas at Austin, and is publicly available on the Institute for Cellular and Molecular Biology web page (http://www.rna.icmb.utexas.edu/). There is also a publicly available rRNA database created and maintained by the University of Antwerp, Belgium at http://www.rrna.uia.ac.be. [0044]
  • These databases have been analyzed to determine regions that are useful as base composition signatures. The characteristics of such regions are: a) between about 80 and 100%, preferably >about 95% identity among species of the particular bioagent of interest, of upstream and downstream nucleotide sequences which serve as sequence amplification primer sites; b) an intervening variable region which exhibits no greater than about 5% identity among species; and c) a separation of between about 30 and 1000 nucleotides, preferably no more than about 50-250 nucleotides, and more preferably no more than about 60-100 nucleotides, between the conserved regions. [0045]
  • Due to their overall conservation, the flanking rRNA primer sequences serve as good “universal” primer binding sites to amplify the region of interest for most, if not all, bacterial species. The intervening region between the sets of primers varies in length and/or composition, and thus provides a unique base composition signature. [0046]
  • It is advantageous to design the “intelligent primers” to be as universal as possible to minimize the number of primers which need to be synthesized, and to allow detection of multiple species using a single pair of primers. These primer pairs can be used to amplify variable regions in these species. Because any variation (due to codon wobble in the [0047] 3rd position) in these conserved regions among species is likely to occur in the third position of a DNA 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 base”. 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 bases include nitroindoles such as 5-nitroindole or 3-nitropyrrole (Loakes et al., Nucleosides and Nucleotides 14:1001-1003, 1995), the degenerate nucleotides dP or dK (Hill et al.), an acyclic nucleoside analog containing 5-nitroindazole (Van Aerschot et al., Nucleosides and Nucleotides 14:1053-1056, 1995) or the purine analog 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide (Sala et al., Nucl. Acids Res. 24:3302-3306, 1996).
  • In another embodiment of the invention, 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 which bind with greater affinity than the unmodified nucleotide. Examples of these analogs are 2,6-diaminopurine which binds to thymine, propyne T which binds to adenine and propyne C and phenoxazines, including G-clamp, which binds to G. Propynes are described in U.S. Pat. Nos. 5,645,985, 5,830.653 and 5,484,908, the entire contents of which are incorporated herein by reference. Phenoxazines are described in U.S. Pat. Nos. 5,502,177, 5,763,588, and 6,005,096, the entire contents of which are incorporated herein by reference. G-clamps are described in U.S. Pat. Nos. 6,007,992 and 6,028,183, the entire contents of which are incorporated herein by reference. [0048]
  • Bacterial biological warfare agents capable of being detected by the present methods include [0049] Bacillus anthracis (anthrax), Yersinia pestis (pneumonic plague), Franciscella tularensis (tularemia), Brucella suis, Brucella abortus, Brucella melitensis (undulant fever), Burkholderia mallei (glanders), Burkholderia pseudomalleii (melioidosis), Salmonella typhi (typhoid fever), Rickettsia typhii (epidemic typhus), Rickettsia prowasekii (endemic typhus) and Coxiella burnetii (Q fever), Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigella flexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila, and Vibrio cholerae.
  • Besides 16S and 23S rRNA, other target regions suitable for use in the present invention for detection of bacteria include 5S rRNA and RNase P (FIG. 3). [0050]
  • Biological warfare fungus biowarfare agents include [0051] coccidioides immitis (Coccidioidomycosis).
  • Biological warfare toxin genes capable of being detected by the methods of the present invention include botulism, T-2 mycotoxins, ricin, staph enterotoxin B, shigatoxin, abrin, aflatoxin, [0052] Clostridium perfringens epsilon toxin, conotoxins, diacetoxyscirpenol, tetrodotoxin and saxitoxin.
  • Biological warfare viral threat agents are mostly RNA viruses (positive-strand and negative-strand), with the exception of smallpox. Every RNA virus is a family of related viruses (quasispecies). These viruses mutate rapidly and the potential for engineered strains (natural or deliberate) is very high. RNA viruses cluster into families that have conserved RNA structural domains on the viral genome (e.g., virion components, accessory proteins) and conserved housekeeping genes that encode core viral proteins including, for single strand positive strand RNA viruses, RNA-dependent RNA polymerase, double stranded RNA helicase, chymotrypsin-like and papain-like proteases and methyltransferases. [0053]
  • Examples of (−)-strand RNA viruses include arenaviruses (e.g., sabia virus, lassa fever, Machupo, Argentine hemorrhagic fever, flexal virus), bunyaviruses (e.g., hantavirus, nairovirus, phlebovirus, hantaan virus, Congo-crimean hemorrhagic fever, rift valley fever), and mononegavirales (e.g., filovirus, paramyxovirus, ebola virus, Marburg, equine morbillivirus). [0054]
  • Examples of (+)-strand RNA viruses include picornaviruses (e.g., coxsackievirus, echovirus, human coxsackievirus A, human echovirus, human enterovirus, human poliovirus, hepatitis A virus, human parechovirus, human rhinovirus), astroviruses (e.g., human astrovirus), calciviruses (e.g., chiba virus, chitta virus, human calcivirus, norwalk virus), nidovirales (e.g., human coronavirus, human torovirus), flaviviruses (e.g., dengue virus 1-4, Japanese encephalitis virus, Kyanasur forest disease virus, Murray Valley encephalitis virus, Rocio virus, St. Louis encephalitis virus, West Nile virus, yellow fever virus, hepatitis c virus) and togaviruses (e.g., Chikugunya virus, Eastern equine encephalitis virus, Mayaro virus, O'nyong-nyong virus, Ross River virus, Venezuelan equine encephalitis virus, Rubella virus, hepatitis E virus). The hepatitis C virus has a 5′-untranslated region of 340 nucleotides, an open reading frame encoding 9 proteins having 3010 amino acids and a 3′-untranslated region of 240 nucleotides. The 5′-UTR and 3′-UTR are 99% conserved in hepatitis C viruses. [0055]
  • In one embodiment, the target gene is an RNA-dependent RNA polymerase or a helicase encoded by (+)-strand RNA viruses, or RNA polymerase from a (−)-strand RNA virus. (+)-strand RNA viruses are double stranded RNA and replicate by RNA-directed RNA synthesis using RNA-dependent RNA polymerase and the positive strand as a template. Helicase unwinds the RNA duplex to allow replication of the single stranded RNA. These viruses include viruses from the family picornaviridae (e.g., poliovirus, coxsackievirus, echovirus), togaviridae (e.g., alphavirus, flavivirus, rubivirus), arenaviridae (e.g., lymphocytic choriomeningitis virus, lassa fever virus), cononaviridae (e.g., human respiratory virus) and Hepatitis A virus. The genes encoding these proteins comprise variable and highly conserved regions which flank the variable regions. [0056]
  • In a preferred embodiment, the detection scheme for the PCR products generated from the bioagent(s) incorporates three features. First, the technique simultaneously detects and differentiates multiple (generally about 6-10) PCR products. Second, the technique provides a BCS that uniquely identifies the bioagent from the possible primer sites. Finally, the detection technique is rapid, allowing multiple PCR reactions to be run in parallel. [0057]
  • In one embodiment, the method can be used to detect the presence of antibiotic resistance and/or toxin genes in a bacterial species. For example, [0058] Bacillus anthracis comprising a tetracycline resistance plasmid and plasmids encoding one or both anthracis toxins (px01 and/or px02) can be detected by using antibiotic resistance primer sets and toxin gene primer sets. If the B. anthracis is positive for tetracycline resistance, then a different antibiotic, for example quinalone, is used.
  • Mass spectrometry (MS)-based detection of PCR products provides all of these features with additional advantages. MS is intrinsically a parallel detection scheme without the need for radioactive or fluorescent labels, since every amplification product with a unique base composition 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. Intact molecular ions can be 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). For example, MALDI of nucleic acids, along with examples of matrices for use in MALDI of nucleic acids, are described in WO 98/54751 (Genetrace, Inc.). [0059]
  • 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. 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), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF, and triple quadrupole. [0060]
  • In general, the mass spectrometric techniques which can be used in the present invention include, but are not limited to, tandem mass spectrometry, infrared multiphoton dissociation and pyrolytic gas chromatography mass spectrometry (PGC-MS). In one embodiment of the invention, the bioagent detection system operates continually in bioagent detection mode using pyrolytic GC-MS without PCR for rapid detection of increases in biomass (for example, increases in fecal contamination of drinking water or of germ warfare agents). To achieve minimal latency, a continuous sample stream flows directly into the PGC-MS combustion chamber. When an increase in biomass is detected, a PCR process is automatically initiated. Bioagent presence produces elevated levels of large molecular fragments from 100-7,000 Da which are observed in the PGC-MS spectrum. The observed mass spectrum is compared to a threshold level and when levels of biomass are determined to exceed a predetermined threshold, the bioagent classification process described hereinabove(combining PCR and MS, preferably FT-ICR MS) is initiated. Optionally, alarms or other processes (halting ventilation flow, physical isolation) are also initiated by this detected biomass level. [0061]
  • The accurate measurement of molecular mass for large DNAs is limited by the adduction of cations from the PCR reaction to each strand, resolution of the isotopic peaks from natural abundance [0062] 13C and 15N isotopes, and assignment of the charge state for any ion. The cations are removed by in-line dialysis using a flow-through chip that brings the solution containing the PCR products into contact with a solution containing ammonium acetate in the presence of an electric field gradient orthogonal to the flow. The latter two problems are addressed by operating with a resolving power of >100,000 and by incorporating isotopically depleted nucleotide triphosphates into the DNA. The resolving power of the instrument is also a consideration. At a resolving power of 10,000, the modeled signal from the [M-14H+]14− charge state of an 84mer PCR product is poorly characterized and assignment of the charge state or exact mass is impossible. At a resolving power of 33,000, the peaks from the individual isotopic components are visible. At a resolving power of 100,000, the isotopic peaks are resolved to the baseline and assignment of the charge state for the ion is straightforward. The [13C, 15N]-depleted triphosphates are obtained, for example, by growing microorganisms on depleted media and harvesting the nucleotides (Batey et al., Nucl. Acids Res. 20:4515-4523, 1992).
  • While mass measurements of intact nucleic acid regions are believed to be adequate to determine most bioagents, tandem mass spectrometry (MS[0063] n) techniques may provide more definitive information pertaining to molecular identity or sequence. Tandem MS involves the coupled use of two or more stages of mass analysis where both the separation and detection steps are based on mass spectrometry. The first stage is used to select an ion or component of a sample from which further structural information is to be obtained. The selected ion is then fragmented using, e.g., blackbody irradiation, infrared multiphoton dissociation, or collisional activation. For example, ions generated by electrospray ionization (ESI) can be fragmented using IR multiphoton dissociation. This activation leads to dissociation of glycosidic bonds and the phosphate backbone, producing two series of fragment ions, called the w-series (having an intact 3′ terminus and a 5′ phosphate following internal cleavage) and the a-Base series(having an intact 5′ terminus and a 3′ furan).
  • The second stage of mass analysis is then used to detect and measure the mass of these resulting fragments of product ions. Such ion selection followed by fragmentation routines can be performed multiple times so as to essentially completely dissect the molecular sequence of a sample. [0064]
  • If there are two or more targets of similar base composition or mass, or if a single amplification reaction results in a product which has the same mass as two or more bioagent reference standards, they can be distinguished by using mass-modifying “tags.” In this embodiment of the invention, a nucleotide analog or “tag” is incorporated during amplification (e.g., a 5-(trifluoromethyl) deoxythymidine triphosphate) which has a different molecular weight than the unmodified base so as to improve distinction of masses. Such tags are described in, for example, PCT WO97/33000. This further limits the number of possible base compositions consistent with any mass. For example, 5-(trifluoromethyl)deoxythymidine triphosphate can be used in place of dTTP in a separate nucleic acid amplification reaction. Measurement of the mass shift between a conventional amplification product and the tagged product is used to quantitate the number of thymidine nucleotides in each of the single strands. Because the strands are complementary, the number of adenosine nucleotides in each strand is also determined. [0065]
  • In another amplification reaction, the number of G and C residues in each strand is determined using, for example, the cytidine analog 5-methylcytosine (5-meC) or propyne C. The combination of the A/T reaction and G/C reaction, followed by molecular weight determination, provides a unique base composition. This method is summarized in FIG. 4 and Table 1. [0066]
    TABLE 1
    Total Total
    Total Base Base base base
    Double Single Δmass info info comp. comp.
    strand strand this this other Top Bottom
    Mass tag sequence sequence strand strand strand strand strand
    T*Δmass T*ACGT*ACGT* T*ACGT*ACGT* 3x 3T 3A 3T 3A
    (T*−T) = x AT*GCAT*GCA 2A 2T
    2C 2C
    2G 2C
    AT*GCAT*GCA 2x 2T 2A
    C*Δmass TAC*GTAC*GT TAC*GTAC*GT 2x 2C 2G
    (C*−C) = y ATGC*ATGC*A
    ATGC*ATGC*A 2x 2C 2G
  • The mass tag phosphorothioate A (A*) was used to distinguish a [0067] Bacillus anthracis cluster. The B. anthracis (A14G9C14T9) had an average MW of 14072.26, and the B. anthracis (A1A*13G9C14T9) had an average molecular weight of 14281.11 and the phosphorothioate A had an average molecular weight of +16.06 as determined by ESI-TOF MS. The deconvoluted spectra are shown in FIG. 5.
  • In another example, assume the measured molecular masses of each strand are 30,000.115 Da and 31,000.115 Da respectively, and the measured number of dT and dA residues are (30,28) and (28,30). If the molecular mass is accurate to 100 ppm, there are 7 possible combinations of dG+dC possible for each strand. However, if the measured molecular mass is accurate to 10 ppm, there are only 2 combinations of dG+dC, and at 1 ppm accuracy there is only one possible base composition for each strand. [0068]
  • Signals from the mass spectrometer may be input to a maximum-likelihood detection and classification algorithm such as is widely used in radar signal processing. The detection processing uses matched filtering of BCS observed in mass-basecount space and allows for detection and subtraction of signatures from known, harmless organisms, and for detection of unknown bioagent threats. Comparison of newly observed bioagents to known bioagents is also possible, for estimation of threat level, by comparing their BCS to those of known organisms and to known forms of pathogenicity enhancement, such as insertion of antibiotic resistance genes or toxin genes. [0069]
  • Processing may end with a Bayesian classifier using log likelihood ratios developed from the observed signals and average background levels. The program 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 (e.g. GenBank) is used to define the mass basecount matched filters. The database contains known threat agents and benign background organisms. The latter is used to estimate and subtract the 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. [0070]
  • In one embodiment, a strategy to “triangulate” each organism by measuring signals from multiple core genes is used to reduce false negative and false positive signals, and enable reconstruction of the origin or hybrid or otherwise engineered bioagents. After identification of multiple core genes, alignments are created from nucleic acid sequence databases. The alignments are then analyzed for regions of conservation and variation, and potential primer binding sites flanking variable regions are identified. Next, amplification target regions for signature analysis are selected which distinguishes organisms based on specific genomic differences (i.e., base composition). For example, detection of signatures for the three part toxin genes typical of [0071] B. anthracis (Bowen, J. E. and C. P. Quinn, 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.
  • The present method can also be used to detect single nucleotide polymorphisms (SNPs), or multiple nucleotide polymorphisms, rapidly and accurately. A SNP is defined as a single base pair site in the genome that is different from one individual to another. The difference can be expressed either as a deletion, an insertion or a substitution, and is frequently linked to a disease state. Because they occur every 100-1000 base pairs, SNPs are the most frequently bound type of genetic marker in the human genome. [0072]
  • For example, sickle cell anemia results from an A-T transition, which encodes a valine rather than a glutamic acid residue. Oligonucleotide primers may be designed such that they bind to sequences which flank a SNP site, followed by nucleotide amplification and mass determination of the amplified product. Because the molecular masses of the resulting product from an individual who does not have sickle cell anemia is different from that of the product from an individual who has the disease, the method can be used to distinguish the two individuals. Thus, the method can be used to detect any known SNP in an individual and thus diagnose or determine increased susceptibility to a disease or condition. [0073]
  • In one embodiment, blood is drawn from an individual and peripheral blood mononuclear cells (PBMC) are isolated and simultaneously tested, preferably in a high-throughput screening method, for one or more SNPs using appropriate primers based on the known sequences which flank the SNP region. The National Center for Biotechnology Information maintains a publicly available database of SNPs (http://www.ncbi.nlm.nih.gov/SNP/). [0074]
  • The method of the present invention can also be used for blood typing. The gene encoding A, B or O blood type can differ by four single nucleotide polymorphisms. If the gene contains the sequence CGTGGTGACCCTT, antigen A results. If the gene contains the sequence CGTCGTCACCGCTA antigen B results. If the gene contains the sequence CGTGGT-ACCCCTT, blood group O results (“−” indicates a deletion). These sequences can be distinguished by designing a single primer pair which flanks these regions, followed by amplification and mass determination. [0075]
  • While the present invention has been described with specificity in accordance with certain of its preferred embodiments, the following examples serve only to illustrate the invention and are not intended to limit the same. [0076]
  • EXAMPLE 1
  • Nucleic Acid Isolation and PCR [0077]
  • In one embodiment, nucleic acid is isolated from the organisms and amplified by PCR using standard methods prior to BCS determination by mass spectrometry. Nucleic acid is isolated, for example, by detergent lysis of bacterial cells, centrifugation and ethanol precipitation. Nucleic acid isolation methods are described in, for example, [0078] Current Protocols in Molecular Biology (Ausubel et al.) and Molecular Cloning; A Laboratory Manual (Sambrook et al.). The nucleic acid is then amplified using standard methodology, such as PCR, with primers which bind to conserved regions of the nucleic acid which contain an intervening variable sequence as described below.
  • EXAMPLE 2
  • Mass Spectrometry [0079]
  • FTICR Instrumentation: [0080]
  • The FTICR instrument is based on a 7 tesla actively shielded superconducting magnet and modified Bruker Daltonics Apex II 70e ion optics and vacuum chamber. The spectrometer is interfaced to a LEAP PAL autosampler and a custom fluidics control system for high throughput screening applications. Samples are analyzed directly from 96-well or 384-well microtiter plates at a rate of about 1 sample/minute. The Bruker data-acquisition platform is supplemented with a lab-built ancillary NT datastation which controls the autosampler and contains an arbitrary waveform generator capable of generating complex rf-excite waveforms (frequency sweeps, filtered noise, stored waveform inverse Fourier transform (SWIFT), etc.) for sophisticated tandem MS experiments. For oligonucleotides in the 20-30-mer regime typical performance characteristics include mass resolving power in excess of 100,000 (FWHM), low ppm mass measurement errors, and an operable m/z range between 50 and 5000 m/z. [0081]
  • Modified ESI Source: [0082]
  • In sample-limited analyses, analyte solutions are delivered at 150 nL/minute to a 30 mm i.d. fused-silica ESI emitter mounted on a 3-D micromanipulator. The ESI ion optics consist of a heated metal capillary, an rf-only hexapole, a skimmer cone, and an auxiliary gate electrode. The 6.2 cm rf-only hexapole is comprised of 1 mm diameter rods and is operated at a voltage of 380 Vpp at a frequency of 5 MHz. A lab-built electromechanical shutter can be employed to prevent the electrospray plume from entering the inlet capillary unless triggered to the “open” position via a TTL pulse from the data station. When in the “closed” position, a stable electrospray plume is maintained between the ESI emitter and the face of the shutter. The back face of the shutter arm contains an elastomeric seal which can be positioned to form a vacuum seal with the inlet capillary. When the seal is removed, a 1 mm gap between the shutter blade and the capillary inlet allows constant pressure in the external ion reservoir regardless of whether the shutter is in the open or closed position. When the shutter is triggered, a “time slice” of ions is allowed to enter the inlet capillary and is subsequently accumulated in the external ion reservoir. The rapid response time of the ion shutter (<25 ms) provides reproducible, user defined intervals during which ions can be injected into and accumulated in the external ion reservoir. [0083]
  • Apparatus for Infrared Multiphoton Dissociation [0084]
  • A 25 watt CW CO[0085] 2 laser operating at 10.6 μm has been interfaced to the spectrometer to enable infrared multiphoton dissociation (IRMPD) for oligonucleotide sequencing and other tandem MS applications. An aluminum optical bench is positioned approximately 1.5 m from the actively shielded superconducting magnet such that the laser beam is aligned with the central axis of the magnet. Using standard IR-compatible mirrors and kinematic mirror mounts, the unfocused 3 mm laser beam is aligned to traverse directly through the 3.5 mm holes in the trapping electrodes of the FTICR trapped ion cell and longitudinally traverse the hexapole region of the external ion guide finally impinging on the skimmer cone. This scheme allows IRMPD to be conducted in an m/z selective manner in the trapped ion cell (e.g. following a SWIFT isolation of the species of interest), or in a broadband mode in the high pressure region of the external ion reservoir where collisions with neutral molecules stabilize IRMPD-generated metastable fragment ions resulting in increased fragment ion yield and sequence coverage.
  • EXAMPLE 3
  • Identification of Bioagents [0086]
  • Table 1 shows a small cross section of a database of calculated molecular masses for over 9 primer sets and approximately 30 organisms. The primer sets were derived from rRNA alignment. Examples of regions from rRNA consensus alignments are shown in FIGS. [0087] 1A-1C. Lines with arrows are examples of regions to which intelligent primer pairs for PCR are designed. The primer pairs are >95% conserved in the bacterial sequence database (currently over 10,000 organisms). The intervening regions are variable in length and/or composition, thus providing the base composition “signature” (BCS) for each organism. Primer pairs were chosen so the total length of the amplified region is less than about 80-90 nucleotides. The label for each primer pair represents the starting and ending base number of the amplified region on the consensus diagram.
  • Included in the short bacterial database cross-section in Table 1 are many well known pathogens/biowarfare agents (shown in bold/red typeface) such as [0088] Bacillus anthracis or Yersinia pestis as well as some of the bacterial organisms found commonly in the natural environment such as Streptomyces. Even closely related organisms can be distinguished from each other by the appropriate choice of primers. For instance, two low G+C organisms, Bacillus anthracis and Staph aureus, can be distinguished from each other by using the primer pair defined by 16S 1337 or 23S855 (ΔM of 4 Da).
    TABLE 2
    Cross Section Of A Database Of Calculated Molecular Masses1
    Primer Regions −−>
    Bug Name 16S_971 16S_1100 16S_1337 16S_1294 16S_1228 23S_1021 23S_855 23S_193 23S_115
    Acinetobacter calcoaceticus 55619.1 55004 28446.7 35854.9 51295.4 30299 42654 39557.5 54999
    Bacillus anthracis 55005 54388 28448 35238 51296 30295 42651 39560 56850
    Bacillus cereus 55622.1 54387.9 28447.6 35854.9 51296.4 30295 42651 39560.5 56850.3
    Bordetella bronchiseptica 56857.3 51300.4 28446.7 35857.9 51307.4 30299 42653 39559.5 51920.5
    Borrelia burgdorferi 56231.2 55621.1 28440.7 35852.9 51295.4 30297 42029.9 38941.4 52524.6
    Brucella abortus 58098 55011 28448 35854 50683
    Campylobacter jejuni 58088.5 54386.9 29061.8 35856.9 50674.3 30294 42032.9 39558.5 45732.5
    Chlamydia pnuemoniae 55000 55007 29063 35855 50676 30295 42036 38941 56230
    Clostridium botulinum 55006 53767 28445 35855 51291 30300 42656 39562 54999
    Clostridium difficile 56855.3 54386.9 28444.7 35853.9 51296.4 30294 41417.8 39556.5 55612.2
    Enterococcus faecalis 55620.1 54387.9 28447.6 35858.9 51296.4 30297 42652 39559.5 56849.3
    Escherichia coli 55622 55009 28445 35857 51301 30301 42656 39562 54999
    Francisella tularensis 53769 54385 28445 35856 51298
    Haemophilus influenzae 55620.1 55006 28444.7 35855.9 51298.4 30298 42656 39560.5 55613.1
    Klebsiella pneumoniae 55622.1 55008 28442.7 35856.9 51297.4 30300 42655 39562.5 55000
    Legionella pneumophila 55618 55626 28446 35857 51303
    Mycobacterium avium 54390.9 55631.1 29064.8 35858.9 51915.5 30298 42656 38942.4 56241.2
    Mycobacterium leprae 54389.9 55629.1 29064.8 35860.9 51917.5 30298 42656 39559.5 56240.2
    Mycobacterium tuberculosis 54390.9 55629.1 29064.8 35860.9 51301.4 30299 42656 39560.5 56243.2
    Mycoplasma genitalium 53143.7 45115.4 29061.8 35854.9 50671.3 30294 43264.1 39558.5 56842.4
    Mycoplasma pneumoniae 53143.7 45118.4 29061.8 35854.9 50673.3 30294 43264.1 39559.5 56843.4
    Neisseria gonorrhoeae 55627.1 54389.9 28445.7 35855.9 51302.4 30300 42649 39561.5 55000
    Pseudomonas aeruginosa 55623 55010 28443 35858 51301 30298 43272 39558 55619
    Rickettsia prowazekii 58093 55621 28448 35853 50677 30293 42650 39559 53139
    Rickettsia rickettsii 58094 55623 28448 35853 50679 30293 42648 39559 53755
    Salmonella typhimurium 55622 55005 28445 35857 51301 30301 42658
    Shigella dysenteriae 55623 55009 28444 35857 51301
    Staphylococcus aureus 56854.3 54386.9 28443.7 35852.9 51294.4 30298 42655 39559.5 57466.4
    Streptomyces 54389.9 59341.6 29063.8 35858.9 51300.4 39563.5 56864.3
    Treponema pallidum 56245.2 55631.1 28445.7 35851.9 51297.4 30299 42034.9 38939.4 57473.4
    Vibrio cholerae 55625 55626 28443 35857 52536 29063 30303 35241 50675
    Vibrio parahaemolyticus 54384.9 55626.1 28444.7 34620.7 50064.2
    Yersinia pestis 55620 55626 28443 35857 51299
  • FIG. 6 shows the use of ESI-FT-ICR MS for measurement of exact mass. The spectra from 46mer PCR products originating at position 1337 of the 16S rRNA from [0089] S. aureus (upper) and B. anthracis (lower) are shown. These data are from the region of the spectrum containing signals from the [M-8H+]8− charge states of the respective 5′-3′ strands. The two strands differ by two (AT→CG) substitutions, and have measured masses of 14206.396 and 14208.373±0.010 Da, respectively. The possible base compositions derived from the masses of the forward and reverse strands for the B. anthracis products are listed in Table 3.
    TABLE 3
    Possible base composition for B. anthracis products
    Calc. Mass Error Base Comp.
    14208.2935 0.079520 A1 G17 C10 T18
    14208.3160 0.056980 A1 G20 C15 T10
    14208.3386 0.034440 A1 G23 C20 T2
    14208.3074 0.065560 A6 G11 C3 T26
    14208.3300 0.043020 A6 G14 C8 T18
    14208.3525 0.020480 A6 G17 C13 T10
    14208.3751 0.002060 A6 G20 C18 T2
    14208.3439 0.029060 A11 G8 C1 T26
    14208.3665 0.006520 A11 G11 C6 T18
    14208.3890 0.016020 A11 G14 C11 T10
    14208.4116 0.038560 A11 G17 C16 T2
    14208.4030 0.029980 A16 G8 C4 T18
    14208.4255 0.052520 A16 G11 C9 T10
    14208.4481 0.075060 A16 G14 C14 T2
    14208.4395 0.066480 A21 G5 C2 T18
    14208.4620 0.089020 A21 G8 C7 T10
    14079.2624 0.080600 A0 G14 C13 T19
    14079.2849 0.058060 A0 G17 C18 T11
    14079.3075 0.035520 A0 G20 C23 T3
    14079.2538 0.089180 A5 G5 C1 T35
    14079.2764 0.066640 A5 G8 C6 T27
    14079.2989 0.044100 A5 G11 C11 T19
    14079.3214 0.021560 A5 G14 C16 T11
    14079.3440 0.000980 A5 G17 C21 T3
    14079.3129 0.030140 A10 G5 C4 T27
    14079.3354 0.007600 A10 G8 C9 T19
    14079.3579 0.014940 A10 G11 C14 T11
    14079.3805 0.037480 A10 G14 C19 T3
    14079.3494 0.006360 A15 G2 C2 T27
    14079.3719 0.028900 A15 G5 C7 T19
    14079.3944 0.051440 A15 G8 C12 T11
    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 compositions for the forward strand and the 18 compositions for the reverse strand that were calculated, only one pair (shown in bold) are complementary, corresponding to the actual base compositions of the [0090] B. anthracis PCR products.
  • EXAMPLE 4
  • BCS of Region from [0091] Bacillus anthracis and Bacillus cereus
  • A conserved Bacillus region from [0092] B. anthracis (A14G9C14T9) and B. cereus (A15G9C13T9) having a C to A base change was synthesized and subjected to ESI-TOF MS. The results are shown in FIG. 7 in which the two regions are clearly distinguished using the method of the present invention (MW=14072.26 vs. 14096.29).
  • EXAMPLE 5 [0093]
  • Identification of Additional Bioagents [0094]
  • In other examples of the present invention, the pathogen [0095] Vibrio cholera can be distinguished from Vibrio parahemolyticus with ΔM>600 Da using one of three 16S primer sets shown in Table 2 (16S 971, 16S 1228 or 16S1294) as shown in Table 4. The two mycoplasma species in the list (M. genitalium and M. pneumoniae) can also be distinguished from each other, as can the three mycobacteriae. While the direct mass measurements of amplified products can identify and distinguish a large number of organisms, measurement of the base composition signature provides dramatically enhanced the base composition signature provides dramatically enhanced resolving power for closely related organisms. In cases such as Bacillus anthracis and Bacillus cereus that are virtually indistinguishable from each other based solely on mass differences, compositional analysis or fragmentation patterns are used to resolve the differences. The single base difference between the two organisms yields different fragmentation patterns, and despite the presence of the ambiguous/unidentified base N at position 20 in B. anthracis, the two organisms can be identified.
  • Tables 4a-b show examples of primer pairs from Table 1 which distinguish pathogens from background. [0096]
    TABLE 4a
    Organism name 23S_855 16S_1337 23S_1021
    Bacillus anthracis 42650.98 28447.65 30294.98
    Staphylococcus aureus 42654.97 28443.67 30297.96
  • [0097]
    TABLE 4b
    Organism name 16S_971 16S_1294 16S_1228
    Vibrio cholerae 55625.09 35856.87 52535.59
    Vibrio parahaemolyticus 54384.91 34620.67 50064.19
  • Table 4 shows the expected molecular weight and base composition of [0098] region 16S1100-1188 in Mycobacterium avium and Streptomyces sp.
    TABLE 5
    Organism Molecular Base
    Region name Length weight comp.
    16S_1100-1188 Mycobacte- 82 25624.1728 A16G32C18T16
    rium avium
    16S_1100-1188 Strepto- 96 29904.871 A17G38C27T14
    myces sp.
  • Table 5 shows base composition (single strand) results for 16S[0099] 1100-1188 primer amplification reactions different species of bacteria. Species which are repeated in the table (e.g., Clostridium botulinum) are different strains which have different base compositions in the 16S1100-1188 region.
    TABLE 6
    Organism name Base comp.
    Mycobacterium A16G32C18T16
    Streptomyces sp. A17G38C27T14
    Ureaplasma urealyticum A18G30C17T17
    Streptomyces sp. A19G36C24T18
    Mycobacterium leprae A20G32C22T16
    M. tuberculosis A 20 G 33 C 21 T 16
    Nocardia asteroides A 20 G 33 C 21 T 16
    Fusobacterium necroforum A21G26C22T18
    Listeria monocytogenes A21G27C19T19
    Clostridium botulinum A21G27C19T21
    Neisseria gonorrhoeae A21G28C21T18
    Bartonella quintana A21G30C22T16
    Enterococcus faecalis A22G27C20T19
    Bacillus megaterium A22G28C20T18
    Bacillus subtilis A22G28C21T17
    Pseudomonas aeruginosa A22G29C23T15
    Legionella pneumophila A22G32C20T16
    Mycoplasma pneumoniae A23G20C14T16
    Clostridium botulinum A23G26C20T19
    Enterococcus faecium A23G26C21T18
    Acinetobacter calcoaceti A23G26C21T19
    Leptospira borgpeterseni A 23 G 26 C 24 T 15
    Leptospira interrogans A 23 G 26 C 24 T 15
    Clostridium perfringens A23G27C19T19
    Bacillus anthracis A 23 G 27 C 20 T 18
    Bacillus cereus A 23 G 27 C 20 nT 18
    Bacillus thuringiensis A 23 G 27 C 20 T 18
    Aeromonas hydrophila A23G29C21T16
    Escherichia coli A23G29C21T16
    Pseudomonas putida A23G29C21T17
    Escherichia coli A 23 G 29 C 22 T 15
    Shigella dysenteriae A 23 G 29 C 22 T 15
    Vibrio cholerae avium A23G30C21T16
    Aeromonas hydrophila A 23 G 31 C 21 T 15
    Aeromonas salmonicida A 23 G 31 C 21 T 15
    Mycoplasma genitalium A24G19C12T18
    Clostridium botulinum A24G25C18T20
    Bordetella bronchiseptica A24G26C19T14
    Francisella tularensis A24G26C19T19
    Bacillus anthracis A 24 G 26 C 20 T 18
    Campylobacter jejuni A 24 G 26 C 20 T 18
    Staphylococcus aureus A 24 G 26 C 20 T 18
    Helicobacter pylori A24G26C20T19
    Helicobacter pylori A24G26C21T18
    Moraxella catarrhalis A24G26C23T16
    Haemophilus influenzae Rd A24G28C20T17
    Chlamydia trachomatis A 24 G 28 C 21 T 16
    Chlamydophila pneumoniae A 24 G 28 C 21 T 16
    C. pneumonia AR39 A 24 G 28 C 21 T 16
    Pseudomonas putida A24G29C21T16
    Proteus vulgaris A 24 G 30 C 21 T 15
    Yersinia pestis A 24 G 30 C 21 T 15
    Yersinia pseudotuberculos A 24 G 30 C 21 T 15
    Clostridium botulinum A25G24C18T21
    Clostridium tetani A25G25C18T20
    Francisella tularensis A25G25C19T19
    Acinetobacter calcoacetic A25G26C20T19
    Bacteriodes fragilis A25G27C16T22
    Chlamydophila psittaci A25G27C21T16
    Borrelia burgdorferi A25G29C17T19
    Streptobacillus monilifor A26G26C20T16
    Rickettsia prowazekii A26G28C18T18
    Rickettsia rickettsii A26G28C20T16
    Mycoplasma mycoides A28G23C16T20
  • The same organism having different base compositions are different strains. Groups of organisms which are highlighted or in italics have the same base compositions in the amplified region. Some of these organisms can be distinguished using multiple primers. For example, [0100] Bacillus anthracis can be distinguished from Bacillus cereus and Bacillus thuringiensis using the primer 16S971-1062 (Table 6). Other primer pairs which produce unique base composition signatures are shown in Table 6 (bold). Clusters containing very similar threat and ubiquitous non-threat organisms (e.g. anthracis cluster) are distinguished at high resolution with focused sets of primer pairs. The known biowarfare agents in Table 6 are Bacillus anthracis, Yersinia pestis, Francisella tularensis and Rickettsia prowazekii.
    TABLE 7
    Organism 16S_971-1062 16S_1228-1310 16S_1100-1188
    Aeromonas A21G29C22T20 A22G27C21T13 A23G31C21T15
    hydrophila
    Aeromonas A21G29C22T20 A22G27C21T13 A23G31C21T15
    salmonicida
    Bacillus anthracis A 21 G 27 C 22 T 22 A24G22C19T18 A23G27C20T18
    Bacillus cereus A22G27C21T22 A24G22C19T18 A23G27C20T18
    Bacillus A22G27C21T22 A24G22C19T18 A23G27C20T18
    thuringiensis
    Chlamydia A 22 G 26 C 20 T 23 A 24 G 23 C 19 T 16 A24G28C21T16
    trachomatis
    Chlamydia A26G23C20T22 A26G22C16T18 A24G28C21T16
    pneumoniae
    AR39
    Leptospira A22G26C20T21 A22G25C21T15 A23G26C24T15
    borgpetersenii
    Leptospira A22G26C20T21 A22G25C21T15 A23G26C24T15
    interrogans
    Mycoplasma A28G23C15T22 A 30 G 18 C 15 T 19 A 24 G 19 C 12 T 18
    genitalium
    Mycoplasma A28G23C15T22 A 27 G 19 C 16 T 20 A 23 G 20 C 14 T 16
    pneumoniae
    Escherichia coli A 22 G 28 C 20 T 22 A24G25C21T13 A23G29C22T15
    Shigella A 22 G 28 C 21 T 21 A24G25C21T13 A23G29C22T15
    dysenteriae
    Proteus vulgaris A 23 G 26 C 22 T 21 A 26 G 24 C 19 T 14 A24G30C21T15
    Yersinia pestis A24G25C21T22 A25G24C20T14 A24G30C21T15
    Yersinia A24G25C21T22 A25G24C20T14 A24G30C21T15
    pseudotubercu-
    losis
    Francisella A 20 G 25 C 21 T 23 A 23 G 26 C 17 T 17 A 24 G 26 C 19 T 19
    tularensis
    Rickettsia A 21 G 26 C 24 T 25 A 24 G 23 C 16 T 19 A 26 G 28 C 18 T 18
    prowazekii
    Rickettsia A 21 G 26 C 25 T 24 A 24 G 24 C 17 T 17 A 26 G 28 C 20 T 16
    rickettsii
  • The sequence of [0101] B. anthracis and B. cereus in region 16S971 is shown below. Shown in bold is the single base difference between the two species which can be detected using the methods of the present invention. B. anthracis has an ambiguous base at position 20.
    B. anthracis_16S_971
    GCGAAGAACCUUACCAGGUNUUGACAUCCUCUGACAACCCUAGAGAUAGGGCUUCUCCUUC (SEQ ID NO:1)
    GGGAGCAGAGUGACAGGUGGUGCAUGGUU
    B. cereus_16S_971
    GCGAAGAACCUUACCAGGUCUUGACAUCCUCUGAAAACCCUAGAGAUAGGGCUUCUCCUUC (SEQ ID NO:2)
    GGGAGCAGAGUGACAGGUGGUGCAUGGUU
  • EXAMPLE 6
  • ESI-TOF MS of sspE 56-mer Plus Calibrant [0102]
  • The mass measurement accuracy that can be obtained using an internal mass standard in the ESI-MS study of PCR products is shown in FIG. 8. The mass standard was a 20-mer phosphorothioate oligonucleotide added to a solution containing a 56-mer PCR product from the [0103] B. anthracis spore coat protein sspE. The mass of the expected PCR product distinguishes B. anthracis from other species of Bacillus such as B. thuringiensis and B. cereus.
  • EXAMPLE 7
  • [0104] B. anthracis ESI-TOF Synthetic 16S1228 Duplex
  • An ESI-TOF MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1228 region of the [0105] B. anthracis 16S rRNA gene. The results (FIG. 9) show that the molecular weights of the forward and reverse strands can be accurately determined and easily distinguish the two strands. The [M-21H+]21− and [M-20H+]20− charge states are shown.
  • EXAMPLE 8
  • ESI-FTICR-MS of [0106] Synthetic B. anthracis 16S1337 46 Base Pair Duplex
  • An ESI-FTICR-MS spectrum was obtained from an aqueous solution containing 5 μM each of synthetic analogs of the expected forward and reverse PCR products from the nucleotide 1337 region of the [0107] B. anthracis 16S rRNA gene. The results (FIG. 10) show that the molecular weights of the strands can be distinguished by this method. The [M-16H+]16− through [M-10H+]10− charge states are shown. The insert highlights the resolution that can be realized on the FTICR-MS instrument, which allows the charge state of the ion to be determined from the mass difference between peaks differing by a single 13C substitution.
  • EXAMPLE 9
  • ESI-TOF MS of 56-mer Oligonucleotide from saspB Gene of [0108] B. anthracis with Internal Mass Standard
  • ESI-TOF MS spectra were obtained on a synthetic 56-mer oligonucleotide (5 μM )from the saspB gene of [0109] B. anthracis containing an internal mass standard at an ESI of 1.7 μL/min as a function of sample consumption. The results (FIG. 11) show that the signal to noise is improved as more scans are summed, and that the standard and the product are visible after only 100 scans.
  • EXAMPLE 10
  • ESI-TOF MS of an Internal Standard with Tributylammonium (TBA)-Trifluoroacetate (TFA) Buffer [0110]
  • An ESI-TOF-MS spectrum of a 20-mer phosphorothioate mass standard was obtained following addition of 5 mM TBA-TFA buffer to the solution. This buffer strips charge from the oligonucleotide and shifts the most abundant charge state from [M-8H[0111] +]8− to [M-3H+]3− (FIG. 12).

