US20090047671A1 - 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 Download PDF

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US20090047671A1
US20090047671A1 US11/842,092 US84209207A US2009047671A1 US 20090047671 A1 US20090047671 A1 US 20090047671A1 US 84209207 A US84209207 A US 84209207A US 2009047671 A1 US2009047671 A1 US 2009047671A1
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Michel G. Bergeron
Marc Quellette
Paul H. Roy
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GeneOhm Sciences Canada Inc
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Definitions

  • Bacteria are classically identified by their ability to utilize different substrates as a source of carbon and nitrogen through the use of biochemical tests such as the API20ETM system. Susceptibility testing of Gram negative bacilli has progressed to microdilution tests. Although the API and the microdilution systems are cost-effective, at least two days are required to obtain preliminary results due to the necessity of two successive overnight incubations to isolate and identify the bacteria from the specimen. Some faster detection methods with sophisticated and expensive apparatus have been developed. For example, the fastest identification system, the autoSCAN-Walk-Away SystemTM identifies both Gram negative and Gram positive from isolated bacterial colonies in 2 hours and susceptibility patterns to antibiotics in only 7 hours.
  • Urine specimens found positive by culture are further characterized using standard biochemical tests to identify the bacterial pathogen and are also tested for susceptibility to antibiotics.
  • DNA probe and DNA amplification technologies offer several advantages over conventional methods. There is no need for subculturing, hence the organism can be detected directly in clinical samples thereby reducing the costs and time associated with isolation of pathogens. DNA-based technologies have proven to be extremely useful for specific applications in the clinical microbiology laboratory. For example, kits for the detection of fastidious organisms based on the use of hybridization probes or DNA amplification for the direct detection of pathogens in clinical specimens are commercially available (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).
  • the present invention is an advantageous alternative to the conventional culture identification methods used in hospital clinical microbiology laboratories and in private clinics for routine diagnosis. Besides being much faster, DNA-based diagnostic tests are more accurate than standard biochemical tests presently used for diagnosis because the bacterial genotype (e.g. DNA level) is more stable than the bacterial phenotype (e.g. biochemical properties).
  • the originality of this invention is that genomic DNA fragments (size of at least 100 base pairs) specific for 12 species of commonly encountered bacterial pathogens were selected from genomic libraries or from data banks.
  • Amplification primers or oligonucleotide probes (both less than 100 nucleotides in length) which are both derived from the sequence of species-specific DNA fragments identified by hybridization from genomic libraries or from selected data bank sequences are used as a basis to develop diagnostic tests. Oligonucleotide primers and probes for the detection of commonly encountered and clinically important bacterial resistance genes are also included. For example, Annexes I and II present a list of suitable oligonucleotide probes and PCR primers which were all derived from the species-specific DNA fragments selected from genomic libraries or from data bank sequences.
  • oligonucleotide sequences appropriate for the specific detection of the above bacterial species other than those listed in Annexes 1 and 2 may be derived from the species-specific fragments or from the selected data bank sequences.
  • the oligonucleotides may be shorter or longer than the ones we have chosen and may be selected anywhere else in the identified species-specific sequences or selected data bank sequences.
  • the oligonucleotides may be designed for use in amplification methods other than PCR.
  • the core of this invention is the identification of species-specific genomic DNA fragments from bacterial genomic DNA libraries and the selection of genomic DNA fragments from data bank sequences which are used as a source of species-specific and ubiquitous oligonucleotides.
  • the selection of oligonucleotides suitable for diagnostic purposes from the sequence of the species-specific fragments or from the selected data bank sequences requires much effort it is quite possible for the individual skilled in the art to derive from our fragments or selected data bank sequences suitable oligonucleotides which are different from the ones we have selected and tested as examples (Annexes I and II).
  • oligonucleotide primers and probes were selected from the highly conserved 16S or 23S rRNA genes to detect all bacterial pathogens possibly encountered in clinical specimens in order to determine whether a clinical specimen is infected or not. This strategy allows rapid screening out of the numerous negative clinical specimens submitted for bacteriological testing.
