CA2199144A1 - 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 - Google Patents

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

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CA2199144A1
CA2199144A1 CA002199144A CA2199144A CA2199144A1 CA 2199144 A1 CA2199144 A1 CA 2199144A1 CA 002199144 A CA002199144 A CA 002199144A CA 2199144 A CA2199144 A CA 2199144A CA 2199144 A1 CA2199144 A1 CA 2199144A1
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bacterial
seq
primers
probe
sample
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CA2199144C (en
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Marc Ouellette
Paul H. Roy
Michel G. Bergeron
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GeneOhm Sciences Canada Inc
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/26—Klebsiella (G)
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/285—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pasteurellaceae (F), e.g. Haemophilus influenza
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
    • C07K14/3156—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci from Streptococcus pneumoniae (Pneumococcus)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Abstract

The present invention relates to DNA-based methods for universal bacterial detection, for specific detection of the pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epiderminis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis as well as for specific detection of commonly encountered and clinically relevant bacterial antibiotic resistance genes directly from clinical specimens or, alternatively, from a bacterial colony. The above bacterial species can account for as much as 80 % of bacterial pathogens isolated in routine microbiology laboratories. The core of this invention consists primarily of the DNA sequences from all species-specific genomic DNA fragments selected by hybridization from genomic libraries or, alternatively, selected from data banks as well as any oligonucleotide sequences derived from these sequences which can be used as probes or amplification primers for PCR or any other nucleic acid amplification methods. This invention also includes DNA sequences from the selected clinically relevant antibiotic resistance genes.

Claims (62)

1. A method using probes and/or amplification primers which are specific, ubiquitous and sensitive for determining the presence and/or amount of nucleic acids:

- from a bacterial antibiotic resistance gene selected from the group consisting of blatem, blarob, blashv, aadB, aacC1, aacC2, aacC3, aacA4, mecA, vanA-vanH-vanX, satA, aacA-aphD, vat, vga, msrA, sul and int, and - from specific bacterial species selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Streptococcus pneumoniea, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Streptococcus pyogenes and Moraxella catarrhalis, in any sample suspected of containing said nucleic acids, wherein each of said nucleic acids or a variant or part thereof comprises a selected target region hybridizable with said probe or primers;

said method comprising the steps of contacting said sample with said probes or primers and detecting the presence and/or amount of hybridized probes or amplified products as an indication of the presence and/or amount of said any bacterial species, specific bacterial species and bacterial antibiotic resistance gene.
2. A method as defined in claim 1, which further comprises the detection of any bacterial species in said sample.
3. The method of claim 1 or 2, which is performed directly on a sample obtained from human patients, animals, environment or food.
4. The method of claim 1 or 2, which is performed directly on a sample consisting of one or more bacterial colonies.
5. The method of any one of claims 1 to 4 wherein said nucleic acids are all simultaneously detected.
6. The method of any one of claims 1 to 5, wherein said nucleic acids are amplified by a method selected from the group consisting of:
a) polymerase chain reaction (PCR), b) ligase chain reaction, c) nucleic acid sequence-based amplification, d) self-sustained sequence replication, e) strand displacement amplification, f) branched DNA signal amplification, g) nested PCR, and h) multiplex PCR.
7. The method of claim 6 wherein said nucleic acids are amplified by PCR.
8. The method of claim 7 wherein the PCR protocol achieves within one hour the determination of the presence of said nucleic acids by performing for each amplification cycle an annealing step of only one second at 55°C and a denaturation step of only one second at 95°C without any time specifically allowed to an elongation step.
9. A method for the detection, identification and/or quantification of Escherichia coli directly from a test sample or from bacterial colonies, which comprises the following steps:
a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of a substantially homogenous population of bacteria isolated from this sample, or inoculating said sample or said substantially homogenous population of bacteria isolated from this sample on an inert support, and lysing in situ said inoculated sample or isolated bacteria to release the bacterial DNA, said bacterial DNA being made in a substantially single stranded form;
b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO:3, SEQ
ID NO: 4, a sequence complementary thereof, a part thereof having at least 12 nucleotides in length, and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Escherichia coli, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, and c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence and/or amount of Escherichia coli in said test sample.
10. A method for detecting the presence and/or amount of Escherichia coli in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Escherichia coli DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, a sequence complementary thereof or a variant thereof;
b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Escherichia coli in said test sample.
11. The method of claim 10, wherein said at least one pair of primers is selected from the group consisting of:
a) SEQ ID NO: 42 and SEQ ID NO: 43, and b) SEQ ID NO: 131 and SEQ ID NO: 132.
12. A method for the detection, identification and/or quantification of Moraxella catarrhalis directly from a test sample or from bacterial colonies, which comprises the following steps:

a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of a substantially homogenous population of bacteria isolated from this sample, or inoculating said sample or said substantially homogenous population of bacteria isolated from this sample on an inert support, and lysing in situ said inoculated sample or isolated bacteria to release the bacterial DNA, said bacterial DNA being made in a substantially single stranded form;
b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 29, a sequence complementary thereof, a part thereof having at least 12 nucleotides in length, and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Moraxella catarrhalis, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed; and c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence and/or amount of Moraxella catarrhalis in said test sample.
13. A method for detecting the presence and/or amount of Moraxella catarrhalis in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Moraxella catarrhalis DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the group consisting of SEQ ID NO: 28, SEQ
ID NO: 29, a sequence complementary thereof and a variant thereof;

b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Moraxella catarrhalis in said test sample.
14. The method of claim 13, wherein said at least one pair of primers is selected from the group consisting of:
a) SEQ ID NO: 112 and SEQ ID NO: 113, b) SEQ ID NO: 118 and SEQ ID NO: 119, and c) SEQ ID NO: 16 0 and SEQ ID NO: 119.
15. A method for the detection, identification and/or quantification of Pseudomonas aeruginosa directly from a test sample or from bacterial colonies, which comprises the following steps:
a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of a substantially homogenous population of bacteria isolated from this sample, or inoculating said sample or said substantially homogenous population of bacteria isolated from this sample on an inert support, and lysing in situ said inoculated sample or isolated bacteria to release the bacterial DNA, said bacterial DNA being made in a substantially single stranded form;
b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 16, SEQ
ID NO: 17, a sequence complementary thereof, a part thereof having at least 12 nucleotides in length, and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Pseudomonas aeruginosa, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed; and c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence and/or amount of Pseudomonas aeruginosa in said test sample.
16. A method for detecting the presence and/or amount of Pseudomonas aeruginosa in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Pseudomonas aeruginosa DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the group consisting of SEQ ID NO: 16, SEQ
ID NO: 17, a sequence complementary thereof and a variant thereof;