Claims (48)

What is claimed is:
1. A method of identifying an unknown bioagent comprising:
(a) contacting nucleic acid from said bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence of the bioagent;
(b) amplifying said variable nucleic acid sequence to produce an amplification product;
(c) determining the molecular mass of said amplification product; and
(d) comparing said molecular mass to one or more molecular masses of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies said unknown bioagent.
2. The method of claim 1, wherein said sequences to which said at least one pair of oligonucleotide primers hybridize are highly conserved.
3. The method of claim 1, wherein said amplifying step comprises polymerase chain reaction.
4. The method of claim 1, wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.
5. The method of claim 1, wherein said bioagent is a bacterium, virus, cell or spore.
6. The method of claim 1, wherein said nucleic acid is ribosomal RNA.
7. The method of claim 1, wherein said nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.
8. The method of claim 1, wherein said amplification product is ionized prior to molecular mass determination.
9. The method of claim 1, further comprising the step of isolating nucleic acid from said bioagent prior to contacting said nucleic acid with said at least one pair of oligonucleotide primers.
10. The method of claim 1, further comprising the step of performing steps (a)-(d) u sing a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification product obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d).
11. The method of claim 1, wherein said one or more molecular masses are contained in a database of molecular masses.
12. The method of claim 1, wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
13. The method of claim 1, wherein said molecular mass is determined by mass spectrometry.
14. The method of claim 11, wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF and triple quadrupole.
15. The method of claim 1, further comprising performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.
16. The method of claim 1, wherein said oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within said primer, wherein said base analog binds with increased affinity to its complement compared to the native base.
17. The method of claim 16, wherein said primer comprises a universal base at position 3 of each triplet within said primer.
18. The method of claim 16, wherein said base analog is selected from the group consisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.
19. The method of claim 16, wherein said universal base is selected from the group consisting of inosine, guanidine uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, and 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
20. A method of identifying an unknown bioagent comprising:
contacting nucleic acid from said bioagent with at least one pair of oligonucleotide primers which hybridize to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence;
amplifying said variable nucleic acid sequence to produce an amplification product;
determining the base composition of said amplification product; and
comparing said base composition to one or more base compositions of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies said unknown bioagent.
21. The method of claim 20, wherein said sequences to which said at least one pair of oligonucleotide primers hybridize are highly conserved.
22. The method of claim 20, wherein said amplifying step comprises polymerase chain reaction.
23. The method of claim 20, wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.
24. The method of claim 20, wherein said bioagent is a bacterium, virus, cell or spore.
25. The method of claim 20, wherein said nucleic acid is ribosomal RNA.
26. The method of claim 20, wherein said nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.
27. The method of claim 20, wherein said amplification product is ionized prior to base composition determination.
28. The method of claim 20, further comprising the step of isolating nucleic acid from said bioagent prior to contacting said nucleic acid with said at least one pair of oligonucleotide primers.
29. The method of claim 20, further comprising the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more base compositions of amplification product obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d).
30. The method of claim 20, wherein said one or more base composition signatures are contained in a database of base composition signatures.
31. The method of claim 20, wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
32. The method of claim 20, wherein said base composition signature is determined by mass spectrometry.
33. The method of claim 32, wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), q-TOF and triple quadrupole.
34. The method of claim 20, further comprising performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.
35. The method of claim 20, wherein said oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within said primer, wherein said base analog binds with increased affinity to its complement compared to the native base.
36. The method of claim 35, wherein said primer comprises a universal base at position 3 of each triplet within said primer.
37. The method of claim 35, wherein said base analog is selected from the group consisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.
38. The method of claim 36, wherein said universal base is selected from the group consisting of inosine, guanidine uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, and 1-(2-deoxy-β-D-ribofuranosyl)-imidazole-4-carboxamide.
39. A method for detecting a single nucleotide polymorphism in an individual, comprising the steps of:
isolating nucleic acid from said individual;
contacting said nucleic acid with oligonucleotide primers which hybridize to regions of said nucleic acid which flank a region comprising said potential polymorphism;
amplifying said region to produce an amplification product;
determining the molecular mass of said amplification product;
comparing said molecular mass to the molecular mass of said region in an individual known to have said polymorphism, wherein if said molecular masses are the same then said individual has said polymorphism.
40. The method of claim 39, wherein said polymorphism is associated with a disease.
41. The method of claim 39, wherein said polymorphism is a blood group antigen.
42. The method of claim 39, wherein said amplification step is the polymerase chain reaction.
43. The method of claim 39, wherein said amplification step is ligase chain reaction or strand displacement amplification.
44. The method of claim 39, wherein said amplification product is ionized prior to mass determination.
45. The method of claim 39, wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.
46. The method of claim 39, wherein said primers hybridize to conserved sequences.
47. The method of claim 39, wherein said molecular mass is determined by mass spectrometry.
48. The method of claim 47, wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF and triple quadrupole.
US09/798,007 2001-03-02 2001-03-02 Method for rapid detection and identification of bioagents Abandoned US20030027135A1 (en)

Priority Applications (54)

Application Number Priority Date Filing Date Title
US09/798,007 US20030027135A1 (en) 2001-03-02 2001-03-02 Method for rapid detection and identification of bioagents
JP2002570692A JP2005504508A (en) 2001-03-02 2002-03-04 Rapid detection and identification of biological materials
NZ527857A NZ527857A (en) 2001-03-02 2002-03-04 Identification of an unknown bacterial bioagent comprising an amplification step followed by determining molecular mass of the amplification product
EP02709785A EP1364064B1 (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bacteria
EP10179791A EP2311992B1 (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bioagents
AU2002244250A AU2002244250B2 (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bioagents
RU2003129269/13A RU2003129269A (en) 2001-03-02 2002-03-04 METHOD FOR QUICK DETECTION AND IDENTIFICATION OF BIOAGENTS
EP10179789.2A EP2333118B1 (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bioagents
CA2853831A CA2853831C (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of single nucleotide polymorphisms
CN201210348178.6A CN102912036B (en) 2001-03-02 2002-03-04 Quickly detection and the method for qualification inanimate object
MX2014010221A MX349763B (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bioagents.
IL15766102A IL157661A0 (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bioagents
PCT/US2002/006763 WO2002070664A2 (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bioagents
ES10179789.2T ES2448770T3 (en) 2001-03-02 2002-03-04 Procedure for rapid detection and identification of bioagents
ES02709785T ES2394731T3 (en) 2001-03-02 2002-03-04 Procedure for rapid detection and identification of bacteria
CN028091221A CN1505685B (en) 2001-03-02 2002-03-04 Methods for rapid detection and identification of bioagents for environmental testing
EP10179795.9A EP2322649B1 (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bioagents
ES10179795.9T ES2451003T3 (en) 2001-03-02 2002-03-04 Procedure for rapid identification and detection of bioagents
CA2439655A CA2439655C (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bioagents
MXPA03007927A MXPA03007927A (en) 2001-03-02 2002-03-04 Method for rapid detection and identification of bioagents.
US10/156,608 US7108974B2 (en) 2001-03-02 2002-05-24 Method for rapid detection and identification of bioagents
US10/319,290 US20030175696A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/318,881 US20030175695A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/319,342 US20030175697A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/326,047 US20030190605A1 (en) 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents for environmental testing
US10/430,253 US20040110169A1 (en) 2001-03-02 2003-05-06 Method for rapid detection and identification of bioagents
US10/435,307 US20040202997A1 (en) 2001-03-02 2003-05-09 Method for rapid detection and identification of bioagents
IL157661A IL157661A (en) 2001-03-02 2003-08-29 Method for rapid detection and identification of bioagents
ZA200306810A ZA200306810B (en) 2001-03-02 2003-09-01 Method for rapid detection and identification of bioagents.
US10/660,122 US7781162B2 (en) 2001-03-02 2003-09-11 Methods for rapid identification of pathogens in humans and animals
US10/660,997 US7226739B2 (en) 2001-03-02 2003-09-12 Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations
US10/660,996 US7255992B2 (en) 2001-03-02 2003-09-12 Methods for rapid detection and identification of bioagents for environmental and product testing
US10/660,998 US7666588B2 (en) 2001-03-02 2003-09-12 Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US10/728,486 US7718354B2 (en) 2001-03-02 2003-12-05 Methods for rapid identification of pathogens in humans and animals
AU2003298030A AU2003298030B2 (en) 2001-03-02 2003-12-05 Methods for rapid identification of pathogens in humans and animals
US11/233,630 US8017322B2 (en) 2001-03-02 2005-09-21 Method for rapid detection and identification of bioagents
US11/331,987 US8017358B2 (en) 2001-03-02 2006-01-13 Method for rapid detection and identification of bioagents
US11/331,978 US7741036B2 (en) 2001-03-02 2006-01-13 Method for rapid detection and identification of bioagents
US11/682,259 US8563250B2 (en) 2001-03-02 2007-03-05 Methods for identifying bioagents
US11/869,449 US20080311558A1 (en) 2001-03-02 2007-10-09 Methods For Rapid Identification Of Pathogens In Humans And Animals
US11/929,707 US8017743B2 (en) 2001-03-02 2007-10-30 Method for rapid detection and identification of bioagents
US11/929,910 US8214154B2 (en) 2001-03-02 2007-10-30 Systems for rapid identification of pathogens in humans and animals
US11/930,017 US8265878B2 (en) 2001-03-02 2007-10-30 Method for rapid detection and identification of bioagents
US11/929,930 US8802372B2 (en) 2001-03-02 2007-10-30 Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US11/930,741 US8815513B2 (en) 2001-03-02 2007-10-31 Method for rapid detection and identification of bioagents in epidemiological and forensic investigations
US12/326,800 US8268565B2 (en) 2001-03-02 2008-12-02 Methods for identifying bioagents
US12/605,628 US20100145626A1 (en) 2001-03-02 2009-10-26 Systems for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
JP2009245976A JP5121803B2 (en) 2001-03-02 2009-10-26 Rapid detection and identification of biological materials
AU2010200893A AU2010200893B2 (en) 2001-03-02 2010-03-10 Methods for rapid identification of pathogens in humans and animals
US13/174,254 US9416424B2 (en) 2001-03-02 2011-06-30 Methods for rapid identification of pathogens in humans and animals
US14/058,723 US20150225780A1 (en) 2001-03-02 2013-10-21 Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations
US14/456,806 US9752184B2 (en) 2001-03-02 2014-08-11 Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
IL238855A IL238855A0 (en) 2001-03-02 2015-05-17 Method for rapid detection and identification of bioagents
US15/237,261 US20170044630A1 (en) 2001-03-02 2016-08-15 Methods For Rapid Identification Of Pathogens In Humans And Animals

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
US09/798,007 US20030027135A1 (en) 2001-03-02 2001-03-02 Method for rapid detection and identification of bioagents

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
US10/156,608 Continuation-In-Part US7108974B2 (en) 2001-03-02 2002-05-24 Method for rapid detection and identification of bioagents
US10/323,438 Continuation-In-Part US20040121310A1 (en) 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents in forensic studies

Related Child Applications (13)

Application Number Title Priority Date Filing Date
US10/156,608 Division US7108974B2 (en) 2001-03-02 2002-05-24 Method for rapid detection and identification of bioagents
US10/319,342 Continuation US20030175697A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/319,290 Continuation US20030175696A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/318,881 Continuation US20030175695A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/323,438 Continuation-In-Part US20040121310A1 (en) 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents in forensic studies
US32318602A Continuation-In-Part 2001-03-02 2002-12-18
US10/326,047 Continuation-In-Part US20030190605A1 (en) 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents for environmental testing
US10/325,527 Continuation-In-Part US20040121335A1 (en) 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents associated with host versus graft and graft versus host rejections
US10/435,307 Continuation US20040202997A1 (en) 2001-03-02 2003-05-09 Method for rapid detection and identification of bioagents
US10/660,122 Continuation-In-Part US7781162B2 (en) 2001-03-02 2003-09-11 Methods for rapid identification of pathogens in humans and animals
US10/660,998 Continuation-In-Part US7666588B2 (en) 2001-03-02 2003-09-12 Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US10/660,996 Continuation-In-Part US7255992B2 (en) 2001-03-02 2003-09-12 Methods for rapid detection and identification of bioagents for environmental and product testing
US10/660,997 Continuation-In-Part US7226739B2 (en) 2001-03-02 2003-09-12 Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations

Publications (1)

Publication Number Publication Date
US20030027135A1 true US20030027135A1 (en) 2003-02-06

Family

ID=25172308

Family Applications (13)

Application Number Title Priority Date Filing Date
US09/798,007 Abandoned US20030027135A1 (en) 2001-03-02 2001-03-02 Method for rapid detection and identification of bioagents
US10/156,608 Expired - Lifetime US7108974B2 (en) 2001-03-02 2002-05-24 Method for rapid detection and identification of bioagents
US10/318,881 Abandoned US20030175695A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/319,290 Abandoned US20030175696A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/319,342 Abandoned US20030175697A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/326,047 Abandoned US20030190605A1 (en) 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents for environmental testing
US10/430,253 Abandoned US20040110169A1 (en) 2001-03-02 2003-05-06 Method for rapid detection and identification of bioagents
US10/435,307 Abandoned US20040202997A1 (en) 2001-03-02 2003-05-09 Method for rapid detection and identification of bioagents
US11/233,630 Expired - Fee Related US8017322B2 (en) 2001-03-02 2005-09-21 Method for rapid detection and identification of bioagents
US11/331,987 Expired - Fee Related US8017358B2 (en) 2001-03-02 2006-01-13 Method for rapid detection and identification of bioagents
US11/331,978 Expired - Fee Related US7741036B2 (en) 2001-03-02 2006-01-13 Method for rapid detection and identification of bioagents
US11/929,707 Expired - Fee Related US8017743B2 (en) 2001-03-02 2007-10-30 Method for rapid detection and identification of bioagents
US11/930,017 Expired - Fee Related US8265878B2 (en) 2001-03-02 2007-10-30 Method for rapid detection and identification of bioagents

Family Applications After (12)

Application Number Title Priority Date Filing Date
US10/156,608 Expired - Lifetime US7108974B2 (en) 2001-03-02 2002-05-24 Method for rapid detection and identification of bioagents
US10/318,881 Abandoned US20030175695A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/319,290 Abandoned US20030175696A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/319,342 Abandoned US20030175697A1 (en) 2001-03-02 2002-12-13 Method for rapid detection and identification of bioagents
US10/326,047 Abandoned US20030190605A1 (en) 2001-03-02 2002-12-18 Methods for rapid detection and identification of bioagents for environmental testing
US10/430,253 Abandoned US20040110169A1 (en) 2001-03-02 2003-05-06 Method for rapid detection and identification of bioagents
US10/435,307 Abandoned US20040202997A1 (en) 2001-03-02 2003-05-09 Method for rapid detection and identification of bioagents
US11/233,630 Expired - Fee Related US8017322B2 (en) 2001-03-02 2005-09-21 Method for rapid detection and identification of bioagents
US11/331,987 Expired - Fee Related US8017358B2 (en) 2001-03-02 2006-01-13 Method for rapid detection and identification of bioagents
US11/331,978 Expired - Fee Related US7741036B2 (en) 2001-03-02 2006-01-13 Method for rapid detection and identification of bioagents
US11/929,707 Expired - Fee Related US8017743B2 (en) 2001-03-02 2007-10-30 Method for rapid detection and identification of bioagents
US11/930,017 Expired - Fee Related US8265878B2 (en) 2001-03-02 2007-10-30 Method for rapid detection and identification of bioagents

Country Status (13)

Country Link
US (13) US20030027135A1 (en)
EP (4) EP1364064B1 (en)
JP (2) JP2005504508A (en)
CN (2) CN102912036B (en)
AU (2) AU2002244250B2 (en)
CA (2) CA2853831C (en)
ES (3) ES2451003T3 (en)
IL (3) IL157661A0 (en)
MX (2) MX349763B (en)
NZ (1) NZ527857A (en)
RU (1) RU2003129269A (en)
WO (1) WO2002070664A2 (en)
ZA (1) ZA200306810B (en)

Cited By (88)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20040161770A1 (en) * 2001-03-02 2004-08-19 Ecker David J. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US20040209260A1 (en) * 2003-04-18 2004-10-21 Ecker David J. Methods and apparatus for genetic evaluation
US20040219517A1 (en) * 2001-03-02 2004-11-04 Ecker David J. Methods for rapid identification of pathogens in humans and animals
WO2005034855A2 (en) * 2003-09-05 2005-04-21 The General Hospital Corporation Photodynamic inactivation of bacterial spores
US20050123992A1 (en) * 2003-12-03 2005-06-09 Palo Alto Research Center Incorporated Concentration and focusing of bio-agents and micron-sized particles using traveling wave grids
US20050130196A1 (en) * 2003-05-13 2005-06-16 Hofstadler Steven A. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US20050142584A1 (en) * 2003-10-01 2005-06-30 Willson Richard C. Microbial identification based on the overall composition of characteristic oligonucleotides
US20050164215A1 (en) * 2003-05-13 2005-07-28 Hofstadler Steven A. Methods for rapid purification of nucleic acids for subsquent analysis by mass spectrometery by solution capture
US20060057603A1 (en) * 2004-03-11 2006-03-16 The Regents Of The University Of California Detecting Bacillus anthracis
US20060121520A1 (en) * 2001-03-02 2006-06-08 Ecker David J Method for rapid detection and identification of bioagents
US20060205040A1 (en) * 2005-03-03 2006-09-14 Rangarajan Sampath Compositions for use in identification of adventitious viruses
US20060240412A1 (en) * 2003-09-11 2006-10-26 Hall Thomas A Compositions for use in identification of adenoviruses
US20070218467A1 (en) * 2005-07-21 2007-09-20 Ecker David J Methods for rapid identification and quantitation of nucleic acid variants
US20070224614A1 (en) * 2003-09-11 2007-09-27 Rangarajan Sampath Compositions for use in identification of bacteria
WO2007140417A2 (en) 2006-05-31 2007-12-06 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US7309410B2 (en) 2003-12-03 2007-12-18 Palo Alto Research Center Incorporated Traveling wave grids and algorithms for biomolecule separation, transport and focusing
US20080096766A1 (en) * 2006-06-16 2008-04-24 Sequenom, Inc. Methods and compositions for the amplification, detection and quantification of nucleic acid from a sample
US20080138808A1 (en) * 2003-09-11 2008-06-12 Hall Thomas A Methods for identification of sepsis-causing bacteria
US20080227087A1 (en) * 2005-10-11 2008-09-18 Ann Huletsky Sequences for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) of MREJ types xi to xx
US20090004643A1 (en) * 2004-02-18 2009-01-01 Isis Pharmaceuticals, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US20090125245A1 (en) * 2004-05-25 2009-05-14 Isis Pharmaceuticals, Inc. Methods For Rapid Forensic Analysis Of Mitochondrial DNA
US20090263809A1 (en) * 2008-03-20 2009-10-22 Zygem Corporation Limited Methods for Identification of Bioagents
US20100035227A1 (en) * 2004-03-03 2010-02-11 Isis Pharmaceuticals, Inc. Compositions for use in identification of alphaviruses
US20100035239A1 (en) * 2003-09-11 2010-02-11 Isis Pharmaceuticals, Inc. Compositions for use in identification of bacteria
US20100035232A1 (en) * 2006-09-14 2010-02-11 Ecker David J Targeted whole genome amplification method for identification of pathogens
US20100075430A1 (en) * 2008-09-16 2010-03-25 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US7714275B2 (en) 2004-05-24 2010-05-11 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US7718354B2 (en) 2001-03-02 2010-05-18 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US20100129811A1 (en) * 2003-09-11 2010-05-27 Ibis Biosciences, Inc. Compositions for use in identification of pseudomonas aeruginosa
US20100136515A1 (en) * 2005-03-03 2010-06-03 Ibis Biosciences, Inc. Compositions for use in identification of papillomavirus
US20100184035A1 (en) * 2007-02-23 2010-07-22 Ibis Bioscience, Inc. Methods for rapid forensic dna analysis
US20100204266A1 (en) * 2007-03-23 2010-08-12 Ibis Biosciences, INC Compositions for use in identification of mixed populations of bioagents
US20100219336A1 (en) * 2009-02-12 2010-09-02 Ibis Biosciences, Inc. Ionization probe assemblies
US20100248298A1 (en) * 2009-02-03 2010-09-30 Bruker Daltonik Gmbh Mass spectrometric identification of microorganisms in complex samples
US7811753B2 (en) 2004-07-14 2010-10-12 Ibis Biosciences, Inc. Methods for repairing degraded DNA
US20100279295A1 (en) * 2009-03-18 2010-11-04 Sequenom, Inc. Use of thermostable endonucleases for generating reporter molecules
US20100291544A1 (en) * 2007-05-25 2010-11-18 Ibis Biosciences, Inc. Compositions for use in identification of strains of hepatitis c virus
US20110014027A1 (en) * 2009-07-17 2011-01-20 Ibis Biosciences, Inc. Lift and mount apparatus
US20110028334A1 (en) * 2009-07-31 2011-02-03 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
US20110045456A1 (en) * 2007-06-14 2011-02-24 Ibis Biosciences, Inc. Compositions for use in identification of adventitious contaminant viruses
US20110065111A1 (en) * 2009-08-31 2011-03-17 Ibis Biosciences, Inc. Compositions For Use In Genotyping Of Klebsiella Pneumoniae
US20110091882A1 (en) * 2009-10-02 2011-04-21 Ibis Biosciences, Inc. Determination of methylation status of polynucleotides
WO2011047307A1 (en) 2009-10-15 2011-04-21 Ibis Biosciences, Inc. Multiple displacement amplification
US20110097704A1 (en) * 2008-01-29 2011-04-28 Ibis Biosciences, Inc. Compositions for use in identification of picornaviruses
US20110143358A1 (en) * 2008-05-30 2011-06-16 Ibis Biosciences, Inc. Compositions for use in identification of tick-borne pathogens
US20110151437A1 (en) * 2008-06-02 2011-06-23 Ibis Biosciences, Inc. Compositions for use in identification of adventitious viruses
US20110166040A1 (en) * 1997-09-05 2011-07-07 Ibis Biosciences, Inc. Compositions for use in identification of strains of e. coli o157:h7
US20110172925A1 (en) * 2001-06-26 2011-07-14 Ibis Biosciences, Inc. Secondary Structure Defining Database And Methods For Determining Identity And Geographic Origin Of An Unknown Bioagent Thereby
US20110177515A1 (en) * 2008-05-30 2011-07-21 Ibis Biosciences, Inc. Compositions for use in identification of francisella
US20110183344A1 (en) * 2008-10-03 2011-07-28 Rangarajan Sampath Compositions for use in identification of clostridium difficile
US20110183345A1 (en) * 2008-10-03 2011-07-28 Ibis Biosciences, Inc. Compositions for use in identification of streptococcus pneumoniae
US20110183346A1 (en) * 2008-10-03 2011-07-28 Ibis Biosciences, Inc. Compositions for use in identification of neisseria, chlamydia, and/or chlamydophila bacteria
US20110183343A1 (en) * 2008-10-03 2011-07-28 Rangarajan Sampath Compositions for use in identification of members of the bacterial class alphaproteobacter
US20110189687A1 (en) * 2008-10-02 2011-08-04 Ibis Bioscience, Inc. Compositions for use in identification of members of the bacterial genus mycoplasma
US20110190170A1 (en) * 2008-10-03 2011-08-04 Ibis Biosciences, Inc. Compositions for use in identification of antibiotic-resistant bacteria
US20110200985A1 (en) * 2008-10-02 2011-08-18 Rangarajan Sampath Compositions for use in identification of herpesviruses
US20110223599A1 (en) * 2010-03-14 2011-09-15 Ibis Biosciences, Inc. Parasite detection via endosymbiont detection
WO2011112718A1 (en) 2010-03-10 2011-09-15 Ibis Biosciences, Inc. Production of single-stranded circular nucleic acid
US20110238316A1 (en) * 2001-06-26 2011-09-29 Ecker David J Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses
US8071309B2 (en) 2002-12-06 2011-12-06 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8088582B2 (en) 2006-04-06 2012-01-03 Ibis Biosciences, Inc. Compositions for the use in identification of fungi
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses
WO2013036603A1 (en) 2011-09-06 2013-03-14 Ibis Biosciences, Inc. Sample preparation methods
US8455184B2 (en) * 2006-04-18 2013-06-04 The United States Of America As Represented By The Secretary Of The Air Force Differential multiplexing with pattern recognition
US8534447B2 (en) 2008-09-16 2013-09-17 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US8550694B2 (en) 2008-09-16 2013-10-08 Ibis Biosciences, Inc. Mixing cartridges, mixing stations, and related kits, systems, and methods
US8563250B2 (en) 2001-03-02 2013-10-22 Ibis Biosciences, Inc. Methods for identifying bioagents
US8652780B2 (en) 2007-03-26 2014-02-18 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
WO2014052590A1 (en) 2012-09-26 2014-04-03 Ibis Biosciences, Inc. Swab interface for a microfluidic device
US8722336B2 (en) 2008-03-26 2014-05-13 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
US8771948B2 (en) 2009-04-03 2014-07-08 Sequenom, Inc. Nucleic acid preparation compositions and methods
GB2510520A (en) * 2009-02-03 2014-08-06 Bruker Daltonik Gmbh Mass Spectrometric Identification of Microorganisms in Complex Samples
US9051608B2 (en) 2006-12-05 2015-06-09 Agena Bioscience, Inc. Detection and quantification of biomolecules using mass spectrometry
US9068017B2 (en) 2010-04-08 2015-06-30 Ibis Biosciences, Inc. Compositions and methods for inhibiting terminal transferase activity
US9080209B2 (en) 2009-08-06 2015-07-14 Ibis Biosciences, Inc. Non-mass determined base compositions for nucleic acid detection
US9194877B2 (en) 2009-07-17 2015-11-24 Ibis Biosciences, Inc. Systems for bioagent indentification
US9393564B2 (en) 2009-03-30 2016-07-19 Ibis Biosciences, Inc. Bioagent detection systems, devices, and methods
US9404150B2 (en) 2007-08-29 2016-08-02 Sequenom, Inc. Methods and compositions for universal size-specific PCR
US9598724B2 (en) 2007-06-01 2017-03-21 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
US9719083B2 (en) 2009-03-08 2017-08-01 Ibis Biosciences, Inc. Bioagent detection methods
US9777335B2 (en) 2001-06-04 2017-10-03 Geneohm Sciences Canada Inc. Method for the detection and identification of methicillin-resistant Staphylococcus aureus
US9970061B2 (en) 2011-12-27 2018-05-15 Ibis Biosciences, Inc. Bioagent detection oligonucleotides
WO2018089978A1 (en) * 2016-11-14 2018-05-17 Wisconsin Alumni Research Foundation Nucleic acid quantification compositions and methods
US10584392B2 (en) 2007-06-01 2020-03-10 Council Of Scientific And Industrial Research Method for simultaneous detection and discrimination of bacterial, fungal, parasitic and viral infections of eye and central nervous system
WO2023077489A1 (en) * 2021-11-06 2023-05-11 江汉大学 Mnp marker combination of yersinia pestis, primer pair combination, kit, and application thereof