  • sequence from genomic DNA fragments (size of at least 100 base pairs and all described in the sequence listing) selected either by hybridization from genomic libraries or from data banks and which are specific for the detection of commonly encountered bacterial pathogens (i.e.
  • Escherichia coli Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis ) in clinical specimens.
  • These bacterial species are associated with approximately 90% of urinary tract infections and with a high percentage of other severe infections including septicemia, meningitis, pneumonia, intraabdominal infections, skin infections and many other severe respiratory tract infections. Overall, the above bacterial species may account for up to 80% of bacterial pathogens isolated in routine microbiology laboratories.
  • Synthetic oligonucleotides for hybridization (probes) or DNA amplification (primers) were derived from the above species-specific DNA fragments (ranging in sizes from 0.25 to 5.0 kilobase pairs (kbp)) or from selected data bank sequences (GenBank and EMBL).
  • Bacterial species for which some of the oligonucleotide probes and amplification primers were derived from selected data bank sequences are Escherichia coli, Enterococcus faecalis, Streptococcus pyogenes and Pseudomonas aeruginosa .
  • hybridization with either fragment or oligonucleotide probes
  • DNA amplification protocols for the detection of pathogens from clinical specimens renders possible a very rapid bacterial identification. This will greatly reduce the time currently required for the identification of pathogens in the clinical laboratory since these technologies can be applied for bacterial detection and identification directly from clinical specimens with minimum pretreatment of any biological specimens to release bacterial DNA.
  • probes and amplification primers allow identification of the bacterial species directly from clinical specimens or, alternatively, from an isolated colony.
  • DNA amplification assays have the added advantages of being faster and more sensitive than hybridization assays, since they allow rapid and exponential in vitro replication of the target segment of DNA from the bacterial genome.
  • Universal probes and amplification primers selected from the 16S or 23S rRNA genes highly conserved among bacteria, which permit the detection of any bacterial pathogens, will serve as a procedure to screen out the numerous negative clinical specimens received in diagnostic laboratories.
  • the use of oligonucleotide probes or primers complementary to characterized bacterial genes encoding resistance to antibiotics to identify commonly encountered and clinically important resistance genes is also under the scope of this invention.
  • DNA fragment probes were developed for the following bacterial species: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Haemophilus influenzae and Moraxella catarrhalis .
  • oligonucleotide sequences were exclusively derived from selected data bank sequences). These species-specific fragments were selected from bacterial genomic libraries by hybridization to DNA from a variety of Gram positive and Gram negative bacterial species (Table 5).
  • the chromosomal DNA from each bacterial species for which probes were seeked was isolated using standard methods. DNA was digested with a frequently cutting restriction enzyme such as Sau3AI and then ligated into the bacterial plasmid vector pGEM3Zf (Promega) linearized by appropriate restriction endonuclease digestion. Recombinant plasmids were then used to transform competent E. coli strain DH5 ⁇ thereby yielding a genomic library. The plasmid content of the transformed bacterial cells was analyzed using standard methods. DNA fragments of target bacteria ranging in size from 0.25 to 5.0 kilobase pairs (kbp) were cut out from the vector by digestion of the recombinant plasmid with various restriction endonucleases. The insert was separated from the vector by agarose gel electrophoresis and purified in low melting point agarose gels. Each of the purified fragments of bacterial genomic DNA was then used as a probe for specificity tests.
  • a frequently cutting restriction enzyme such as
  • the gel-purified restriction fragments of unknown coding potential were labeled with the radioactive nucleotide ⁇ 32P(dATP) which was incorporated into the DNA fragment by the random priming labeling reaction.
  • Non-radioactive modified nucleotides could also be incorporated into the DNA by this method to serve as a label.
  • Each DNA fragment probe i.e. a segment of bacterial genomic DNA of at least 100 bp in length cut out from clones randomly selected from the genomic library
  • the double-stranded labeled DNA probe was heat-denatured to yield labeled single-stranded DNA which could then hybridize to any single-stranded target DNA fixed onto a solid support or in solution.