b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Pseudomonas aeruginosa in said test sample.
17. The method of claim 16, wherein said at least one pair of primers is selected from the group consisting of:
a) SEQ ID NO: 83 and SEQ ID NO: 84, and b) SEQ ID NO: 85 and SEQ ID NO: 86.
18. A method for the detection, identification and/or quantification of Staphylococcus epidermidis directly from a test sample or from bacterial colonies, which comprises the following steps:
a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of a substantially homogenous population of bacteria isolated from this sample, or inoculating said sample or said substantially homogenous population of bacteria isolated from this sample on an inert support, and lysing in situ said inoculated sample or isolated bacteria to release the bacterial DNA, said bacterial DNA being made in a substantially single stranded form;
b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 36, a sequence complementary thereof, a part thereof having at least 12 nucleotides in length, and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Staphylococcus epidermidis, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed; and c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence and/or amount of Staphylococcus epidermidis in said test sample.
19. A method for detecting the presence and/or amount of Staphylococcus epidermidis in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Staphylococcus epidermidis DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the group consisting of SEQ ID NO: 36, a sequence complementary thereof and a variant thereof;
b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Staphylococcus epidermidis in said test sample.
20. The method of claim 19, wherein said at least one pair of primers is selected from the group consisting of:
a) SEQ ID NO: 145 and SEQ ID NO: 146, and b) SEQ ID NO: 147 and SEQ ID NO: 148.
21. A method for the detection, identification and/or quantification of Staphylococcus aureus directly from a test sample or from bacterial colonies, which comprises the following steps:
a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of a substantially homogenous population of bacteria isolated from this sample, or inoculating said sample or said substantially homogenous population of bacteria isolated from this sample on an inert support, and lysing in situ said inoculated sample or isolated bacteria to release the bacterial DNA, said bacterial DNA being made in a substantially single stranded form;
b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 37, a sequence complementary thereof, a part thereof having at least 12 nucleotides in length, and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Staphylococcus aureus, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed, said complex being detected by labelling means, the label being present on said probe or the label being present on a first reactive member of said labelling means, said first reactive member reacting with a second reactive member present on said probe; and c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence and/or amount of Staphylococcus aureus in said test sample.
22. A method for detecting the presence and/or amount of Staphylococcus aureus in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Staphylococcus aureus DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the group consisting of SEQ ID NO: 37, a sequence complementary thereof and a variant thereof;
b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Staphylococcus aureus in said test sample.
23. The method of claim 22, wherein said at least one pair of primers is selected from the group consisting of:
a) SEQ ID NO: 149 and SEQ ID NO: 150, b) SEQ ID NO: 149 and SEQ ID NO: 151, and c) SEQ ID NO: 152 and SEQ ID NO: 153.
24. A method for the detection, identification and/or quantification of Streptococcus pneumoniae directly from a test sample or from bacterial colonies, which comprises the following steps:
a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of a substantially homogenous population of bacteria isolated from this sample, or inoculating said sample or said substantially homogenous population of bacteria isolated from this sample on an inert support, and lysing in situ said inoculated sample or isolated bacteria to release the bacterial DNA, said bacterial DNA being made in a substantially single stranded form;
b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 35, a sequence complementary thereof, a part thereof having at least 12 nucleotides in length, and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Streptococcus pneumoniae, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed; and c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence and/or amount of Streptococcus pneumoniae in said test sample.
25. A method for detecting the presence and/or amount of Streptococcus pneumoniae in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Streptococcus pneumoniae DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the group consisting of SEQ ID NO: 35, a sequence complementary thereof and a variant thereof;
b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Streptococcus pneumoniae in said test sample.
26. The method of claim 25, wherein said pair of primers is defined in SEQ ID NO: 158 and SEQ ID NO: 159.
27. A method for the detection, identification and/or quantification of Streptococcus pyogenes directly from a test sample or from bacterial colonies, which comprises the following steps:
a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of a substantially homogenous population of bacteria isolated from this sample, or inoculating said sample or said substantially homogenous population of bacteria isolated from this sample on an inert support, and lysing in situ said inoculated sample or isolated bacteria to release the bacterial DNA, said bacterial DNA being made in a substantially single stranded form;

b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 32, SEQ
ID NO: 33, a sequence complementary thereof, a part thereof having at least 12 nucleotides in length, and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Streptococcus pyogenes, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed; and c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence and/or amount of Streptococcus pyogenes in said test sample.
28. A method for detecting the presence and/or amount of Streptococcus pyogenes in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Streptococcus pyogenes DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the group consisting of SEQ ID NO: 32, SEQ
ID NO: 33, a sequence complementary thereof and a variant thereof;
b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Streptococcus pyogenes in said test sample.
29. The method of claim 28, wherein said at least one pair of primers is selected from the group consisting of:

a) SEQ ID NO: 141 and SEQ ID NO: 142, and b) SEQ ID NO: 143 and SEQ ID NO: 144.
30. A method for the detection, identification and/or quantification of Enterococcus faecalis directly from a test sample or from bacterial colonies, which comprises the following steps:
a) depositing and fixing on an inert support or leaving in solution the bacterial DNA of the sample or of a substantially homogenous population of bacteria isolated from this sample, or inoculating said sample or said substantially homogenous population of bacteria isolated from this sample on an inert support, and lysing in situ said inoculated sample or isolated bacteria to release the bacterial DNA, said bacterial DNA being made in a substantially single stranded form;
b) contacting said single stranded DNA with a probe, said probe comprising at least one single stranded nucleic acid which nucleotidic sequence is selected from the group consisting of SEQ ID NO: 1, SEQ