Families Citing this family (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2003298030B2 (en) * 2001-03-02 2009-12-10 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
AU2003279664A1 (en) * 2002-11-15 2004-06-15 Biotage Ab A method for inter-species differentiation and identification of a gram-positive bacteria
CA2560521C (en) * 2004-02-18 2012-01-03 Isis Pharmaceuticals, Inc. Compositions for use in identification of bacteria
US7888011B2 (en) * 2004-10-18 2011-02-15 U.S. Genomics, Inc. Methods for isolation of nucleic acids from prokaryotic spores
US20070003996A1 (en) * 2005-02-09 2007-01-04 Hitt Ben A Identification of bacteria and spores
WO2007086904A2 (en) * 2005-04-13 2007-08-02 Isis Pharmaceuticals, Inc. Compositions for use in identification of adenoviruses
GB0601302D0 (en) * 2006-01-23 2006-03-01 Semikhodskii Andrei Diagnostic methods and apparatus
US8076104B2 (en) * 2007-01-25 2011-12-13 Rogan Peter K Rapid and comprehensive identification of prokaryotic organisms
US7811766B2 (en) * 2007-03-28 2010-10-12 Thinkvillage, Llc Genetic identification and validation of Echinacea species
US8527207B2 (en) * 2007-05-15 2013-09-03 Peter K. Rogan Accurate identification of organisms based on individual information content
EP2179041A4 (en) * 2007-06-22 2010-12-22 Ibis Biosciences Inc Compositions and methods for identification of subspecies characteristics of mycobacterium tuberculosis
US7964843B2 (en) 2008-07-18 2011-06-21 The George Washington University Three-dimensional molecular imaging by infrared laser ablation electrospray ionization mass spectrometry
US8901487B2 (en) 2007-07-20 2014-12-02 George Washington University Subcellular analysis by laser ablation electrospray ionization mass spectrometry
US8067730B2 (en) 2007-07-20 2011-11-29 The George Washington University Laser ablation electrospray ionization (LAESI) for atmospheric pressure, In vivo, and imaging mass spectrometry
EP2358905A1 (en) * 2008-12-19 2011-08-24 Abbott Laboratories Diagnostic test for mutations in codons 12-13 of human k-ras
WO2010080616A1 (en) * 2008-12-19 2010-07-15 Abbott Laboratories Molecular assay for diagnosis of malaria
WO2011152899A1 (en) 2010-06-02 2011-12-08 Johns Hopkins University System and methods for determining drug resistance in microorganisms
EA027558B1 (en) 2011-05-19 2017-08-31 Эйджена Байосайенс, Инк. Process for multiplex nucleic acid identification
ES2644839T3 (en) * 2011-06-15 2017-11-30 Grifols Therapeutics Inc. Procedure, compositions and kits to determine the human immunodeficiency virus (HIV)
EP2732457A4 (en) 2011-07-14 2015-09-16 Univ George Washington Plume collimation for laser ablation electrospray ionization mass spectrometry
EP2753932A4 (en) * 2011-09-07 2015-07-15 Alpha Biotech Ab Determination of bacterial infections of the genus rickettsia and possibly borrelia, in patients exhibiting symptoms of disease and being blood donors
CN107267615A (en) * 2011-09-23 2017-10-20 霍夫曼-拉罗奇有限公司 G-shaped clamp is used for the purposes of improved ApoE gene
WO2013096838A2 (en) 2011-12-22 2013-06-27 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids
WO2013096799A1 (en) 2011-12-22 2013-06-27 Ibis Biosciences, Inc. Systems and methods for isolating nucleic acids from cellular samples
US9855559B2 (en) 2011-12-30 2018-01-02 Abbott Molecular Inc. Microorganism nucleic acid purification from host samples
EP2834370B1 (en) 2012-04-03 2019-01-02 The Regents Of The University Of Michigan Biomarker associated with irritable bowel syndrome and crohn's disease
US20140272967A1 (en) 2013-03-13 2014-09-18 Abbott Molecular Inc. Systems and methods for isolating nucleic acids
US9701999B2 (en) 2013-03-14 2017-07-11 Abbott Molecular, Inc. Multiplex methylation-specific amplification systems and methods
WO2014146025A1 (en) * 2013-03-15 2014-09-18 Bio-Rad Laboratories, Inc. Digital assays with associated targets
WO2014186874A1 (en) 2013-05-23 2014-11-27 Yyz Pharmatech, Inc. Methods and compositions for enzyme linked immuno and hybridization mass spectrometric assay
CN103344695B (en) * 2013-06-18 2015-06-10 中国疾病预防控制中心传染病预防控制所 Kit for rapid mass spectrometric detection of leptospira
CN103667109B (en) * 2013-10-23 2016-02-03 大连市水产技术推广总站 A kind of Rhodobacter capsulatus and application thereof
EP3161490B1 (en) 2014-06-27 2019-10-02 Abbott Laboratories Compositions and methods for detecting human pegivirus 2 (hpgv-2)
RU2580412C1 (en) * 2015-02-02 2016-04-10 Федеральное государственное автономное образовательное учреждение высшего образования "Национальный исследовательский Томский политехнический университет" Method for quantitative determination of t-2 toxin by differential voltammetry
CN114540470A (en) 2015-04-24 2022-05-27 基纳生物技术有限公司 Multiplexing method for identification and quantification of minor alleles and polymorphisms
CN107787371B (en) 2015-04-24 2022-02-01 基纳生物技术有限公司 Parallel method for detecting and quantifying minor variants
CN107085034A (en) * 2017-04-19 2017-08-22 中国人民解放军第三〇二医院 MALDI TOF MS identify shigella sonnei
CN107988341B (en) * 2018-01-03 2019-04-23 北京毅新博创生物科技有限公司 The method and product of Mass Spectrometric Identification Typing of Vibrio Cholerae
CN108048540B (en) * 2018-01-03 2021-06-08 北京毅新博创生物科技有限公司 Method for preparing fingerprint atlas database for detecting vibrio cholerae typing
JP7378712B2 (en) 2019-08-07 2023-11-14 学校法人常翔学園 Method for testing xenograft material for pathogen infection, method for producing test kit and xenograft material product evaluated for pathogen infection
CN112861170B (en) * 2020-08-03 2023-03-31 德能森智能科技(成都)有限公司 Smart park management system capable of protecting privacy
CN113219115A (en) * 2021-05-18 2021-08-06 广东省科学院微生物研究所(广东省微生物分析检测中心) MALDI-TOF MS-based method for rapidly identifying bacillus cereus and bacillus thuringiensis

Citations (76)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5484909A (en) * 1993-09-10 1996-01-16 Amoco Corporation Nucleic acid probes for the detection of bacteria of the genera Pediococcus and Lactobacillus and methods for the detection of the bacterial agents causing spoilage of beer
US5484908A (en) * 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5502177A (en) * 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization
US5527675A (en) * 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
US5622824A (en) * 1993-03-19 1997-04-22 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5625184A (en) * 1995-05-19 1997-04-29 Perseptive Biosystems, Inc. Time-of-flight mass spectrometry analysis of biomolecules
US5645985A (en) * 1991-11-26 1997-07-08 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5686242A (en) * 1991-09-05 1997-11-11 Isis Pharmaceuticals, Inc. Determination of oligonucleotides for therapeutics, diagnostics and research reagents
US5759771A (en) * 1990-10-17 1998-06-02 The Perkin-Elmer Corporation Method of determining a genotype by comparing the nucleotide sequence of members of a gene family and kit therefor
US5770367A (en) * 1993-07-30 1998-06-23 Oxford Gene Technology Limited Tag reagent and assay method
US5777324A (en) * 1996-09-19 1998-07-07 Sequenom, Inc. Method and apparatus for maldi analysis
US5830655A (en) * 1995-05-22 1998-11-03 Sri International Oligonucleotide sizing using cleavable primers
US5830653A (en) * 1991-11-26 1998-11-03 Gilead Sciences, Inc. Methods of using oligomers containing modified pyrimidines
US5864137A (en) * 1996-10-01 1999-01-26 Genetrace Systems, Inc. Mass spectrometer
US5869242A (en) * 1995-09-18 1999-02-09 Myriad Genetics, Inc. Mass spectrometry to assess DNA sequence polymorphisms
US5871697A (en) * 1995-10-24 1999-02-16 Curagen Corporation Method and apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing
US5876936A (en) * 1997-01-15 1999-03-02 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators
US5928906A (en) * 1996-05-09 1999-07-27 Sequenom, Inc. Process for direct sequencing during template amplification
US5981176A (en) * 1992-06-17 1999-11-09 City Of Hope Method of detecting and discriminating between nucleic acid sequences
US5994066A (en) * 1995-09-11 1999-11-30 Infectio Diagnostic, Inc. Species-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
US6046005A (en) * 1997-01-15 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group
US6051378A (en) * 1996-03-04 2000-04-18 Genetrace Systems Inc. Methods of screening nucleic acids using mass spectrometry
US6054278A (en) * 1997-05-05 2000-04-25 The Perkin-Elmer Corporation Ribosomal RNA gene polymorphism based microorganism identification
US6074823A (en) * 1993-03-19 2000-06-13 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6090558A (en) * 1997-09-19 2000-07-18 Genetrace Systems, Inc. DNA typing by mass spectrometry with polymorphic DNA repeat markers
US6104028A (en) * 1998-05-29 2000-08-15 Genetrace Systems Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry
US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6153389A (en) * 1999-02-22 2000-11-28 Haarer; Brian K. DNA additives as a mechanism for unambiguously marking biological samples
US6194144B1 (en) * 1993-01-07 2001-02-27 Sequenom, Inc. DNA sequencing by mass spectrometry
US6218118B1 (en) * 1998-07-09 2001-04-17 Agilent Technologies, Inc. Method and mixture reagents for analyzing the nucleotide sequence of nucleic acids by mass spectrometry
US6235480B1 (en) * 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids
US6235476B1 (en) * 1996-08-20 2001-05-22 Dako A/S Process for detecting nucleic acids by mass determination
US6238927B1 (en) * 1998-10-05 2001-05-29 Mosaic Technologies, Incorporated Reverse displacement assay for detection of nucleic acid sequences
US6268129B1 (en) * 1995-03-03 2001-07-31 Imperial Cancer Research Technology Limited Method of nucleic acid analysis
US6268146B1 (en) * 1998-03-13 2001-07-31 Promega Corporation Analytical methods and materials for nucleic acid detection
US6268131B1 (en) * 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids
US6270974B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Exogenous nucleic acid detection
US6270973B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection
US6277578B1 (en) * 1998-03-13 2001-08-21 Promega Corporation Deploymerization method for nucleic acid detection of an amplified nucleic acid target
US6312893B1 (en) * 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6312902B1 (en) * 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection
US6361940B1 (en) * 1996-09-24 2002-03-26 Qiagen Genomics, Inc. Compositions and methods for enhancing hybridization and priming specificity
US6372424B1 (en) * 1995-08-30 2002-04-16 Third Wave Technologies, Inc Rapid detection and identification of pathogens
US20020045178A1 (en) * 2000-06-13 2002-04-18 The Trustees Of Boston University Use of nucleotide analogs in the analysis of oligonucleotide mixtures and in highly multiplexed nucleic acid sequencing
US6391551B1 (en) * 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids
US6428955B1 (en) * 1995-03-17 2002-08-06 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6432651B1 (en) * 1998-07-10 2002-08-13 Cetek Corporation Method to detect and analyze tight-binding ligands in complex biological samples using capillary electrophoresis and mass spectrometry
US6436640B1 (en) * 1999-03-18 2002-08-20 Exiqon A/S Use of LNA in mass spectrometry
US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids
US20020137057A1 (en) * 2000-07-27 2002-09-26 Wold Barbara J. Rapid, quantitative method for the mass spectrometric analysis of nucleic acids for gene expression and genotyping
US6458533B1 (en) * 1997-12-19 2002-10-01 High Throughput Genomics, Inc. High throughput assay system for monitoring ESTs
US20020150927A1 (en) * 1999-04-30 2002-10-17 Matray Tracy J. Methods for detecting a plurality of analytes by mass spectrometry
US6475736B1 (en) * 2000-05-23 2002-11-05 Variagenics, Inc. Methods for genetic analysis of DNA using biased amplification of polymorphic sites
US6479239B1 (en) * 1998-03-10 2002-11-12 Large Scale Biology Corporation Detection and characterization of microorganisms
US20030017487A1 (en) * 2001-06-06 2003-01-23 Pharmacogenetics, Ltd. Method for detecting single nucleotide polymorphisms (SNP'S) and point mutations
US20030039976A1 (en) * 2001-08-14 2003-02-27 Haff Lawrence A. Methods for base counting
US20030064483A1 (en) * 1993-09-03 2003-04-03 Duke University. Method of nucleic acid sequencing
US20030073112A1 (en) * 2000-01-13 2003-04-17 Jing Zhang Universal nucleic acid amplification system for nucleic acids in a sample
US6558902B1 (en) * 1998-05-07 2003-05-06 Sequenom, Inc. Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of macromolecules
US6566055B1 (en) * 1996-09-19 2003-05-20 Sequenom, Inc. Methods of preparing nucleic acids for mass spectrometric analysis
US6582916B1 (en) * 1998-07-13 2003-06-24 Aventis Research & Technologies Gmbh & Co. Kg Metal ion-binding mass markers for nucleic acids
US20030129589A1 (en) * 1996-11-06 2003-07-10 Hubert Koster Dna diagnostics based on mass spectrometry
US20030134312A1 (en) * 2001-11-15 2003-07-17 Whatman, Inc. Methods and materials for detecting genetic material
US20030148284A1 (en) * 2001-12-17 2003-08-07 Vision Todd J. Solid phase detection of nucleic acid molecules
US6613509B1 (en) * 1999-03-22 2003-09-02 Regents Of The University Of California Determination of base (nucleotide) composition in DNA oligomers by mass spectrometry
US20030175729A1 (en) * 1999-12-29 2003-09-18 Van Eijk Michael Josephus Theresia Method for generating oligonucleotides, in particular for the detection of amplified restriction fragments obtained using aflp
US20030203398A1 (en) * 1999-12-16 2003-10-30 Bramucci Michael G. Nucleic acid fragments for the identification of bacteria in industrial wastewater bioreactors
US20030220844A1 (en) * 2002-05-24 2003-11-27 Marnellos Georgios E. Method and system for purchasing genetic data
US20040005555A1 (en) * 2000-08-31 2004-01-08 Rothman Richard E. Molecular diagnosis of bactermia
US6682889B1 (en) * 2000-11-08 2004-01-27 Becton, Dickinson And Company Amplification and detection of organisms of the Chlamydiaceae family
US20040038206A1 (en) * 2001-03-14 2004-02-26 Jia Zhang Method for high throughput assay of genetic analysis
US20040038385A1 (en) * 2002-08-26 2004-02-26 Langlois Richard G. System for autonomous monitoring of bioagents
US20040038234A1 (en) * 2000-06-30 2004-02-26 Gut Ivo Glynne Sample generation for genotyping by mass spectrometry