  • the target DNAs consisted of total cellular DNA from an array of bacterial species found in clinical samples (Table 5). Each target DNA was released from the bacterial cells and denatured by conventional methods and then irreversibly fixed onto a solid support (e.g. nylon or nitrocellulose membranes) or free in solution.
  • Pre-hybridization, hybridization and post-hybridization conditions were as follows: (i) Pre-hybridization; in 1 M NaCl+10% dextran sulfate+1% SDS (sodium dodecyl sulfate)+1 .mu.g/ml salmon sperm DNA at 650.degree. C. for 15 min. (ii) Hybridization; in fresh pre-hybridization solution containing the labeled probe at 650.degree. C. overnight.
  • Species-specific DNA fragments selected from various bacterial genomic libraries ranging in size from 0.25 to 5.0 kbp were isolated for 10 common bacterial pathogens (Table 6) based on hybridization to chromosomal DNAs from a variety of bacteria performed as described above. All of the bacterial species tested (66 species listed in Table 5) were likely to be pathogens associated with common infections or potential contaminants which can be isolated from clinical specimens. A DNA fragment probe was considered specific only when it hybridized solely to the pathogen from which it was isolated. DNA fragment probes found to be specific were subsequently tested for their ubiquity (i.e. ubiquitous probes recognized most isolates of the target species) by hybridization to bacterial DNAs from approximately 10 to 80 clinical isolates of the species of interest (Table 6). The DNAs were denatured, fixed onto nylon membranes and hybridized as described above.
  • nucleotide sequence of the totality or of a portion of the species-specific DNA fragments isolated was determined using the dideoxynucleotide termination sequencing method which was performed using SequenaseTM (USB Biochemicals) or T7 DNA polymerase (Pharmacia). These nucleotide sequences are shown in the sequence listing. Alternatively, sequences selected from data banks (GenBank and EMBL) were used as sources of oligonucleotides for diagnostic purposes for Escherichia coli, Enterococcus faecalis, Streptococcus pyogenes and Pseudomonas aeruginosa .
  • oligonucleotide primers or probes derived from a variety of genomic DNA fragments (size of more than 100 bp) selected from data banks was tested for their specificity and ubiquity in PCR and hybridization assays as described later. It is important to note that the data bank sequences were selected based on their potential of being species-specific according to available sequence information. Only data bank sequences from which species-specific oligonucleotides could be derived are included in this invention.
  • Oligonucleotide probes and amplification primers derived from species-specific fragments selected from the genomic libraries or from data bank sequences were synthesized using an automated DNA synthesizer (Millipore). Prior to synthesis, all oligonucleotides (probes for hybridization and primers for DNA amplification) were evaluated for their suitability for hybridization or DNA amplification by polymerase chain reaction (PCR) by computer analysis using standard programs (e.g. Genetics Computer Group (GCG) and OligoTM 4.0 (National Biosciences)).
  • GCG Genetics Computer Group
  • OligoTM 4.0 National Biosciences
  • PCR primer pairs were also evaluated prior to the synthesis by verifying the absence of unwanted features such as long stretches of one nucleotide, a high proportion of G or C residues at the 3′ end and a 3′-terminal T residue (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).
  • oligonucleotides size less than 100 nucleotides
  • have some advantages over DNA fragment probes for the detection of bacteria such as ease of preparation in large quantities, consistency in results from batch to batch and chemical stability.
  • oligonucleotides were 5′ end-labeled with the radionucleotide ⁇ 32P(ATP) using T4 polynucleotide kinase (Pharmacia).
  • T4 polynucleotide kinase Pharmacia
  • the unincorporated radionucleotide was removed by passing the labeled single-stranded oligonucleotide through a Sephadex G50 column.
  • oligonucleotides were labeled with biotin, either enzymatically at their 3′ ends or incorporated directly during synthesis at their 5′ ends, or with digoxigenin. It will be appreciated by the person skilled in the art that labeling means other than the three above labels may be used.
  • the target DNA was denatured, fixed onto a solid support and hybridized as previously described for the DNA fragment probes.
  • Conditions for pre-hybridization and hybridization were as described earlier.