ID NO: 2, a sequence complementary thereof, a part thereof at least 12 nucleotides in length, and a variant thereof, which specifically and ubiquitously anneals with strains or representatives of Enterococcus faecalis, under conditions such that the nucleic acid of said probe can selectively hybridize with said bacterial DNA, whereby a hybridization complex is formed; and c) detecting the presence of said hybridization complex on said inert support or in said solution as an indication of the presence and/or amount of Enterococcus faecalis in said test sample.
31. A method for detecting the presence and/or amount of Enterococcus faecalis in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing at least one pair of oligonucleotide primers having at least 12 nucleotides in length, one of said primers being capable of hybridizing selectively with one of the two complementary strands of Enterococcus faecalis DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template, said at least one pair of primers being chosen from within the group consisting of SEQ ID NO: 1, SEQ ID
NO: 2, a sequence complementary thereof and a variant thereof;
b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of Enterococcus faecalis in said test sample.
32. The method of claim 31, wherein said at least one pair of primers is selected from the group consisting of:
a) SEQ ID NO: 38 and SEQ ID NO: 39, and b) SEQ ID NO: 40 and SEQ ID NO: 41.
33. A method for detecting the presence and/or amount of any bacterial species in a test sample which comprises the following steps:
a) treating said sample with an aqueous solution containing a pair of universal primers which sequence is defined in SEQ ID NO: 126 and SEQ ID NO: 127, one of said primers being capable of hybridizing selectively with one of the two complementary strands of said any bacterial species DNA that contains a target sequence, and the other of said primers being capable of hybridizing with the other of said strands so as to form an extension product which contains the target sequence as a template;
b) synthesizing an extension product of each of said primers which extension products contain the target sequence, and amplifying said target sequence, if any, to a detectable level; and c) detecting the presence and/or amount of said amplified target sequence as an indication of the presence and/or amount of said any bacterial species in said test sample.
34. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene blatem directly from a test sample or from bacterial colonies, which comprises the steps of:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 161, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene blatem.
35. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene blarob directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 162, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene balrod.
36. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene blashv directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 163, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene blashv.
37. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aadB directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 164, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene aadB.
38. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC1 directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 165, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene aacC1.
39. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC2 directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 166, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene aacC2.
40. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacC3 directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 167, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene aacC3.
41. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacA4 directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 168, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene accA4.
42. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene mecA directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 169, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene mecA.
43. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to vancomycin mediated by the bacterial antibiotic resistance genes vanH, vanA and vanX directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 170, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance genes vanH, vanA, vanX.
44. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to streptogramin A mediated by the bacterial antibiotic resistance gene satA directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 173, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene satA.
45. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to aminoglycoside antibiotics mediated by the bacterial antibiotic resistance gene aacA-aphD directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 174, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene accA- aphD.
46. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vat directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 175, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene vat.
47. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to virginiamycin mediated by the bacterial antibiotic resistance gene vga directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 176, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene vga.
48. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to erythromycin mediated by the bacterial antibiotic resistance gene msrA directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 177, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene msrA.
49. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to b-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene int directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 171, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene int.
50. A method as defined in any one of claims 1 to 4, which comprises the evaluation of the presence of a bacterial resistance to .beta.-lactams, aminoglycosides, chloramphenicol and/or trimethoprim mediated by the bacterial antibiotic resistance gene sul directly from a test sample or from bacterial colonies, which comprises the following steps:
a) contacting the bacterial DNA with a probe or with amplification primers, said probe or primers comprising at least one single stranded nucleic acid which nucleotidic sequence having at least 12 nucleotides in length selected from within the group consisting of SEQ ID NO: 172, a sequence complementary thereof, and a variant thereof, which specifically hybridizes with said bacterial antibiotic resistance gene; and b) detecting the presence of a hybridization complex or of an amplification product as an indication of a bacterial resistance to .beta.-lactam antibiotics mediated by the bacterial antibiotic resistance gene sul.
51. A nucleic acid having the nucleotide sequence of any one of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 29, SEQ ID
NO: 36, SEQ ID NO: 37, a part thereof and variants thereof which, when in single stranded form, ubiquitously and specifically hybridize with a target bacterial DNA as a probe or as a primer.
52. An oligonucleotide having a nucleotidic sequence of any one of SEQ ID NOs: 38 to 43, SEQ ID NOs: 83 to 86, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 118, SEQ ID
NO: 119, SEQ ID NO: 127, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NOs: 141 to 153, SEQ ID NO: 158, SEQ ID NO: 159 and SEQ ID NO: 160.
53. A recombinant plasmid comprising a nucleic acid as defined in claim 51.
54. A recombinant host which has been transformed by a recombinant plasmid according to claim 53.
55. A recombinant host according to claim 54 wherein said host is Escherichia coli.
56. A diagnostic kit for the detection and/or quantification of the nucleic acids of any combination of the bacterial species defined in any one of claims 9, 12, 15, 18, 21, 24, 27 and 30, comprising any combination of probes defined therein.
57. A diagnostic kit for the detection and/or quantification of the nucleic acids of any combination of the bacterial species defined in any one of claims 10, 11, 13, 14, 16, 17, 19, 20, 22, 23, 25, 26, 28, 29, 31 and 32, comprising any combination of pairs of primers defined therein.
58. A diagnostic kit for the detection and/or quantification of the nucleic acids of any combination of the bacterial resistance genes defined in any one of claims 34 to 50 comprising any combination of probes defined therein.
59. A diagnostic kit for the detection and/or quantification of nucleic acids of any bacterial species comprising the primers defined in SEQ ID Nos. 126 and 127.
60. A diagnostic kit for the simultaneous detection and/or quantification of nucleic acids of any bacterial antibiotic resistance genes selected from the group consisting of : blatem, blarob, blashv, aadB, aacC1, aacC2, aacC3, aacA4, mecA, vanA, vanH, vanX, satA, aacA-aphD, vat, vga, msrA, sul and int, and of any combination of the bacterial species defined in claim 56, comprising any combination of the bacterial probes defined in claim 56 and any combination of the probes to the antibiotic resistance genes defined in any one of SEQ ID NOs: 161 to 177 in whole or in part.
61. A diagnostic kit for the simultaneous detection and/or quantification of nucleic acids of any antibiotic resistance genes selected from the group consisting of :

blatem, blarob, blashv, aadB, aacC1, aacC2, aacC3, aacA4, mecA, vanA, vanH, vanX, satA, aacA-aphD, vat, vga, msrA, sul and int, and of any combination of the bacterial species defined in claim 57, comprising any combination of the pairs of primers defined in claim 57 and any combination of pairs of primers that anneal to the antibiotic resistance genes defined in any one of SEQ ID
NOs: 161 to 177.
62. A diagnostic kit for the simultaneous detection and/or quantification of any bacterial species, of any bacterial antibiotic resistance genes selected from the group consisting of: blatem, blarob, blashv, aadB, aacC1, aacC2, aacC3, aacA4, mecA, vanA, vanH, vanX, satA, aacA-aphD, vat, vga, msrA, sul and int, and of any combination of the bacterial species defined in claim 57, comprising any combination of pairs of primers defined in claim 57, the pairs of primers defined in SEQ
ID Nos. 126 and 127 and any combination of pairs of primers that anneal to the antibiotic resistance genes defined in any one of SEQ ID Nos.: 161 to 177.
CA2199144A 1994-09-12 1995-09-12 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 Expired - Lifetime CA2199144C (en)

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