Family Cites Families (351)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4075475A (en) * 1976-05-03 1978-02-21 Chemetron Corporation Programmed thermal degradation-mass spectrometry analysis method facilitating identification of a biological specimen
US5288611A (en) * 1983-01-10 1994-02-22 Gen-Probe Incorporated Method for detecting, identifying, and quantitating organisms and viruses
WO1984002721A1 (en) * 1983-01-10 1984-07-19 Gen Probe Inc Method for detecting, identifying, and quantitating organisms and viruses
US5612183A (en) 1983-01-10 1997-03-18 Gen-Probe Incorporated Method for determining the effect of antimicrobial agents on growth using ribosomal nucleic acid subunit subsequence specific probes
US4965188A (en) 1986-08-22 1990-10-23 Cetus Corporation Process for amplifying, detecting, and/or cloning nucleic acid sequences using a thermostable enzyme
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
WO1988003957A1 (en) 1986-11-24 1988-06-02 Gen-Probe Incorporated Nucleic acid probes for detection and/or quantitation of non-viral organisms
WO1988006633A1 (en) 1987-03-02 1988-09-07 Arnold Lyle John Jr Polycationic supports for nucleic acid purification, separation and hybridization
US5188963A (en) 1989-11-17 1993-02-23 Gene Tec Corporation Device for processing biological specimens for analysis of nucleic acids
US5270030A (en) 1988-12-29 1993-12-14 Bio-Technology General Corp. Fibrin binding domain polypeptide and method of producing
US5198543A (en) 1989-03-24 1993-03-30 Consejo Superior Investigaciones Cientificas PHI29 DNA polymerase
IE81145B1 (en) * 1989-04-20 2000-05-03 Thomas Gerard Barry Generation of specific probes for target nucleotide sequences
ATE127530T1 (en) 1989-05-31 1995-09-15 Amoco Corp UNIVERSAL NUCLEIC ACID PROBE FOR EUBACTERIA AND METHODS.
US5219727A (en) 1989-08-21 1993-06-15 Hoffmann-Laroche Inc. Quantitation of nucleic acids using the polymerase chain reaction
US5192659A (en) * 1989-08-25 1993-03-09 Genetype Ag Intron sequence analysis method for detection of adjacent and remote locus alleles as haplotypes
US5213961A (en) * 1989-08-31 1993-05-25 Brigham And Women's Hospital Accurate quantitation of RNA and DNA by competetitive polymerase chain reaction
EP0667900B1 (en) 1990-01-12 2001-05-23 HSC Research Development Corporation Introns and exons of the cystic fibrosis gene and mutations at various positions of the gene
US5770029A (en) 1996-07-30 1998-06-23 Soane Biosciences Integrated electrophoretic microdevices
US5143905A (en) 1990-05-03 1992-09-01 The Regents Of The University Of California Method and means for extending the host range of insecticidal proteins
US5015845A (en) * 1990-06-01 1991-05-14 Vestec Corporation Electrospray method for mass spectrometry
US5712125A (en) * 1990-07-24 1998-01-27 Cemv Bioteknik Ab Competitive PCR for quantitation of DNA
DE4030262A1 (en) 1990-09-25 1992-03-26 Suedzucker Ag METHOD FOR PRODUCING RHAMNOSE FROM RHAMNOLIPID
WO1992009703A1 (en) 1990-11-26 1992-06-11 Cbr Laboratories, Inc. Testing for spirochetal nucleic acid sequences in samples
US5072115A (en) 1990-12-14 1991-12-10 Finnigan Corporation Interpretation of mass spectra of multiply charged ions of mixtures
WO1992013629A1 (en) 1991-01-31 1992-08-20 Wayne State University A method for analyzing an organic sample
US5866429A (en) * 1991-04-03 1999-02-02 Bloch; Will Precision and accuracy of anion-exchange separation of nucleic acids
US5472843A (en) 1991-04-25 1995-12-05 Gen-Probe Incorporated Nucleic acid probes to Haemophilus influenzae
US5213796A (en) 1991-05-06 1993-05-25 Dana Farber Cancer Institute Assay for polyomavirus in humans and uses thereof
US6055487A (en) 1991-07-30 2000-04-25 Margery; Keith S. Interactive remote sample analysis system
CA2116543C (en) 1991-07-31 2007-10-02 Kay S. Greisen Methods and reagents for detection of bacteria in cerebrospinal fluid
DE69233285T2 (en) 1991-08-02 2004-11-25 bioMérieux B.V. Quantification of nucleic acids
ATE241704T1 (en) 1991-08-27 2003-06-15 Hoffmann La Roche PRIMERS AND BUT FOR DETECTING HEPATITIS C
WO1993008297A1 (en) 1991-10-23 1993-04-29 Baylor College Of Medicine Fingerprinting bacterial strains using repetitive dna sequence amplification
FR2683827B1 (en) * 1991-11-15 1994-03-04 Institut Nal Sante Recherc Medic METHOD FOR DETERMINING THE QUANTITY OF A FRAGMENT OF DNA OF INTEREST BY AN ENZYMATIC AMPLIFICATION METHOD.
US6235887B1 (en) * 1991-11-26 2001-05-22 Isis Pharmaceuticals, Inc. Enhanced triple-helix and double-helix formation directed by oligonucleotides containing modified pyrimidines
IL103935A0 (en) 1991-12-04 1993-05-13 Du Pont Method for the identification of microorganisms by the utilization of directed and arbitrary dna amplification
EP0746857A4 (en) 1992-03-13 2001-01-03 Thermomicroscopes Corp Scanning probe microscope
US6303297B1 (en) 1992-07-17 2001-10-16 Incyte Pharmaceuticals, Inc. Database for storage and analysis of full-length sequences
FR2694754B1 (en) 1992-08-12 1994-09-16 Bio Merieux Mycobacteria DNA fragments, amplification primers, hybridization probes, reagents and method for detecting detection of mycobacteria.
AU5128593A (en) 1992-09-16 1994-04-12 University Of Tennessee Research Corporation, The Antigen of hybrid m protein and carrier for group a streptococcal vaccine
EP0672187A4 (en) * 1992-10-08 1999-11-17 Univ California Pcr assays to determine the presence and concentration of a target.
FR2701961B1 (en) 1993-02-24 1995-04-21 Bio Merieux Method for destabilizing an intracatenary secondary structure of a single-stranded polynucleotide, and for capturing said nucleotide.
US5639606A (en) 1993-04-06 1997-06-17 The University Of Rochester Method for quantitative measurement of gene expression using multiplex competitive reverse transcriptase-polymerase chain reaction
US6323041B1 (en) * 1993-06-11 2001-11-27 Pfizer Inc. Screening novel human phosphodiesterase IV isozymes for compounds which modify their enzymatic activity
JPH0775585A (en) 1993-06-14 1995-03-20 Immuno Japan:Kk Hepatitis c virus-related oligonucleotide and method for judging virus gene type
US5830853A (en) 1994-06-23 1998-11-03 Astra Aktiebolag Systemic administration of a therapeutic preparation
US6001584A (en) 1993-07-19 1999-12-14 The Regents Of The University Of California Oncoprotein protein kinase
AU7551594A (en) 1993-07-29 1995-02-28 MURASHIGE, Kate H. Method for recognition of the nucleotide sequence of a purified dna segment
AU7679394A (en) 1993-09-03 1995-03-22 Duke University A method of nucleic acid sequencing
WO1995011996A1 (en) 1993-10-27 1995-05-04 Cornell Research Foundation, Inc. Detection assay for listeria and erwinia microorganisms
US5504327A (en) * 1993-11-04 1996-04-02 Hv Ops, Inc. (H-Nu) Electrospray ionization source and method for mass spectrometric analysis
DE4338119A1 (en) 1993-11-08 1995-05-11 Bayer Ag Specific gene probes and methods for the quantitative detection of methicillin-resistant staphylococci
NL9301957A (en) 1993-11-11 1995-06-01 U Gene Research Bv Method for identifying microorganisms, and useful tools.
US5928905A (en) 1995-04-18 1999-07-27 Glaxo Group Limited End-complementary polymerase reaction
JP3396942B2 (en) 1994-02-21 2003-04-14 三菱化学株式会社 Method for producing aromatic polycarbonate
US5849492A (en) 1994-02-28 1998-12-15 Phylogenetix Laboratories, Inc. Method for rapid identification of prokaryotic and eukaryotic organisms
US5608217A (en) * 1994-03-10 1997-03-04 Bruker-Franzen Analytik Gmbh Electrospraying method for mass spectrometric analysis
DE4444229C2 (en) * 1994-03-10 1996-07-25 Bruker Franzen Analytik Gmbh Methods and devices for electrospray ionization for storage mass spectrometers
US5976798A (en) 1994-03-30 1999-11-02 Mitokor Methods for detecting mitochondrial mutations diagnostic for Alzheimer's disease and methods for determining heteroplasmy of mitochondrial nucleic acid
AU7242994A (en) 1994-05-20 1995-12-18 United States Of America, As Represented By The Secretary Of The Army, The Model for testing immunogenicity of peptides
US5814442A (en) 1994-06-10 1998-09-29 Georgetown University Internally controlled virion nucleic acid amplification reaction for quantitation of virion and virion nucleic acid
DE4421901A1 (en) * 1994-06-23 1996-01-04 Bayer Ag A rapid DNA test for the detection of quinolone-resistant Staphylococcus aureus pathogens in clinical specimens
US5541405A (en) 1994-07-27 1996-07-30 Parker-Hannifin Corporation Method and device for continuous pattern sensing using fiber optics
GB9417211D0 (en) 1994-08-25 1994-10-12 Solicitor For The Affairs Of H Nucleotide sequencing method
US6001564A (en) 1994-09-12 1999-12-14 Infectio Diagnostic, Inc. Species specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
US20020055101A1 (en) 1995-09-11 2002-05-09 Michel G. Bergeron Specific and universal probes and amplification primers to rapidly detect and identify common bacterial pathogens and antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
CA2118048C (en) * 1994-09-30 2003-04-08 James W. Schumm Multiplex amplification of short tandem repeat loci
US5753489A (en) * 1994-11-10 1998-05-19 Immuno Ag Method for producing viruses and vaccines in serum-free culture
US5654141A (en) 1994-11-18 1997-08-05 Thomas Jefferson University Amplification based detection of bacterial infection
KR100399813B1 (en) 1994-12-14 2004-06-09 가부시키가이샤 니콘 Exposure apparatus
US6180339B1 (en) 1995-01-13 2001-01-30 Bayer Corporation Nucleic acid probes for the detection and identification of fungi
US5707802A (en) 1995-01-13 1998-01-13 Ciba Corning Diagnostics Corp. Nucleic acid probes for the detection and identification of fungi
US5763169A (en) 1995-01-13 1998-06-09 Chiron Diagnostics Corporation Nucleic acid probes for the detection and identification of fungi
US5702895A (en) 1995-01-19 1997-12-30 Wakunaga Seiyaku Kabushiki Kaisha Method and kit for detecting methicillin-resistant Staphylococcus aureus
US5484808A (en) * 1995-02-09 1996-01-16 Eli Lilly And Company Methods of inhibiting cell-cell adhesion
EP0830460A1 (en) 1995-04-11 1998-03-25 Trustees Of Boston University Solid phase sequencing of biopolymers
US5932220A (en) 1995-05-08 1999-08-03 Board Of Regents University Of Texas System Diagnostic tests for a new spirochete, Borrelia lonestari sp. nov.
US5700642A (en) 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
US5630653A (en) * 1995-06-02 1997-05-20 Polka; John G. Wheel cover mounting bracket
EP0837933A4 (en) 1995-06-07 2003-05-21 Commw Scient Ind Res Org Optimized minizymes and miniribozymes and uses thereof
US5856174A (en) * 1995-06-29 1999-01-05 Affymetrix, Inc. Integrated nucleic acid diagnostic device
US6218529B1 (en) * 1995-07-31 2001-04-17 Urocor, Inc. Biomarkers and targets for diagnosis, prognosis and management of prostate, breast and bladder cancer
US6146854A (en) 1995-08-31 2000-11-14 Sequenom, Inc. Filtration processes, kits and devices for isolating plasmids
US5727202A (en) 1995-10-18 1998-03-10 Palm Computing, Inc. Method and apparatus for synchronizing information on two different computer systems
US5972693A (en) 1995-10-24 1999-10-26 Curagen Corporation Apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing
US5716825A (en) * 1995-11-01 1998-02-10 Hewlett Packard Company Integrated nucleic acid analysis system for MALDI-TOF MS
GB9602028D0 (en) 1996-02-01 1996-04-03 Amersham Int Plc Nucleoside analogues
US6852487B1 (en) * 1996-02-09 2005-02-08 Cornell Research Foundation, Inc. Detection of nucleic acid sequence differences using the ligase detection reaction with addressable arrays
WO1997037041A2 (en) 1996-03-18 1997-10-09 Sequenom, Inc. Dna sequencing by mass spectrometry
WO1997034909A1 (en) 1996-03-20 1997-09-25 Bio Merieux Nucleic acid isolation
JP3365198B2 (en) 1996-03-21 2003-01-08 ミノルタ株式会社 Image forming device
US5745751A (en) 1996-04-12 1998-04-28 Nelson; Robert W. Civil site information system
US6214555B1 (en) * 1996-05-01 2001-04-10 Visible Genetics Inc. Method compositions and kit for detection
CA2257866A1 (en) 1996-06-10 1997-12-18 University Of Utah Research Foundation Rapid, accurate identification of dna sequence variants by electrospray mass spectrometry
AU4042597A (en) 1996-07-19 1998-02-10 Hybridon, Inc. Method for sequencing nucleic acids using matrix-assisted laser desorption ionization time-of-flight mass spectrometry
US6563025B1 (en) 1996-07-26 2003-05-13 Board Of Trustees Of The University Of Illinois Nucleotide sequences encoding anthranilate synthase
US5831046A (en) * 1996-08-05 1998-11-03 Prolinx, Incorporated Boronic acid-contaning nucleic acid monomers
CA2301875A1 (en) 1996-09-19 1998-03-26 Genetrace Systems Methods of preparing nucleic acids for mass spectrometric analysis
GB9620769D0 (en) 1996-10-04 1996-11-20 Brax Genomics Ltd Nucleic acid sequencing
US5885775A (en) 1996-10-04 1999-03-23 Perseptive Biosystems, Inc. Methods for determining sequences information in polynucleotides using mass spectrometry
US6110710A (en) 1996-10-15 2000-08-29 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Sequence modification of oligonucleotide primers to manipulate non-templated nucleotide addition
WO1998020020A2 (en) 1996-11-06 1998-05-14 Sequenom, Inc. High density immobilization of nucleic acids
US5900481A (en) * 1996-11-06 1999-05-04 Sequenom, Inc. Bead linkers for immobilizing nucleic acids to solid supports
US6133436A (en) 1996-11-06 2000-10-17 Sequenom, Inc. Beads bound to a solid support and to nucleic acids
US7285422B1 (en) 1997-01-23 2007-10-23 Sequenom, Inc. Systems and methods for preparing and analyzing low volume analyte array elements
US6024925A (en) * 1997-01-23 2000-02-15 Sequenom, Inc. Systems and methods for preparing low volume analyte array elements
WO1998021066A1 (en) 1996-11-12 1998-05-22 Hp-Chemie Pelzer Research And Development Ltd. Reusable shaped floor carpets
US6060246A (en) 1996-11-15 2000-05-09 Avi Biopharma, Inc. Reagent and method for isolation and detection of selected nucleic acid sequences
BR9713417A (en) * 1996-11-28 2000-04-18 New Japan Chem Co Ltd Composed of sugar, gelling agent, gelling agent composition, processes for its preparations and gel composition.
US5822824A (en) 1996-12-03 1998-10-20 Dion; William D. Mountable washing device
CA2274587A1 (en) 1996-12-10 1998-06-18 Genetrace Systems Inc. Releasable nonvolatile mass-label molecules
US5981190A (en) 1997-01-08 1999-11-09 Ontogeny, Inc. Analysis of gene expression, methods and reagents therefor
DE69838519T2 (en) 1997-01-15 2008-07-03 Xzillion Gmbh & Co. Kg Mass-labeled hybridization probes
WO1998035057A1 (en) 1997-02-06 1998-08-13 The National University Of Singapore Diagnosis of plasmodium infection by analysis of extrachromosomal genetic material
US5858062A (en) * 1997-02-10 1999-01-12 Litton Systems, Inc. Oxygen concentrator
US6727061B2 (en) * 1997-02-20 2004-04-27 Cabtec, Inc. Methods for identifying species or Shigella and E. coli using operon sequence analysis
US5828062A (en) 1997-03-03 1998-10-27 Waters Investments Limited Ionization electrospray apparatus for mass spectrometry
US6553317B1 (en) 1997-03-05 2003-04-22 Incyte Pharmaceuticals, Inc. Relational database and system for storing information relating to biomolecular sequences and reagents
DE19710166C1 (en) 1997-03-12 1998-12-10 Bruker Franzen Analytik Gmbh Two-step method of DNA amplification for MALDI-TOF measurements
AU6553498A (en) 1997-03-14 1998-09-29 Hybridon, Inc. Method for sequencing of modified nucleic acids using electrospray ionization-fourier transform mass spectrometry
US5849497A (en) 1997-04-03 1998-12-15 The Research Foundation Of State University Of New York Specific inhibition of the polymerase chain reaction using a non-extendable oligonucleotide blocker
US6018713A (en) 1997-04-09 2000-01-25 Coli; Robert D. Integrated system and method for ordering and cumulative results reporting of medical tests
DE19717085C2 (en) 1997-04-23 1999-06-17 Bruker Daltonik Gmbh Processes and devices for extremely fast DNA multiplication using polymerase chain reactions (PCR)
US20010039263A1 (en) 1997-05-02 2001-11-08 Max-Delbruck-Centrum Fur Molekulare Medizin Chimeric oligonucleotides and the use thereof
WO1998054571A1 (en) 1997-05-28 1998-12-03 The Walter And Eliza Hall Institute Of Medical Research Nucleic acid diagnostics based on mass spectrometry or mass separation and base specific cleavage
US6159681A (en) 1997-05-28 2000-12-12 Syntrix Biochip, Inc. Light-mediated method and apparatus for the regional analysis of biologic material
WO1998054751A1 (en) 1997-05-30 1998-12-03 Genetrace Systems, Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry
US6061686A (en) 1997-06-26 2000-05-09 Digital Equipment Corporation Updating a copy of a remote document stored in a local computer system
DE69814629T2 (en) 1997-07-22 2004-03-25 Qiagen Genomics, Inc., Bothell METHOD AND CONNECTIONS FOR ANALYZING NUCLEIC ACIDS BY MASS SPECTROMETRY
DE19732086C2 (en) 1997-07-25 2002-11-21 Univ Leipzig Method for the quantitative determination of eubacteria
US6207370B1 (en) 1997-09-02 2001-03-27 Sequenom, Inc. Diagnostics based on mass spectrometric detection of translated target polypeptides
GB9719044D0 (en) 1997-09-08 1997-11-12 Inst Of Ophthalmology Assay
US6063031A (en) 1997-10-14 2000-05-16 Assurance Medical, Inc. Diagnosis and treatment of tissue with instruments
US6111096A (en) 1997-10-31 2000-08-29 Bbi Bioseq, Inc. Nucleic acid isolation and purification
JP3423597B2 (en) 1997-11-05 2003-07-07 三井農林株式会社 Bacterial identification method
US6007992A (en) 1997-11-10 1999-12-28 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US6028183A (en) 1997-11-07 2000-02-22 Gilead Sciences, Inc. Pyrimidine derivatives and oligonucleotides containing same
CA2312052A1 (en) 1997-12-05 1999-06-17 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Method for identifying nucleic acids by means of matrix-assisted laser desorption/ionisation mass spectrometry
US6914137B2 (en) 1997-12-06 2005-07-05 Dna Research Innovations Limited Isolation of nucleic acids
US20030096232A1 (en) 1997-12-19 2003-05-22 Kris Richard M. High throughput assay system
JP3793342B2 (en) 1997-12-26 2006-07-05 ソニーケミカル株式会社 Acrylic emulsion and adhesive tape
DE19802905C2 (en) 1998-01-27 2001-11-08 Bruker Daltonik Gmbh Process for the preferred production of only one strand of selected genetic material for mass spectrometric measurements
US6428956B1 (en) 1998-03-02 2002-08-06 Isis Pharmaceuticals, Inc. Mass spectrometric methods for biomolecular screening
US6261769B1 (en) 1998-03-31 2001-07-17 The United States Of America As Represented By The Secretary Of Agriculture Intergenic spacer target sequence for detecting and distinguishing Chlamydial species or strains
US7321828B2 (en) * 1998-04-13 2008-01-22 Isis Pharmaceuticals, Inc. System of components for preparing oligonucleotides
US20030228597A1 (en) 1998-04-13 2003-12-11 Cowsert Lex M. Identification of genetic targets for modulation by oligonucleotides and generation of oligonucleotides for gene modulation
US6223186B1 (en) 1998-05-04 2001-04-24 Incyte Pharmaceuticals, Inc. System and method for a precompiled database for biomolecular sequence information
DE19822108A1 (en) 1998-05-12 2000-02-03 Schering Ag Method for the detection of microorganisms in products, in particular in medicines and cosmetics
US6221587B1 (en) 1998-05-12 2001-04-24 Isis Pharmceuticals, Inc. Identification of molecular interaction sites in RNA for novel drug discovery
US6468743B1 (en) 1998-05-18 2002-10-22 Conagra Grocery Products Company PCR techniques for detecting microbial contaminants in foodstuffs
DE19922161A1 (en) 1998-05-18 1999-12-09 Fraunhofer Ges Forschung Anti-adhesion coating for e.g. soldering/welding tools and electric contacts
DE19824280B4 (en) 1998-05-29 2004-08-19 Bruker Daltonik Gmbh Mutation analysis using mass spectrometry
GB2339905A (en) 1998-06-24 2000-02-09 Bruker Daltonik Gmbh Use of mass-specrometry for detection of mutations
WO2000001850A2 (en) 1998-07-02 2000-01-13 Gen-Probe Incorporated Molecular torches
US6074831A (en) 1998-07-09 2000-06-13 Agilent Technologies, Inc. Partitioning of polymorphic DNAs
US6605433B1 (en) 1998-08-20 2003-08-12 The Johns Hopkins University Mitochondrial dosimeter
US6146144A (en) 1998-09-29 2000-11-14 Fowler; Ernest R. Rug hooking kit and method for handicapped
US6610492B1 (en) 1998-10-01 2003-08-26 Variagenics, Inc. Base-modified nucleotides and cleavage of polynucleotides incorporating them
IT1303330B1 (en) 1998-11-05 2000-11-06 Ilpea Ind Spa GASKET FOR FROGORIFERI WITH SHAPED COUNTERPORT.
DE19852167C2 (en) 1998-11-12 2000-12-14 Bruker Saxonia Analytik Gmbh Simple SNP analysis using mass spectrometry
WO2000032750A1 (en) 1998-11-24 2000-06-08 Regents Of The University Of Minnesota Transgenic circulating endothelial cells
US6994962B1 (en) * 1998-12-09 2006-02-07 Massachusetts Institute Of Technology Methods of identifying point mutations in a genome
DE19859723A1 (en) 1998-12-23 2000-06-29 Henkel Kgaa Preparations for coloring keratinous fibers
US6503718B2 (en) 1999-01-10 2003-01-07 Exact Sciences Corporation Methods for detecting mutations using primer extension for detecting disease
US6638714B1 (en) 1999-02-03 2003-10-28 Ortho-Clinical Diagnostics, Inc. Oligonucleotide primers for efficient detection of hepatitis C virus (HCV) and methods of use thereof
EP1035219A1 (en) 1999-02-25 2000-09-13 Universiteit Gent Gastric helicobacter 16 S rDNA sequences from cattle and pigs and their use for detection and typing of Helicobacter strains
DE19910592A1 (en) * 1999-03-10 2000-09-14 Volkswagen Ag Restraint
CA2370656A1 (en) 1999-04-21 2000-10-26 Mitchell T. Gore Magnetic dna extraction kit for plants
US6140067A (en) 1999-04-30 2000-10-31 Mitokor Indicators of altered mitochondrial function in predictive methods for determining risk of type 2 diabetes mellitus
AU778230B2 (en) 1999-05-03 2004-11-25 Gen-Probe Incorporated Polynucleotide matrix-based method of identifying microorganisms
US20020086289A1 (en) 1999-06-15 2002-07-04 Don Straus Genomic profiling: a rapid method for testing a complex biological sample for the presence of many types of organisms
WO2001000828A2 (en) 1999-06-30 2001-01-04 Corixa Corporation Compositions and methods for the therapy and diagnosis of lung cancer