  • Post-hybridization washing conditions were as follows: twice in 3 ⁇ SSC containing 1% SDS, twice in 2 ⁇ SSC containing 1% SDS and twice in 1 ⁇ SSC containing 1% SDS (all of these washes were at 65° C. for 15 min), and a final wash in 0.1 ⁇ SSC containing 1% SDS at 25° C. for 15 min.
  • For probes labeled with radioactive labels the detection of hybrids was by autoradiography as described earlier.
  • For non-radioactive labels detection may be calorimetric or by chemiluminescence.
  • the oligonucleotide probes may be derived from either strand of the duplex DNA.
  • the probes may consist of the bases A, G, C, or T or analogs.
  • the probes may be of any suitable length and may be selected anywhere within the species-specific genomic DNA fragments selected from the genomic libraries or from data bank sequences.
  • primer pairs were derived either from the sequenced species-specific DNA fragments or from data bank sequences or, alternatively, were shortened versions of oligonucleotide probes. Prior to synthesis, the potential primer pairs were analyzed by using the program OligoTM4.0 (National Biosciences) to verify that they are likely candidates for PCR amplifications.
  • Clinical specimens or bacterial colonies were added directly to the 50 ⁇ L PCR reaction mixtures containing 50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl 2 , 0.4 ⁇ M of each of the two primers, 200 ⁇ M of each of the four dNTPs and 1.25 Units of Taq DNA polymerase (Perkin Elmer). PCR reactions were then subjected to thermal cycling (3 min at 95° C. followed by 30 cycles of 1 second at 95° C. and 1 second at 55° C.) using a Perkin Elmer 480TM thermal cycler and subsequently analyzed by standard ethidium bromide-stained agarose gel electrophoresis.
  • glycerol or dimethyl sulfoxide (DMSO) or other related solvents can be used to increase the sensitivity of the PCR and to overcome problems associated with the amplification of target with a high GC content or with strong secondary structures.
  • concentration ranges for glycerol and DMSO are 5-15% (v/v) and 3-10% (v.backslash.v), respectively.
  • concentration ranges for the amplification primers and the MgCl 2 are 0.1-1.0 ⁇ M and 1.5-3.5 mM, respectively. Modifications of the standard PCR protocol using external and nested primers (i.e. nested PCR) or using more than one primer pair (i.e.
  • multiplex PCR may also be used (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). For more details about the PCR protocols and amplicon detection methods see examples 7 and 8.
  • LCR ligase chain reaction
  • TAS transcription-based amplification systems
  • NASBA self-sustained sequence replication
  • SDA strand displacement amplification
  • bDNA branched DNA
  • oligonucleotide probes derived either from the sequenced species-specific fragments or from data bank sequences, was tested by hybridization to DNAs from the array of bacterial species listed in Table 5 as previously described. Oligonucleotides found to be specific were subsequently tested for their ubiquity by hybridization to bacterial DNAs from approximately 80 isolates of the target species as described for fragment probes. Probes were considered ubiquitous when they hybridized specifically with the DNA from at least 80% of the isolates. Results for specificity and ubiquity tests with the oligonucleotide probes are summarized in Table 6. The specificity and ubiquity of the amplification primer pairs were tested directly from cultures (see example 7) of the same bacterial strains.
  • PCR assays were performed directly from bacterial colonies of approximately 80 isolates of the target species. Results are summarized in Table 7. All specific and ubiquitous oligonucleotide probes and amplification primers for each of the 12 bacterial species investigated are listed in Annexes I and II, respectively. Divergence in the sequenced DNA fragments can occur and, insofar as the divergence of these sequences or a part thereof does not affect the specificity of the probes or amplification primers, variant bacterial DNA is under the scope of this invention.
  • oligonucleotides and amplification primers were therefore synthesized from highly conserved portions of bacterial 16S or 23S ribosomal RNA gene sequences available in data banks (Annexes III and IV).
  • a pool of seven oligonucleotides (Annex I; Table 6) hybridized strongly to DNA from all bacterial species listed in Table 5.
  • This pool of universal probes labeled with radionucleotides or with any other modified nucleotides is consequently very useful for detection of bacteria in urine samples with a sensitivity range of ⁇ 10 7 CFU/L. These probes can also be applied for bacterial detection in other clinical samples.
  • Amplification primers also derived from the sequence of highly conserved ribosomal RNA genes were used as an alternative strategy for universal bacterial detection directly from clinical specimens (Annex IV; Table 7).
  • the DNA amplification strategy was developed to increase the sensitivity and the rapidity of the test. This amplification test was ubiquitous since it specifically amplified DNA from 23 different bacterial species encountered in clinical specimens.
  • ribosomal RNA genes could also be good candidates for universal bacterial detection directly from clinical specimens. Such genes may be associated with processes essential for bacterial survival (e.g. protein synthesis, DNA synthesis, cell division or DNA repair) and could therefore be highly conserved during evolution. We are working on these candidate genes to develop new rapid tests for the universal detection of bacteria directly from clinical specimens.
  • Antimicrobial resistance complicates treatment and often leads to therapeutic failures. Furthermore, overuse of antibiotics inevitably leads to the emergence of bacterial resistance. Our goal is to provide the clinicians, within one hour, the needed information to prescribe optimal treatments. Besides the rapid identification of negative clinical specimens with DNA-based tests for universal bacterial detection and the identification of the presence of a specific pathogen in the positive specimens with DNA-based tests for specific bacterial detection, the clinicians also need timely information about the ability of the bacterial pathogen to resist antibiotic treatments. We feel that the most efficient strategy to evaluate rapidly bacterial resistance to antimicrobials is to detect directly from the clinical specimens the most common and important antibiotic resistance genes (i.e. DNA-based tests for the detection of antibiotic resistance genes).
  • any recombinant plasmids and corresponding transformed host cells are under the scope of this invention.
  • the plasmid content of the transformed bacterial cells was analyzed using standard methods. DNA fragments from target bacteria ranging in size from 0.25 to 5.0 kbp were cut out from the vector by digestion of the recombinant plasmid with various restriction endonucleases. The insert was separated from the vector by agarose gel electrophoresis and purified in a low melting point agarose gel. Each of the purified fragments was then used for specificity tests.
  • the label used was ⁇ 32P(dATP), a radioactive nucleotide which can be incorporated enzymatically into a double-stranded DNA molecule.
  • the fragment of interest is first denatured by heating at 95° C. for 5 min, then a mixture of random primers is allowed to anneal to the strands of the fragments. These primers, once annealed, provide a starting point for synthesis of DNA.
  • DNA polymerase usually the Klenow fragment, is provided along with the four nucleotides, one of which is radioactive. When the reaction is terminated, the mixture of new DNA molecules is once again denatured to provide radioactive single-stranded DNA molecules (i.e. the probe).
  • other modified nucleotides may be used to label the probes.
  • Species-specific DNA fragments ranging in size from 0.25 to 5.0 kbp were isolated for 10 common bacterial pathogens (Table 6) based on hybridization to chromosomal DNAs from a variety of bacteria. Samples of whole cell DNA for each bacterial strain listed in Table 5 were transferred onto a nylon membrane using a dot blot apparatus, washed and denatured before being irreversibly fixed. Hybridization conditions were as described earlier. A DNA fragment probe was considered specific only when it hybridized solely to the pathogen from which it was isolated. Labeled DNA fragments hybridizing specifically only to target bacterial species (i.e.
  • testing of the strains is by colony hybridization.
  • the bacterial strains were inoculated onto a nylon membrane placed on nutrient agar. The membranes were incubated at 37° C. for two hours and then bacterial lysis and DNA denaturation were carried out according to standard procedures. DNA hybridization was performed as described earlier.
  • Nucleotide sequencing of DNA fragments The nucleotide sequence of the totality or a portion of each fragment found to be specific and ubiquitous (Example 1) was determined using the dideoxynucleotide termination sequencing method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA. 74:5463-5467). These DNA sequences are shown in the sequence listing. Oligonucleotide probes and amplification primers were selected from these nucleotide sequences, or alternatively, from selected data banks sequences and were then synthesized on an automated Biosearch synthesizer (MilliporeTM) using phosphoramidite chemistry.