AU5371099A (en) 1999-07-22 2001-02-13 Artus Gesellschaft Fur Molekularbiologische Diagnostik Und Entwicklung Mbh Method for the species-specific detection of organisms
US6723505B1 (en) 1999-08-13 2004-04-20 Nye Colifast As Method for identification of the indicators of contamination in liquid samples
US6266144B1 (en) 1999-08-26 2001-07-24 Taiwan Semiconductor Manufacturing Company Stepper and scanner new exposure sequence with intra-field correction
DE19943374A1 (en) 1999-09-10 2001-03-29 Max Planck Gesellschaft Method for binding nucleic acids to a solid phase
US7005274B1 (en) 1999-09-15 2006-02-28 Migenix Corp. Methods and compositions for diagnosing and treating arthritic disorders and regulating bone mass
WO2001023608A2 (en) 1999-09-27 2001-04-05 Merck Sharp & Dohme De Espana, S.A.E. Hybridization probes which specifically detect strains of the genera microbispora, microtetraspora, nonomuria and planobispora
CA2811455C (en) 1999-09-28 2015-12-08 Geneohm Sciences Canada Inc. Nucleic acids and methods for the detection of coagulase-negative staphylococcus
US6296188B1 (en) * 1999-10-01 2001-10-02 Perfect Plastic Printing Corporation Transparent/translucent financial transaction card including an infrared light filter
KR20020064298A (en) * 1999-10-13 2002-08-07 시쿼넘, 인코포레이티드 Methods for generating databases and databases for identifying polymorphic genetic markers
US6787302B2 (en) 1999-10-25 2004-09-07 Genprime, Inc. Method and apparatus for prokaryotic and eukaryotic cell quantitation
EP1228244A4 (en) 1999-11-04 2005-02-09 California Inst Of Techn Methods and apparatuses for analyzing polynucleotide sequences
US6286146B1 (en) 1999-11-15 2001-09-11 Debra Rocker Method of wearing weighted training vest while listening to audio equipment
US6856914B1 (en) * 1999-11-19 2005-02-15 The University Of British Columbia Method, apparatus, media and signals for identifying associated cell signaling proteins
AU781961B2 (en) 1999-11-29 2005-06-23 Aventis Pharma S.A. Method for obtaining nucleic acids from an environment sample, resulting nucleic acids and use in synthesis of novel compounds
US6936414B2 (en) 1999-12-22 2005-08-30 Abbott Laboratories Nucleic acid isolation method and kit
SE0000061D0 (en) 2000-01-10 2000-01-10 Bjoern Herrmann A method for detection of pathogenic organisms
WO2001053917A2 (en) 2000-01-24 2001-07-26 Sanjay Chadha Hand-held personal computing device with microdisplay
US20020009727A1 (en) 2000-02-02 2002-01-24 Schultz Gary A. Detection of single nucleotide polymorphisms
CA2298181C (en) 2000-02-02 2006-09-19 Dayan Burke Goodnough Non-targeted complex sample analysis
US6453244B1 (en) 2000-02-10 2002-09-17 Stanford University Detection of polymorphisms by denaturing high-performance liquid chromatography
UA73967C2 (en) 2000-02-14 2005-10-17 First Opinion Corp Structure-based automatic processing for diagnostics (variants)
US6393367B1 (en) 2000-02-19 2002-05-21 Proteometrics, Llc Method for evaluating the quality of comparisons between experimental and theoretical mass data
DE10015797B4 (en) 2000-03-26 2006-02-02 Bruker Daltonik Gmbh Multiplex analysis of DNA mixtures using photolytically readable DNA chips
DE10015262A1 (en) 2000-03-28 2001-10-04 Basf Ag Paper coating composition useful for off set printing, contains a binding agent prepared by radical polymerization of ethylenically unsaturated compounds
AU6052001A (en) 2000-03-29 2001-10-08 Ct For The Applic Of Molecular Methods for genotyping by hybridization analysis
ES2632538T3 (en) 2000-04-10 2017-09-14 Taxon Biosciences, Inc. Methods for the study and genetic analysis of populations
US6716634B1 (en) * 2000-05-31 2004-04-06 Agilent Technologies, Inc. Increasing ionization efficiency in mass spectrometry
US6507837B1 (en) 2000-06-08 2003-01-14 Hyperphrase Technologies, Llc Tiered and content based database searching
EP1287028A2 (en) 2000-06-09 2003-03-05 Corixa Corporation Compositions and methods for the therapy and diagnosis of colon cancer
FR2811321A1 (en) 2000-07-04 2002-01-11 Bio Merieux New oligonucleotide primers, useful for identifying bacteria, particularly in cases of septicemia, provide amplification of bacterial 16S ribosomal nucleic acid
US6504021B2 (en) * 2000-07-05 2003-01-07 Edge Biosystems, Inc. Ion exchange method for DNA purification
SG144729A1 (en) 2000-07-06 2008-08-28 Bio Merieux Method for controlling the microbiological quality of an aqueous medium and kit therefor
US6783939B2 (en) 2000-07-07 2004-08-31 Alphavax, Inc. Alphavirus vectors and virosomes with modified HIV genes for use in vaccines
AUPQ909000A0 (en) 2000-07-28 2000-08-24 University Of Sydney, The A method of detecting microorganisms
US20030190635A1 (en) 2002-02-20 2003-10-09 Mcswiggen James A. RNA interference mediated treatment of Alzheimer's disease using short interfering RNA
GB0021286D0 (en) 2000-08-30 2000-10-18 Gemini Genomics Ab Identification of drug metabolic capacity
US6813615B1 (en) 2000-09-06 2004-11-02 Cellomics, Inc. Method and system for interpreting and validating experimental data with automated reasoning
US20020120408A1 (en) * 2000-09-06 2002-08-29 Kreiswirth Barry N. System and method for tracking and controlling infections
US7349808B1 (en) * 2000-09-06 2008-03-25 Egenomics, Inc. System and method for tracking and controlling infections
AU2001288762A1 (en) 2000-09-08 2002-03-22 Large Scale Proteomics Corporation Detection and characterization of microorganisms
SE0003286D0 (en) 2000-09-15 2000-09-15 Ulf Gyllensten Method and kit for human identification
DE60139026D1 (en) 2000-09-25 2009-07-30 Polymun Scient Immunbio Forsch LIVING INFLUENZA VACCINE AND METHOD FOR ITS MANUFACTURE
WO2002028901A2 (en) 2000-10-05 2002-04-11 Millennium Pharmaceuticals, Inc. Seven-transmembrane (7tm) receptors and uses thereof
US6996472B2 (en) 2000-10-10 2006-02-07 The United States Of America As Represented By The Department Of Health And Human Services Drift compensation method for fingerprint spectra
WO2002031747A1 (en) 2000-10-13 2002-04-18 Irm Llc High throughput processing system and method of using
ATE380883T1 (en) 2000-10-24 2007-12-15 Univ Leland Stanford Junior DIRECT MULTIPLEX CHARACTERIZATION OF GENOMIC DNA
US6906316B2 (en) 2000-10-27 2005-06-14 Fuji Electric Co., Ltd. Semiconductor device module
AU2002222614A1 (en) 2000-12-12 2002-07-01 Chugai Seiyaku Kabushiki Kaisha Method of detecting polymorphism in dna by using mass spectroscopy
US6800289B2 (en) 2000-12-21 2004-10-05 Her Majesty The Queen In Right Of Canada, As Represented By The Minister Of National Defence Strain of the western equine encephalitis virus
US6586584B2 (en) 2001-01-29 2003-07-01 Becton, Dickinson And Company Sequences and methods for detection of Hepatitis C virus
DE10108453B4 (en) 2001-02-22 2005-10-20 Bruker Daltonik Gmbh Mass spectrometric mutation analysis with photolytically cleavable primers
EP1404868A2 (en) 2001-02-28 2004-04-07 Chondrogene Inc. Compositions and methods relating to osteoarthritis
AU2002305941A1 (en) 2001-03-01 2002-09-19 The Johns Hopkins University Quantitative assay for the simultaneous detection and speciation of bacterial infections
AU2003298030B2 (en) 2001-03-02 2009-12-10 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US7666588B2 (en) 2001-03-02 2010-02-23 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US20040121310A1 (en) 2002-12-18 2004-06-24 Ecker David J. Methods for rapid detection and identification of bioagents in forensic studies
US7226739B2 (en) 2001-03-02 2007-06-05 Isis Pharmaceuticals, Inc Methods for rapid detection and identification of bioagents in epidemiological and forensic investigations
US20040121314A1 (en) 2002-12-06 2004-06-24 Ecker David J. Methods for rapid detection and identification of bioagents in containers
US20040121313A1 (en) 2002-12-06 2004-06-24 Ecker David J. Methods for rapid detection and identification of bioagents in organs for transplantation
US7718354B2 (en) 2001-03-02 2010-05-18 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US20030027135A1 (en) 2001-03-02 2003-02-06 Ecker David J. Method for rapid detection and identification of bioagents
US20030104410A1 (en) 2001-03-16 2003-06-05 Affymetrix, Inc. Human microarray
AU2002306777C1 (en) 2001-03-19 2008-04-24 President And Fellows Of Harvard College Evolving new molecular function
AU2001258719B2 (en) 2001-03-28 2007-09-06 Council Of Scientific And Industrial Research Universal primers for wildlife identification
US7630836B2 (en) 2001-05-30 2009-12-08 The Kitasato Institute Polynucleotides
CA2348042A1 (en) 2001-06-04 2002-12-04 Ann Huletsky Sequences for detection and identification of methicillin-resistant staphylococcus aureus
EP1392824B1 (en) 2001-06-06 2008-08-20 DSM IP Assets B.V. Improved isoprenoid production
US20020187490A1 (en) 2001-06-07 2002-12-12 Michigan State University Microbial identification chip based on DNA-DNA hybridization
GB0113907D0 (en) 2001-06-07 2001-08-01 Univ London Virus detection using degenerate PCR primers
GB0113908D0 (en) 2001-06-07 2001-08-01 Univ London Designing degenerate PCR primers
US8073627B2 (en) 2001-06-26 2011-12-06 Ibis Biosciences, Inc. System for indentification of pathogens
US7217510B2 (en) 2001-06-26 2007-05-15 Isis Pharmaceuticals, Inc. Methods for providing bacterial bioagent characterizing information
DE10132147B4 (en) 2001-07-03 2004-04-15 Universität Leipzig Method for the rapid quantitative determination of Eu bacteria
GB0117054D0 (en) 2001-07-12 2001-09-05 Plant Bioscience Ltd Methods and means for modification of plant characteristics
AU2002354838B2 (en) 2001-07-19 2007-11-29 Infectio Diagnostic (I.D.I.) Inc. Universal method and composition for the rapid lysis of cells for the release of nucleic acids and their detection
US20040191769A1 (en) 2001-07-24 2004-09-30 Transgenomic, Inc. Methods, compositions, and kits for mutation detection in mitochondrial DNA
WO2003012074A2 (en) 2001-07-30 2003-02-13 Den Kgl. Veterinær- Og Landbohøjskole Bacterial strains belonging to lactobacillus species and their use in food and feed industry
US7115385B2 (en) 2001-08-02 2006-10-03 North Carolina State University Media and methods for cultivation of microorganisms
AT411174B (en) 2001-08-09 2003-10-27 Lambda Labor Fuer Molekularbio METHOD AND CHIP FOR ANALYZING NUCLEIC ACIDS
US20040175715A1 (en) 2001-08-21 2004-09-09 Burgoyne Leigh A. Method and device for simultaneously molecularly cloning and polylocus profiling of genomes or genomes mixtures
US7105296B2 (en) 2001-08-29 2006-09-12 E. I. Du Pont De Nemours And Company Genes encoding Baeyer-Villiger monooxygenases
US7049286B2 (en) 2001-08-30 2006-05-23 Diatos, S.A. Insulin conjugates and methods of use thereof
WO2003020739A2 (en) 2001-09-04 2003-03-13 Exiqon A/S Novel lna compositions and uses thereof
US20040101809A1 (en) 2001-09-21 2004-05-27 Weiss Ervin I Device, method and materials for mobilizing substances into dentinal tubules in root canal treatment
DE10150121B4 (en) 2001-10-11 2005-12-01 Bernhard-Nocht-Institut für Tropenmedizin Real-time detection of DNA amplification products
US7297485B2 (en) 2001-10-15 2007-11-20 Qiagen Gmbh Method for nucleic acid amplification that results in low amplification bias
US6977148B2 (en) 2001-10-15 2005-12-20 Qiagen Gmbh Multiple displacement amplification
US20040029129A1 (en) * 2001-10-25 2004-02-12 Liangsu Wang Identification of essential genes in microorganisms
DE10152821B4 (en) 2001-10-25 2006-11-16 Bruker Daltonik Gmbh Mass spectra without electronic noise
EP1308506A1 (en) 2001-11-06 2003-05-07 Eidgenössische Technische Hochschule Zürich Mixtures of Propionibacterium jensenii and Lactobacillus sp. with antimicrobial activities for use as a natural preservation system
CA3066428C (en) 2001-11-13 2022-05-24 The Trustees Of The University Of Pennsylvania A method of detecting and/or identifying adeno-associated virus (aav) sequences and isolating novel sequences identified thereby
US20040023209A1 (en) * 2001-11-28 2004-02-05 Jon Jonasson Method for identifying microorganisms based on sequencing gene fragments
JP3692067B2 (en) 2001-11-30 2005-09-07 株式会社東芝 Polishing slurry for copper CMP and method of manufacturing semiconductor device using the same
TW509116U (en) 2001-12-18 2002-11-01 Ind Tech Res Inst Device for clipping and tightening spindle of honing and milling machine
US20030175709A1 (en) 2001-12-20 2003-09-18 Murphy George L. Method and system for depleting rRNA populations
US7468185B2 (en) 2001-12-21 2008-12-23 Pfizer Inc. Vaccine for periodontal disease
KR100444230B1 (en) 2001-12-27 2004-08-16 삼성전기주식회사 Nonreducible dielectric ceramic composition
US20060088826A1 (en) 2001-12-28 2006-04-27 Van Eijk Michael Josephus Ther Discrimination and detection of target nucleotide sequences using mass spectrometry
EP1333101B1 (en) 2002-02-01 2007-03-28 Bruker Daltonik GmbH Mutation analysis by PCR and Mass spectrometry
CN1202204C (en) 2002-02-27 2005-05-18 财团法人工业技术研究院 Organic electroluminescent light-emitting compound and module and device with it
KR100600988B1 (en) 2002-03-13 2006-07-13 주식회사 엘지생명과학 Method for enhancing immune responses by codelivering influenza NP DNA in DNA immunization
US7024370B2 (en) * 2002-03-26 2006-04-04 P) Cis, Inc. Methods and apparatus for early detection of health-related events in a population
US6897027B2 (en) 2002-03-27 2005-05-24 Decode Genetics Ehf. Method for desalting nucleic acids
US20030228571A1 (en) 2002-04-01 2003-12-11 Ecker David J. Method for rapid detection and identification of viral bioagents
WO2003087993A2 (en) 2002-04-09 2003-10-23 Beattie Kenneth L Oligonucleotide probes for genosensor chips
JP2004000200A (en) 2002-04-19 2004-01-08 Menicon Co Ltd Method for detecting microorganism
FR2838740A1 (en) 2002-04-22 2003-10-24 Centre Nat Rech Scient Detecting primate T cell lymphoma/leukemia viruses, useful e.g. for diagnosis and drug screening, using degenerate oligonucleotides based on conserved regions of envelope protein
GB0209812D0 (en) 2002-04-30 2002-06-05 Renovo Ltd Genetic testing
DE10222632B4 (en) 2002-05-17 2006-03-09 Con Cipio Gmbh Microsatellite markers for genetic analysis and for the differentiation of roses
US6906319B2 (en) 2002-05-17 2005-06-14 Micromass Uk Limited Mass spectrometer
EP1365031A1 (en) 2002-05-21 2003-11-26 MTM Laboratories AG Method for detection of somatic mutations using mass spectometry
WO2003099840A1 (en) * 2002-05-24 2003-12-04 Isis Pharmaceuticals, Inc. Oligonucleotides having modified nucleoside units
AU2003240150B2 (en) 2002-05-29 2008-10-30 Aresa Biodetection Aps Reporter system for plants
GB0212666D0 (en) 2002-05-31 2002-07-10 Secr Defence Immunogenic sequences
AU2003238930A1 (en) 2002-06-07 2003-12-22 Incyte Corporation Enzymes
WO2004005458A2 (en) 2002-06-13 2004-01-15 Regulome Corporation Functional sites
KR100484144B1 (en) * 2002-06-20 2005-04-18 삼성전자주식회사 Remote management server and the method thereof
WO2004003511A2 (en) 2002-07-01 2004-01-08 Wayne State University Methods and compositions for the identification of antibiotics that are not susceptible to antibiotic resistance
WO2004009849A1 (en) 2002-07-19 2004-01-29 Isis Pharmaceuticals, Inc. Methods for mass spectrometry analysis utilizing an integrated microfluidics sample platform
US6916483B2 (en) * 2002-07-22 2005-07-12 Biodynamics, Llc Bioabsorbable plugs containing drugs
GB0217434D0 (en) 2002-07-27 2002-09-04 Royal Vetinary College Biological material
US20040022764A1 (en) * 2002-07-31 2004-02-05 Hanan Polansky Inhibition of microcompetition with a foreign polynucleotide as treatment of chronic disease
US7172868B2 (en) 2002-08-01 2007-02-06 The Regents Of The University Of California Nucleotide sequences specific to Francisella tularensis and methods for the detection of Francisella tularensis
CN102344960B (en) * 2002-09-06 2014-06-18 波士顿大学信托人 Quantification of gene expression
US20040110138A1 (en) 2002-11-01 2004-06-10 University Of Ottawa Method for the detection of multiple genetic targets
EP1560932A2 (en) 2002-11-12 2005-08-10 Genolife One step real-time rt pcr kits for the universal detection of organisms in industrial products
US7250496B2 (en) 2002-11-14 2007-07-31 Rosetta Genomics Ltd. Bioinformatically detectable group of novel regulatory genes and uses thereof
EP1572977B1 (en) 2002-11-15 2010-03-03 Gen-Probe Incorporated Assay and compositions for detection of bacillus anthracis nucleic acid
US6680476B1 (en) * 2002-11-22 2004-01-20 Agilent Technologies, Inc. Summed time-of-flight mass spectrometry utilizing thresholding to reduce noise
EP1578399A4 (en) 2002-12-06 2007-11-28 Isis Pharmaceuticals Inc Methods for rapid identification of pathogens in humans and animals
JP2004201679A (en) 2002-12-10 2004-07-22 Kao Corp Primer for detecting fusobacterium nucleatum by pcr process and method for detecting the same
US20040117354A1 (en) 2002-12-16 2004-06-17 Azzaro Steven Hector Process for tagging and measuring quality
US20040121312A1 (en) 2002-12-18 2004-06-24 Ecker David J. Methods for rapid detection and identification of the absence of bioagents
US20040121340A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent associated with host versus graft and graft versus host rejections thereby
US20040121315A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in containers thereby
US20040122857A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in forensic studies thereby
US20040121329A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in blood, bodily fluids, and bodily tissues thereby
US20040122598A1 (en) 2002-12-18 2004-06-24 Ecker David J. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent in food products and cosmetics thereby
US9487823B2 (en) 2002-12-20 2016-11-08 Qiagen Gmbh Nucleic acid amplification
JP2004201641A (en) 2002-12-26 2004-07-22 Mitsubishi Kagaku Bio-Clinical Laboratories Inc Detection of eumycetes
US20040170981A1 (en) 2003-02-10 2004-09-02 Mckenney Keith Real-time polymerase chain reaction using large target amplicons
US20040170954A1 (en) 2003-02-10 2004-09-02 Mckenney Keith Pathogen inactivation assay
US20040185438A1 (en) 2003-03-10 2004-09-23 Ecker David J. Methods of detection and notification of bioagent contamination
US20050065813A1 (en) * 2003-03-11 2005-03-24 Mishelevich David J. Online medical evaluation system
US8046171B2 (en) 2003-04-18 2011-10-25 Ibis Biosciences, Inc. Methods and apparatus for genetic evaluation
WO2004097369A2 (en) 2003-04-25 2004-11-11 Sequenom, Inc. Fragmentation-based methods and systems for de novo sequencing
US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses
WO2005009202A2 (en) 2003-05-12 2005-02-03 Isis Pharmaceuticals, Inc. Automatic identification of bioagents
US7964343B2 (en) 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US8158354B2 (en) 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
EP1644520B1 (en) 2003-07-03 2007-09-19 Danmarks og Gronlands Geologiske Undersogelse Method for selective detection of a target nucleic acid
AU2003257013A1 (en) * 2003-07-29 2005-03-07 Sigma Aldrich Co. Methods and compositions for amplification of dna
JP4304292B2 (en) * 2003-07-30 2009-07-29 日本電気株式会社 Mobile communication system, mobile communication terminal, power control method used therefor, and program thereof
KR100632429B1 (en) 2003-08-01 2006-10-09 프로테온 주식회사 Screening system of reassortant influenza viruses using primer dependent multiplex RT-PCR
US20120122103A1 (en) 2003-09-11 2012-05-17 Rangarajan Sampath Compositions for use in identification of bacteria
US20060240412A1 (en) 2003-09-11 2006-10-26 Hall Thomas A Compositions for use in identification of adenoviruses
US20050142584A1 (en) 2003-10-01 2005-06-30 Willson Richard C. Microbial identification based on the overall composition of characteristic oligonucleotides
WO2005036369A2 (en) 2003-10-09 2005-04-21 Isis Pharmaceuticals, Inc. Database for microbial investigations
FR2861743B1 (en) 2003-11-04 2007-10-19 Univ Aix Marseille Ii MOLECULAR IDENTIFICATION OF BACTERIA OF THE GENUS CORYNEBACTERIUM
JP3861871B2 (en) 2003-11-26 2006-12-27 サンケン電気株式会社 Switching power supply
US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses
WO2005062770A2 (en) 2003-12-19 2005-07-14 Novakoff James L Method for conducting pharmacogenomics-based studies
DE602005024234D1 (en) 2004-02-10 2010-12-02 Roche Diagnostics Gmbh NEW PRIMERS AND PROBES TO DETECT PARVOVIRUS B19
CA2560521C (en) 2004-02-18 2012-01-03 Isis Pharmaceuticals, Inc. Compositions for use in identification of bacteria
US7666592B2 (en) * 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US8119336B2 (en) 2004-03-03 2012-02-21 Ibis Biosciences, Inc. Compositions for use in identification of alphaviruses
US7312036B2 (en) * 2004-03-22 2007-12-25 Isis Pharmaceuticals, Inc. Compositions for use in identification of viral hemorrhagic fever viruses
US20050266411A1 (en) 2004-05-25 2005-12-01 Hofstadler Steven A Methods for rapid forensic analysis of mitochondrial DNA
US7627437B2 (en) 2005-01-14 2009-12-01 Idaho Research Foundation Categorization of microbial communities
DE102005008583B4 (en) 2005-02-24 2007-10-25 Johannes-Gutenberg-Universität Mainz A method of typing an individual by short tandem repeat (STR) loci of the genomic DNA
WO2006094238A2 (en) 2005-03-03 2006-09-08 Isis Pharmaceuticals, Inc. Compositions for use in identification of adventitious viruses
WO2007086904A2 (en) 2005-04-13 2007-08-02 Isis Pharmaceuticals, Inc. Compositions for use in identification of adenoviruses
EP1882045B1 (en) 2005-04-21 2012-08-29 Ibis Biosciences, Inc. COMPOSITION FOR IDENTIFICATION OF Staphylococcus aureus
AU2006272776B2 (en) * 2005-07-21 2012-01-19 Ibis Biosciences, Inc. Methods for rapid identification and quantitation of nucleic acid variants
US20070026435A1 (en) 2005-07-28 2007-02-01 Polysciences, Inc. Hydroxysilane functionalized magnetic particles and nucleic acid separation method
WO2008104002A2 (en) 2007-02-23 2008-08-28 Ibis Biosciences, Inc. Methods for rapid forensic dna analysis
WO2008118809A1 (en) 2007-03-23 2008-10-02 Ibis Biosciences, Inc. Compositions for use in identification of mixed populations of bioagents
JP5067239B2 (en) 2008-03-28 2012-11-07 富士通株式会社 Cable management device and electronic device device

Patent Citations (99)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5759771A (en) * 1990-10-17 1998-06-02 The Perkin-Elmer Corporation Method of determining a genotype by comparing the nucleotide sequence of members of a gene family and kit therefor
US5686242A (en) * 1991-09-05 1997-11-11 Isis Pharmaceuticals, Inc. Determination of oligonucleotides for therapeutics, diagnostics and research reagents
US5645985A (en) * 1991-11-26 1997-07-08 Gilead Sciences, Inc. Enhanced triple-helix and double-helix formation with oligomers containing modified pyrimidines
US5484908A (en) * 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines
US5830653A (en) * 1991-11-26 1998-11-03 Gilead Sciences, Inc. Methods of using oligomers containing modified pyrimidines
US5981176A (en) * 1992-06-17 1999-11-09 City Of Hope Method of detecting and discriminating between nucleic acid sequences
US5503980A (en) * 1992-11-06 1996-04-02 Trustees Of Boston University Positional sequencing by hybridization
US6436635B1 (en) * 1992-11-06 2002-08-20 Boston University Solid phase sequencing of double-stranded nucleic acids
US6194144B1 (en) * 1993-01-07 2001-02-27 Sequenom, Inc. DNA sequencing by mass spectrometry
US6238871B1 (en) * 1993-01-07 2001-05-29 Sequenom, Inc. DNA sequences by mass spectrometry
US5691141A (en) * 1993-01-07 1997-11-25 Sequenom, Inc. DNA sequencing by mass spectrometry
US5605798A (en) * 1993-01-07 1997-02-25 Sequenom, Inc. DNA diagnostic based on mass spectrometry
US5547835A (en) * 1993-01-07 1996-08-20 Sequenom, Inc. DNA sequencing by mass spectrometry
US6225450B1 (en) * 1993-01-07 2001-05-01 Sequenom, Inc. DNA sequencing by mass spectrometry
US5622824A (en) * 1993-03-19 1997-04-22 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6074823A (en) * 1993-03-19 2000-06-13 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US5770367A (en) * 1993-07-30 1998-06-23 Oxford Gene Technology Limited Tag reagent and assay method
US5527675A (en) * 1993-08-20 1996-06-18 Millipore Corporation Method for degradation and sequencing of polymers which sequentially eliminate terminal residues
US20030064483A1 (en) * 1993-09-03 2003-04-03 Duke University. Method of nucleic acid sequencing
US5484909A (en) * 1993-09-10 1996-01-16 Amoco Corporation Nucleic acid probes for the detection of bacteria of the genera Pediococcus and Lactobacillus and methods for the detection of the bacterial agents causing spoilage of beer
US5502177A (en) * 1993-09-17 1996-03-26 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US5763588A (en) * 1993-09-17 1998-06-09 Gilead Sciences, Inc. Pyrimidine derivatives for labeled binding partners
US6268129B1 (en) * 1995-03-03 2001-07-31 Imperial Cancer Research Technology Limited Method of nucleic acid analysis
US6268144B1 (en) * 1995-03-17 2001-07-31 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6277573B1 (en) * 1995-03-17 2001-08-21 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6043031A (en) * 1995-03-17 2000-03-28 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6258538B1 (en) * 1995-03-17 2001-07-10 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US20020150903A1 (en) * 1995-03-17 2002-10-17 Hubert Koster Diagnostics based on mass spectrometry
US6235478B1 (en) * 1995-03-17 2001-05-22 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6602662B1 (en) * 1995-03-17 2003-08-05 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6428955B1 (en) * 1995-03-17 2002-08-06 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6589485B2 (en) * 1995-03-17 2003-07-08 Sequenom, Inc. Solid support for mass spectrometry
US6221601B1 (en) * 1995-03-17 2001-04-24 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6300076B1 (en) * 1995-03-17 2001-10-09 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6221605B1 (en) * 1995-03-17 2001-04-24 Sequenom, Inc. DNA diagnostics based on mass spectrometry
US6197498B1 (en) * 1995-03-17 2001-03-06 Sequenom, Inc DNA diagnostics based on mass spectrometry
US5625184A (en) * 1995-05-19 1997-04-29 Perseptive Biosystems, Inc. Time-of-flight mass spectrometry analysis of biomolecules
US5830655A (en) * 1995-05-22 1998-11-03 Sri International Oligonucleotide sizing using cleavable primers
US6372424B1 (en) * 1995-08-30 2002-04-16 Third Wave Technologies, Inc Rapid detection and identification of pathogens
US5994066A (en) * 1995-09-11 1999-11-30 Infectio Diagnostic, Inc. Species-specific and universal DNA probes and amplification primers to rapidly detect and identify common bacterial pathogens and associated antibiotic resistance genes from clinical specimens for routine diagnosis in microbiology laboratories
US5869242A (en) * 1995-09-18 1999-02-09 Myriad Genetics, Inc. Mass spectrometry to assess DNA sequence polymorphisms
US5871697A (en) * 1995-10-24 1999-02-16 Curagen Corporation Method and apparatus for identifying, classifying, or quantifying DNA sequences in a sample without sequencing
US6312893B1 (en) * 1996-01-23 2001-11-06 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6623928B2 (en) * 1996-01-23 2003-09-23 Qiagen Genomics, Inc. Methods and compositions for determining the sequence of nucleic acid molecules
US6468748B1 (en) * 1996-03-04 2002-10-22 Sequenom, Inc. Methods of screening nucleic acids using volatile salts in mass spectrometry
US20030113745A1 (en) * 1996-03-04 2003-06-19 Monforte Joseph A. Methods of screening nucleic acids using mass spectrometry
US6051378A (en) * 1996-03-04 2000-04-18 Genetrace Systems Inc. Methods of screening nucleic acids using mass spectrometry
US5928906A (en) * 1996-05-09 1999-07-27 Sequenom, Inc. Process for direct sequencing during template amplification
US6235476B1 (en) * 1996-08-20 2001-05-22 Dako A/S Process for detecting nucleic acids by mass determination
US6111251A (en) * 1996-09-19 2000-08-29 Sequenom, Inc. Method and apparatus for MALDI analysis
US5777324A (en) * 1996-09-19 1998-07-07 Sequenom, Inc. Method and apparatus for maldi analysis
US6566055B1 (en) * 1996-09-19 2003-05-20 Sequenom, Inc. Methods of preparing nucleic acids for mass spectrometric analysis
US6423966B2 (en) * 1996-09-19 2002-07-23 Sequenom, Inc. Method and apparatus for maldi analysis
US6361940B1 (en) * 1996-09-24 2002-03-26 Qiagen Genomics, Inc. Compositions and methods for enhancing hybridization and priming specificity
US5864137A (en) * 1996-10-01 1999-01-26 Genetrace Systems, Inc. Mass spectrometer
US20030129589A1 (en) * 1996-11-06 2003-07-10 Hubert Koster Dna diagnostics based on mass spectrometry
US6140053A (en) * 1996-11-06 2000-10-31 Sequenom, Inc. DNA sequencing by mass spectrometry via exonuclease degradation
US6046005A (en) * 1997-01-15 2000-04-04 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators comprising a cleavable linking group
US5876936A (en) * 1997-01-15 1999-03-02 Incyte Pharmaceuticals, Inc. Nucleic acid sequencing with solid phase capturable terminators
US6054278A (en) * 1997-05-05 2000-04-25 The Perkin-Elmer Corporation Ribosomal RNA gene polymorphism based microorganism identification
US6090558A (en) * 1997-09-19 2000-07-18 Genetrace Systems, Inc. DNA typing by mass spectrometry with polymorphic DNA repeat markers
US6268131B1 (en) * 1997-12-15 2001-07-31 Sequenom, Inc. Mass spectrometric methods for sequencing nucleic acids
US6458533B1 (en) * 1997-12-19 2002-10-01 High Throughput Genomics, Inc. High throughput assay system for monitoring ESTs
US6479239B1 (en) * 1998-03-10 2002-11-12 Large Scale Biology Corporation Detection and characterization of microorganisms
US20030194699A1 (en) * 1998-03-13 2003-10-16 Promega Corporation Multiplex method for nucleic acid detection
US6270974B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Exogenous nucleic acid detection
US6312902B1 (en) * 1998-03-13 2001-11-06 Promega Corporation Nucleic acid detection
US6277578B1 (en) * 1998-03-13 2001-08-21 Promega Corporation Deploymerization method for nucleic acid detection of an amplified nucleic acid target
US6391551B1 (en) * 1998-03-13 2002-05-21 Promega Corporation Detection of nucleic acid hybrids
US6268146B1 (en) * 1998-03-13 2001-07-31 Promega Corporation Analytical methods and materials for nucleic acid detection
US6235480B1 (en) * 1998-03-13 2001-05-22 Promega Corporation Detection of nucleic acid hybrids
US6270973B1 (en) * 1998-03-13 2001-08-07 Promega Corporation Multiplex method for nucleic acid detection
US6558902B1 (en) * 1998-05-07 2003-05-06 Sequenom, Inc. Infrared matrix-assisted laser desorption/ionization mass spectrometric analysis of macromolecules
US6265716B1 (en) * 1998-05-29 2001-07-24 Genetrace Systems, Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry
US6104028A (en) * 1998-05-29 2000-08-15 Genetrace Systems Inc. Volatile matrices for matrix-assisted laser desorption/ionization mass spectrometry
US6218118B1 (en) * 1998-07-09 2001-04-17 Agilent Technologies, Inc. Method and mixture reagents for analyzing the nucleotide sequence of nucleic acids by mass spectrometry
US6432651B1 (en) * 1998-07-10 2002-08-13 Cetek Corporation Method to detect and analyze tight-binding ligands in complex biological samples using capillary electrophoresis and mass spectrometry
US6582916B1 (en) * 1998-07-13 2003-06-24 Aventis Research & Technologies Gmbh & Co. Kg Metal ion-binding mass markers for nucleic acids
US6238927B1 (en) * 1998-10-05 2001-05-29 Mosaic Technologies, Incorporated Reverse displacement assay for detection of nucleic acid sequences
US6153389A (en) * 1999-02-22 2000-11-28 Haarer; Brian K. DNA additives as a mechanism for unambiguously marking biological samples
US6436640B1 (en) * 1999-03-18 2002-08-20 Exiqon A/S Use of LNA in mass spectrometry
US6613509B1 (en) * 1999-03-22 2003-09-02 Regents Of The University Of California Determination of base (nucleotide) composition in DNA oligomers by mass spectrometry
US20020150927A1 (en) * 1999-04-30 2002-10-17 Matray Tracy J. Methods for detecting a plurality of analytes by mass spectrometry
US20030203398A1 (en) * 1999-12-16 2003-10-30 Bramucci Michael G. Nucleic acid fragments for the identification of bacteria in industrial wastewater bioreactors
US20030175729A1 (en) * 1999-12-29 2003-09-18 Van Eijk Michael Josephus Theresia Method for generating oligonucleotides, in particular for the detection of amplified restriction fragments obtained using aflp
US20030073112A1 (en) * 2000-01-13 2003-04-17 Jing Zhang Universal nucleic acid amplification system for nucleic acids in a sample
US6475736B1 (en) * 2000-05-23 2002-11-05 Variagenics, Inc. Methods for genetic analysis of DNA using biased amplification of polymorphic sites
US20020045178A1 (en) * 2000-06-13 2002-04-18 The Trustees Of Boston University Use of nucleotide analogs in the analysis of oligonucleotide mixtures and in highly multiplexed nucleic acid sequencing
US20040038234A1 (en) * 2000-06-30 2004-02-26 Gut Ivo Glynne Sample generation for genotyping by mass spectrometry
US20020137057A1 (en) * 2000-07-27 2002-09-26 Wold Barbara J. Rapid, quantitative method for the mass spectrometric analysis of nucleic acids for gene expression and genotyping
US20040005555A1 (en) * 2000-08-31 2004-01-08 Rothman Richard E. Molecular diagnosis of bactermia
US6682889B1 (en) * 2000-11-08 2004-01-27 Becton, Dickinson And Company Amplification and detection of organisms of the Chlamydiaceae family
US20040038206A1 (en) * 2001-03-14 2004-02-26 Jia Zhang Method for high throughput assay of genetic analysis
US20030017487A1 (en) * 2001-06-06 2003-01-23 Pharmacogenetics, Ltd. Method for detecting single nucleotide polymorphisms (SNP'S) and point mutations
US20030039976A1 (en) * 2001-08-14 2003-02-27 Haff Lawrence A. Methods for base counting
US20030134312A1 (en) * 2001-11-15 2003-07-17 Whatman, Inc. Methods and materials for detecting genetic material
US20030148284A1 (en) * 2001-12-17 2003-08-07 Vision Todd J. Solid phase detection of nucleic acid molecules
US20030220844A1 (en) * 2002-05-24 2003-11-27 Marnellos Georgios E. Method and system for purchasing genetic data
US20040038385A1 (en) * 2002-08-26 2004-02-26 Langlois Richard G. System for autonomous monitoring of bioagents

Cited By (176)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20110166040A1 (en) * 1997-09-05 2011-07-07 Ibis Biosciences, Inc. Compositions for use in identification of strains of e. coli o157:h7
US8017743B2 (en) 2001-03-02 2011-09-13 Ibis Bioscience, Inc. Method for rapid detection and identification of bioagents
US8017358B2 (en) 2001-03-02 2011-09-13 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents
US8268565B2 (en) 2001-03-02 2012-09-18 Ibis Biosciences, Inc. Methods for identifying bioagents
US8265878B2 (en) 2001-03-02 2012-09-11 Ibis Bioscience, Inc. Method for rapid detection and identification of bioagents
US8214154B2 (en) 2001-03-02 2012-07-03 Ibis Biosciences, Inc. Systems for rapid identification of pathogens in humans and animals
US20040219517A1 (en) * 2001-03-02 2004-11-04 Ecker David J. Methods for rapid identification of pathogens in humans and animals
US8017322B2 (en) 2001-03-02 2011-09-13 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents
US8563250B2 (en) 2001-03-02 2013-10-22 Ibis Biosciences, Inc. Methods for identifying bioagents
US8802372B2 (en) 2001-03-02 2014-08-12 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US20060121520A1 (en) * 2001-03-02 2006-06-08 Ecker David J Method for rapid detection and identification of bioagents
US8815513B2 (en) 2001-03-02 2014-08-26 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents in epidemiological and forensic investigations
US9416424B2 (en) 2001-03-02 2016-08-16 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US9752184B2 (en) 2001-03-02 2017-09-05 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US7781162B2 (en) 2001-03-02 2010-08-24 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US20040161770A1 (en) * 2001-03-02 2004-08-19 Ecker David J. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US7741036B2 (en) 2001-03-02 2010-06-22 Ibis Biosciences, Inc. Method for rapid detection and identification of bioagents
US20100145626A1 (en) * 2001-03-02 2010-06-10 Ecker David J Systems for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US7718354B2 (en) 2001-03-02 2010-05-18 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US7666588B2 (en) 2001-03-02 2010-02-23 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA and characterization of mitochondrial DNA heteroplasmy
US20090182511A1 (en) * 2001-03-02 2009-07-16 Ibis Biosciences, Inc. Methods For Rapid Forensic Analysis Of Mitochondrial DNA And Characterization Of Mitochondrial DNA Heteroplasmy
US20090148836A1 (en) * 2001-03-02 2009-06-11 Ibis Biosciences, Inc. Method for Rapid Detection and Identification of Bioagents
US20090148837A1 (en) * 2001-03-02 2009-06-11 Ibis Biosciences, Inc. Method for Rapid Detection and Identification of Bioagents
US20080311558A1 (en) * 2001-03-02 2008-12-18 Isis Pharmaceuticals, Inc. Methods For Rapid Identification Of Pathogens In Humans And Animals
US9777335B2 (en) 2001-06-04 2017-10-03 Geneohm Sciences Canada Inc. Method for the detection and identification of methicillin-resistant Staphylococcus aureus
US10577664B2 (en) 2001-06-04 2020-03-03 Geneohm Sciences Canada, Inc. Method for the detection and identification of methicillin-resistant Staphylococcus aureus
US10801074B2 (en) 2001-06-04 2020-10-13 Geneohm Sciences Canada, Inc. Method for the detection and identification of methicillin-resistant Staphylococcus aureus
US20110238316A1 (en) * 2001-06-26 2011-09-29 Ecker David J Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US8298760B2 (en) 2001-06-26 2012-10-30 Ibis Bioscience, Inc. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US8921047B2 (en) 2001-06-26 2014-12-30 Ibis Biosciences, Inc. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US8380442B2 (en) 2001-06-26 2013-02-19 Ibis Bioscience, Inc. Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US20110172925A1 (en) * 2001-06-26 2011-07-14 Ibis Biosciences, Inc. Secondary Structure Defining Database And Methods For Determining Identity And Geographic Origin Of An Unknown Bioagent Thereby
US8073627B2 (en) 2001-06-26 2011-12-06 Ibis Biosciences, Inc. System for indentification of pathogens
US9725771B2 (en) 2002-12-06 2017-08-08 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8071309B2 (en) 2002-12-06 2011-12-06 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8822156B2 (en) 2002-12-06 2014-09-02 Ibis Biosciences, Inc. Methods for rapid identification of pathogens in humans and animals
US8046171B2 (en) * 2003-04-18 2011-10-25 Ibis Biosciences, Inc. Methods and apparatus for genetic evaluation
US20040209260A1 (en) * 2003-04-18 2004-10-21 Ecker David J. Methods and apparatus for genetic evaluation
US8057993B2 (en) 2003-04-26 2011-11-15 Ibis Biosciences, Inc. Methods for identification of coronaviruses
US20050130196A1 (en) * 2003-05-13 2005-06-16 Hofstadler Steven A. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US8476415B2 (en) 2003-05-13 2013-07-02 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US8158354B2 (en) 2003-05-13 2012-04-17 Ibis Biosciences, Inc. Methods for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
US20050164215A1 (en) * 2003-05-13 2005-07-28 Hofstadler Steven A. Methods for rapid purification of nucleic acids for subsquent analysis by mass spectrometery by solution capture
US7964343B2 (en) 2003-05-13 2011-06-21 Ibis Biosciences, Inc. Method for rapid purification of nucleic acids for subsequent analysis by mass spectrometry by solution capture
WO2005034855A3 (en) * 2003-09-05 2005-07-14 Gen Hospital Corp Photodynamic inactivation of bacterial spores
US20060223729A1 (en) * 2003-09-05 2006-10-05 Hamblin Michael R Photodynamic inactivation of bacterial spores
WO2005034855A2 (en) * 2003-09-05 2005-04-21 The General Hospital Corporation Photodynamic inactivation of bacterial spores
US20060240412A1 (en) * 2003-09-11 2006-10-26 Hall Thomas A Compositions for use in identification of adenoviruses
US7956175B2 (en) 2003-09-11 2011-06-07 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US8288523B2 (en) 2003-09-11 2012-10-16 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US8394945B2 (en) 2003-09-11 2013-03-12 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US8097416B2 (en) 2003-09-11 2012-01-17 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US20100035239A1 (en) * 2003-09-11 2010-02-11 Isis Pharmaceuticals, Inc. Compositions for use in identification of bacteria
US8013142B2 (en) 2003-09-11 2011-09-06 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US8546082B2 (en) 2003-09-11 2013-10-01 Ibis Biosciences, Inc. Methods for identification of sepsis-causing bacteria
US20070224614A1 (en) * 2003-09-11 2007-09-27 Rangarajan Sampath Compositions for use in identification of bacteria
US20100129811A1 (en) * 2003-09-11 2010-05-27 Ibis Biosciences, Inc. Compositions for use in identification of pseudomonas aeruginosa
US8242254B2 (en) * 2003-09-11 2012-08-14 Ibis Biosciences, Inc. Compositions for use in identification of bacteria
US20080138808A1 (en) * 2003-09-11 2008-06-12 Hall Thomas A Methods for identification of sepsis-causing bacteria
US20070248969A1 (en) * 2003-09-11 2007-10-25 Rangarajan Sampath Compositions for use in identification of bacteria
US20050142584A1 (en) * 2003-10-01 2005-06-30 Willson Richard C. Microbial identification based on the overall composition of characteristic oligonucleotides
US7309410B2 (en) 2003-12-03 2007-12-18 Palo Alto Research Center Incorporated Traveling wave grids and algorithms for biomolecule separation, transport and focusing
US7163611B2 (en) 2003-12-03 2007-01-16 Palo Alto Research Center Incorporated Concentration and focusing of bio-agents and micron-sized particles using traveling wave grids
US20050123992A1 (en) * 2003-12-03 2005-06-09 Palo Alto Research Center Incorporated Concentration and focusing of bio-agents and micron-sized particles using traveling wave grids
US8163895B2 (en) 2003-12-05 2012-04-24 Ibis Biosciences, Inc. Compositions for use in identification of orthopoxviruses
US20090004643A1 (en) * 2004-02-18 2009-01-01 Isis Pharmaceuticals, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US7666592B2 (en) 2004-02-18 2010-02-23 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US9447462B2 (en) 2004-02-18 2016-09-20 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US8187814B2 (en) 2004-02-18 2012-05-29 Ibis Biosciences, Inc. Methods for concurrent identification and quantification of an unknown bioagent
US20100035227A1 (en) * 2004-03-03 2010-02-11 Isis Pharmaceuticals, Inc. Compositions for use in identification of alphaviruses
US8119336B2 (en) 2004-03-03 2012-02-21 Ibis Biosciences, Inc. Compositions for use in identification of alphaviruses
US20060057603A1 (en) * 2004-03-11 2006-03-16 The Regents Of The University Of California Detecting Bacillus anthracis
US8173957B2 (en) 2004-05-24 2012-05-08 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US8987660B2 (en) 2004-05-24 2015-03-24 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US9449802B2 (en) 2004-05-24 2016-09-20 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US20100127165A1 (en) * 2004-05-24 2010-05-27 Ibis Biosciences, Inc. Mass spectromety with selective ion filtration by digital thresholding
US7714275B2 (en) 2004-05-24 2010-05-11 Ibis Biosciences, Inc. Mass spectrometry with selective ion filtration by digital thresholding
US8407010B2 (en) 2004-05-25 2013-03-26 Ibis Biosciences, Inc. Methods for rapid forensic analysis of mitochondrial DNA
US20090125245A1 (en) * 2004-05-25 2009-05-14 Isis Pharmaceuticals, Inc. Methods For Rapid Forensic Analysis Of Mitochondrial DNA
US7811753B2 (en) 2004-07-14 2010-10-12 Ibis Biosciences, Inc. Methods for repairing degraded DNA
US9873906B2 (en) 2004-07-14 2018-01-23 Ibis Biosciences, Inc. Methods for repairing degraded DNA
US20060205040A1 (en) * 2005-03-03 2006-09-14 Rangarajan Sampath Compositions for use in identification of adventitious viruses
US20100136515A1 (en) * 2005-03-03 2010-06-03 Ibis Biosciences, Inc. Compositions for use in identification of papillomavirus
US8182992B2 (en) 2005-03-03 2012-05-22 Ibis Biosciences, Inc. Compositions for use in identification of adventitious viruses
US8084207B2 (en) 2005-03-03 2011-12-27 Ibis Bioscience, Inc. Compositions for use in identification of papillomavirus
US8551738B2 (en) 2005-07-21 2013-10-08 Ibis Biosciences, Inc. Systems and methods for rapid identification of nucleic acid variants
US20070218467A1 (en) * 2005-07-21 2007-09-20 Ecker David J Methods for rapid identification and quantitation of nucleic acid variants
US8026084B2 (en) 2005-07-21 2011-09-27 Ibis Biosciences, Inc. Methods for rapid identification and quantitation of nucleic acid variants
US20100070194A1 (en) * 2005-07-21 2010-03-18 Ecker David J Methods for rapid identification and quantitation of nucleic acid variants
US20080227087A1 (en) * 2005-10-11 2008-09-18 Ann Huletsky Sequences for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) of MREJ types xi to xx
US11834720B2 (en) 2005-10-11 2023-12-05 Geneohm Sciences, Inc. Sequences for detection and identification of methicillin-resistant Staphylococcus aureus (MRSA) of MREJ types xi to xx
US8088582B2 (en) 2006-04-06 2012-01-03 Ibis Biosciences, Inc. Compositions for the use in identification of fungi
US8455184B2 (en) * 2006-04-18 2013-06-04 The United States Of America As Represented By The Secretary Of The Air Force Differential multiplexing with pattern recognition
EP3617321A2 (en) 2006-05-31 2020-03-04 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
WO2007140417A2 (en) 2006-05-31 2007-12-06 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US20100297710A1 (en) * 2006-05-31 2010-11-25 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
EP2602321A1 (en) 2006-05-31 2013-06-12 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US8679741B2 (en) 2006-05-31 2014-03-25 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US9453257B2 (en) 2006-05-31 2016-09-27 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US10662421B2 (en) 2006-05-31 2020-05-26 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
EP3260556A1 (en) 2006-05-31 2017-12-27 Sequenom, Inc. Methods and compositions for the extraction and amplification of nucleic acid from a sample
US20080096766A1 (en) * 2006-06-16 2008-04-24 Sequenom, Inc. Methods and compositions for the amplification, detection and quantification of nucleic acid from a sample
US9149473B2 (en) 2006-09-14 2015-10-06 Ibis Biosciences, Inc. Targeted whole genome amplification method for identification of pathogens
US20100035232A1 (en) * 2006-09-14 2010-02-11 Ecker David J Targeted whole genome amplification method for identification of pathogens
US9051608B2 (en) 2006-12-05 2015-06-09 Agena Bioscience, Inc. Detection and quantification of biomolecules using mass spectrometry
US20100184035A1 (en) * 2007-02-23 2010-07-22 Ibis Bioscience, Inc. Methods for rapid forensic dna analysis
US8871471B2 (en) 2007-02-23 2014-10-28 Ibis Biosciences, Inc. Methods for rapid forensic DNA analysis
US20100204266A1 (en) * 2007-03-23 2010-08-12 Ibis Biosciences, INC Compositions for use in identification of mixed populations of bioagents
US8652780B2 (en) 2007-03-26 2014-02-18 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
US20100291544A1 (en) * 2007-05-25 2010-11-18 Ibis Biosciences, Inc. Compositions for use in identification of strains of hepatitis c virus
US9598724B2 (en) 2007-06-01 2017-03-21 Ibis Biosciences, Inc. Methods and compositions for multiple displacement amplification of nucleic acids
US10584392B2 (en) 2007-06-01 2020-03-10 Council Of Scientific And Industrial Research Method for simultaneous detection and discrimination of bacterial, fungal, parasitic and viral infections of eye and central nervous system
US20110045456A1 (en) * 2007-06-14 2011-02-24 Ibis Biosciences, Inc. Compositions for use in identification of adventitious contaminant viruses
US9404150B2 (en) 2007-08-29 2016-08-02 Sequenom, Inc. Methods and compositions for universal size-specific PCR
US20110097704A1 (en) * 2008-01-29 2011-04-28 Ibis Biosciences, Inc. Compositions for use in identification of picornaviruses
US20090263809A1 (en) * 2008-03-20 2009-10-22 Zygem Corporation Limited Methods for Identification of Bioagents
US8722336B2 (en) 2008-03-26 2014-05-13 Sequenom, Inc. Restriction endonuclease enhanced polymorphic sequence detection
US20110143358A1 (en) * 2008-05-30 2011-06-16 Ibis Biosciences, Inc. Compositions for use in identification of tick-borne pathogens
US20110177515A1 (en) * 2008-05-30 2011-07-21 Ibis Biosciences, Inc. Compositions for use in identification of francisella
US20110151437A1 (en) * 2008-06-02 2011-06-23 Ibis Biosciences, Inc. Compositions for use in identification of adventitious viruses
US8609430B2 (en) 2008-09-16 2013-12-17 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US8550694B2 (en) 2008-09-16 2013-10-08 Ibis Biosciences, Inc. Mixing cartridges, mixing stations, and related kits, systems, and methods
US8148163B2 (en) 2008-09-16 2012-04-03 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US20100075430A1 (en) * 2008-09-16 2010-03-25 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US8252599B2 (en) 2008-09-16 2012-08-28 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US8534447B2 (en) 2008-09-16 2013-09-17 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods
US9027730B2 (en) 2008-09-16 2015-05-12 Ibis Biosciences, Inc. Microplate handling systems and related computer program products and methods
US9023655B2 (en) 2008-09-16 2015-05-05 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
US20110189687A1 (en) * 2008-10-02 2011-08-04 Ibis Bioscience, Inc. Compositions for use in identification of members of the bacterial genus mycoplasma
US20110200985A1 (en) * 2008-10-02 2011-08-18 Rangarajan Sampath Compositions for use in identification of herpesviruses
US20110190170A1 (en) * 2008-10-03 2011-08-04 Ibis Biosciences, Inc. Compositions for use in identification of antibiotic-resistant bacteria
US20110183343A1 (en) * 2008-10-03 2011-07-28 Rangarajan Sampath Compositions for use in identification of members of the bacterial class alphaproteobacter
US20110183344A1 (en) * 2008-10-03 2011-07-28 Rangarajan Sampath Compositions for use in identification of clostridium difficile
US20110183345A1 (en) * 2008-10-03 2011-07-28 Ibis Biosciences, Inc. Compositions for use in identification of streptococcus pneumoniae
US20110183346A1 (en) * 2008-10-03 2011-07-28 Ibis Biosciences, Inc. Compositions for use in identification of neisseria, chlamydia, and/or chlamydophila bacteria
GB2510520B (en) * 2009-02-03 2014-11-05 Bruker Daltonik Gmbh Mass spectrometric identification of microorganisms in complex samples
GB2510520A (en) * 2009-02-03 2014-08-06 Bruker Daltonik Gmbh Mass Spectrometric Identification of Microorganisms in Complex Samples
US20100248298A1 (en) * 2009-02-03 2010-09-30 Bruker Daltonik Gmbh Mass spectrometric identification of microorganisms in complex samples
US9165740B2 (en) 2009-02-12 2015-10-20 Ibis Biosciences, Inc. Ionization probe assemblies
US20100219336A1 (en) * 2009-02-12 2010-09-02 Ibis Biosciences, Inc. Ionization probe assemblies
US8796617B2 (en) 2009-02-12 2014-08-05 Ibis Biosciences, Inc. Ionization probe assemblies
US8158936B2 (en) 2009-02-12 2012-04-17 Ibis Biosciences, Inc. Ionization probe assemblies
US9719083B2 (en) 2009-03-08 2017-08-01 Ibis Biosciences, Inc. Bioagent detection methods
US20100279295A1 (en) * 2009-03-18 2010-11-04 Sequenom, Inc. Use of thermostable endonucleases for generating reporter molecules
US9393564B2 (en) 2009-03-30 2016-07-19 Ibis Biosciences, Inc. Bioagent detection systems, devices, and methods
US10053685B2 (en) 2009-04-03 2018-08-21 Sequenom, Inc. Nucleic acid preparation compositions and methods
US10858645B2 (en) 2009-04-03 2020-12-08 Sequenom, Inc. Nucleic acid preparation compositions and methods
US8771948B2 (en) 2009-04-03 2014-07-08 Sequenom, Inc. Nucleic acid preparation compositions and methods
US9850480B2 (en) 2009-04-03 2017-12-26 Sequenom, Inc. Nucleic acid preparation compositions and methods
US9580741B2 (en) 2009-04-03 2017-02-28 Sequenom, Inc. Nucleic acid preparation compositions and methods
US9194877B2 (en) 2009-07-17 2015-11-24 Ibis Biosciences, Inc. Systems for bioagent indentification
US8950604B2 (en) 2009-07-17 2015-02-10 Ibis Biosciences, Inc. Lift and mount apparatus
US20110014027A1 (en) * 2009-07-17 2011-01-20 Ibis Biosciences, Inc. Lift and mount apparatus
US9416409B2 (en) 2009-07-31 2016-08-16 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
US20110028334A1 (en) * 2009-07-31 2011-02-03 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
US10119164B2 (en) 2009-07-31 2018-11-06 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
US9080209B2 (en) 2009-08-06 2015-07-14 Ibis Biosciences, Inc. Non-mass determined base compositions for nucleic acid detection
US20110065111A1 (en) * 2009-08-31 2011-03-17 Ibis Biosciences, Inc. Compositions For Use In Genotyping Of Klebsiella Pneumoniae
US20110091882A1 (en) * 2009-10-02 2011-04-21 Ibis Biosciences, Inc. Determination of methylation status of polynucleotides
US9890408B2 (en) 2009-10-15 2018-02-13 Ibis Biosciences, Inc. Multiple displacement amplification
WO2011047307A1 (en) 2009-10-15 2011-04-21 Ibis Biosciences, Inc. Multiple displacement amplification
EP2957641A1 (en) 2009-10-15 2015-12-23 Ibis Biosciences, Inc. Multiple displacement amplification
EP3225695A1 (en) 2009-10-15 2017-10-04 Ibis Biosciences, Inc. Multiple displacement amplification
US20110118151A1 (en) * 2009-10-15 2011-05-19 Ibis Biosciences, Inc. Multiple displacement amplification
WO2011112718A1 (en) 2010-03-10 2011-09-15 Ibis Biosciences, Inc. Production of single-stranded circular nucleic acid
US20110223599A1 (en) * 2010-03-14 2011-09-15 Ibis Biosciences, Inc. Parasite detection via endosymbiont detection
US9758840B2 (en) 2010-03-14 2017-09-12 Ibis Biosciences, Inc. Parasite detection via endosymbiont detection
US9068017B2 (en) 2010-04-08 2015-06-30 Ibis Biosciences, Inc. Compositions and methods for inhibiting terminal transferase activity
US9752173B2 (en) 2010-04-08 2017-09-05 Ibis Biosciences, Inc. Compositions and methods for inhibiting terminal transferase activity
EP3170831A1 (en) 2011-09-06 2017-05-24 Ibis Biosciences, Inc. Sample preparation methods
WO2013036603A1 (en) 2011-09-06 2013-03-14 Ibis Biosciences, Inc. Sample preparation methods
US10662485B2 (en) 2011-12-27 2020-05-26 Ibis Biosciences, Inc. Bioagent detection oligonucleotides
US9970061B2 (en) 2011-12-27 2018-05-15 Ibis Biosciences, Inc. Bioagent detection oligonucleotides
WO2014052590A1 (en) 2012-09-26 2014-04-03 Ibis Biosciences, Inc. Swab interface for a microfluidic device
WO2018089978A1 (en) * 2016-11-14 2018-05-17 Wisconsin Alumni Research Foundation Nucleic acid quantification compositions and methods
WO2023077489A1 (en) * 2021-11-06 2023-05-11 江汉大学 Mnp marker combination of yersinia pestis, primer pair combination, kit, and application thereof

Also Published As

Publication number Publication date
US8017322B2 (en) 2011-09-13
MX349763B (en) 2017-08-10
US20040110169A1 (en) 2004-06-10
EP2322649B1 (en) 2013-12-18
US20030175696A1 (en) 2003-09-18
MXPA03007927A (en) 2004-10-15
EP2333118A1 (en) 2011-06-15
CA2439655A1 (en) 2002-09-12
US8017358B2 (en) 2011-09-13
CN1505685B (en) 2012-11-14
CN1505685A (en) 2004-06-16
IL157661A0 (en) 2004-03-28
US7741036B2 (en) 2010-06-22
RU2003129269A (en) 2005-04-10
CA2853831C (en) 2018-01-23
US20030124556A1 (en) 2003-07-03
US20030175697A1 (en) 2003-09-18
US20090148836A1 (en) 2009-06-11
ES2394731T3 (en) 2013-02-05
IL238855A0 (en) 2015-06-30
NZ527857A (en) 2005-08-26
US8265878B2 (en) 2012-09-11
US20030190605A1 (en) 2003-10-09
EP2333118B1 (en) 2013-12-18
US20030175695A1 (en) 2003-09-18
CN102912036A (en) 2013-02-06
EP2311992B1 (en) 2013-02-13
WO2002070664A2 (en) 2002-09-12
AU2010200893A1 (en) 2010-04-01
US20060275788A1 (en) 2006-12-07
JP2005504508A (en) 2005-02-17
EP2311992A1 (en) 2011-04-20
US7108974B2 (en) 2006-09-19
JP5121803B2 (en) 2013-01-16
US20090148837A1 (en) 2009-06-11
US20040202997A1 (en) 2004-10-14
EP1364064A4 (en) 2005-11-23
ES2448770T3 (en) 2014-03-17
CA2853831A1 (en) 2002-09-12
EP1364064A2 (en) 2003-11-26
ZA200306810B (en) 2004-09-06
CA2439655C (en) 2014-12-09
EP2322649A1 (en) 2011-05-18
IL157661A (en) 2015-06-30
US8017743B2 (en) 2011-09-13
AU2010200893B2 (en) 2013-09-19
CN102912036B (en) 2016-08-03
EP1364064B1 (en) 2012-08-01
JP2010057495A (en) 2010-03-18
US20080160512A1 (en) 2008-07-03
AU2002244250B2 (en) 2006-11-09
US20060121520A1 (en) 2006-06-08
WO2002070664A3 (en) 2002-12-19
ES2451003T3 (en) 2014-03-26

Similar Documents

Publication Publication Date Title
US8017358B2 (en) Method for rapid detection and identification of bioagents
US8298760B2 (en) Secondary structure defining database and methods for determining identity and geographic origin of an unknown bioagent thereby
US20040121314A1 (en) Methods for rapid detection and identification of bioagents in containers
US20040121335A1 (en) Methods for rapid detection and identification of bioagents associated with host versus graft and graft versus host rejections
US20040121310A1 (en) Methods for rapid detection and identification of bioagents in forensic studies
US20040121312A1 (en) Methods for rapid detection and identification of the absence of bioagents
AU2002244250A1 (en) Method for rapid detection and identification of bioagents

Legal Events

Date Code Title Description
AS Assignment

Owner name: ISIS PHARMACEUTICALS, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ECKER, DAVID J.;GRIFFEY, RICHARD;SAMPATH, RANGARAJAN;AND OTHERS;REEL/FRAME:012047/0790;SIGNING DATES FROM 20010621 TO 20010702

STCB Information on status: application discontinuation

Free format text: EXPRESSLY ABANDONED -- DURING EXAMINATION

AS Assignment

Owner name: IBIS BIOSCIENCES, INC., CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ISIS PHARMACEUTICALS, INC.;REEL/FRAME:019691/0371

Effective date: 20070814

Owner name: IBIS BIOSCIENCES, INC.,CALIFORNIA

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:ISIS PHARMACEUTICALS, INC.;REEL/FRAME:019691/0371

Effective date: 20070814