  • oligonucleotide was 5′ end-labeled with ⁇ 32P-ATP by the T4 polynucleotide kinase (Pharmacia) as described earlier.
  • the label could also be non-radioactive.
  • oligonucleotide probes Specificity test for oligonucleotide probes. All labeled oligonucleotide probes were tested for their specificity by hybridization to DNAs from a variety of Gram positive and Gram negative bacterial species as described earlier. Species-specific probes were those hybridizing only to DNA from the bacterial species from which it was isolated. Oligonucleotide probes found to be specific were submitted to ubiquity tests as follows.
  • Ubiquity test for oligonucleotide probes were then used in ubiquity tests with approximately 80 strains of the target species. Chromosomal DNAs from the isolates were transferred onto nylon membranes and hybridized with labeled oligonucleotide probes as described for specificity tests. The batteries of approximately 80 isolates constructed for each target species contain reference ATCC strains as well as a variety of clinical isolates obtained from various sources. Ubiquitous probes were those hybridizing to at least 80% of DNAs from the battery of clinical isolates of the target species. Examples of specific and ubiquitous oligonucleotide probes are listed in Annex I.
  • a pool of specific oligonucleotide probes is used for bacterial identification (i) to increase sensitivity and assure 100% ubiquity or (ii) to identify simultaneously more than one bacterial species. Bacterial identification could be done from isolated colonies or directly from clinical specimens
  • PCR amplification The technique of PCR was used to increase sensitivity and rapidity of the tests.
  • the PCR primers used were often shorter derivatives of the extensive sets of oligonucleotides previously developed for hybridization assays (Table 6).
  • the sets of primers were tested in PCR assays performed directly from a bacterial colony or from a bacterial suspension (see Example 7) to determine their specificity and ubiquity (Table 7). Examples of specific and ubiquitous PCR primer pairs are listed in annex II.
  • PCR assays were performed either directly from a bacterial colony or from a bacterial suspension, the latter being adjusted to a standard McFarland 0.5 (corresponds to 1.5 times.10.sup.8 bacteria/mL).
  • McFarland 0.5 corresponds to 1.5 times.10.sup.8 bacteria/mL.
  • direct amplification from a colony a portion of the colony was transferred directly to a 50 ⁇ L PCR reaction mixture (containing 50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl 2 , 0.4 ⁇ M of each of the two primers, 200 ⁇ M of each of the four dNTPs and 1.25 Unit of Taq DNA polymerase (Perkin Elmer)) using a plastic rod.
  • PCR amplification products were then analyzed by standard agarose gel (2%) electrophoresis. Amplification products were visualized in agarose gels containing 2.5 ⁇ g/mL of ethidium bromide under UV at 254 mm. The entire PCR assay can be completed in approximately one hour.
  • amplification from bacterial cultures was performed as described above but using a “hot start” protocol.
  • an initial reaction mixture containing the target DNA, primers and dNTPs was heated at 85° C. prior to the addition of the other components of the PCR reaction mixture.
  • the final concentration of all reagents was as described above.
  • the PCR reactions were submitted to thermal cycling and analysis as described above.
  • samples were routinely diluted in lysis buffer containing detergent(s). Subsequently, the lysate was added directly to the PCR reaction mixture. Heat treatments of the lysates, prior to DNA amplification, using the thermocycler or a microwave oven could also be performed to increase the efficiency of cell lysis.
  • PCR has the advantage of being compatible with crude DNA preparations. For example, blood, cerebrospinal fluid and sera may be used directly in PCR assays after a brief heat treatment. We intend to use such rapid and simple strategies to develop fast protocols for DNA amplification from a variety of clinical specimens.
  • Detection of antibiotic resistance genes The presence of specific antibiotic resistance genes which are frequently encountered and clinically relevant is identified using the PCR amplification or hybridization protocols described in previous sections. Specific oligonucleotides used as a basis for the DNA-based tests are selected from the antibiotic resistance gene sequences. These tests can be performed either directly from clinical specimens or from a bacterial colony and should complement diagnostic tests for specific bacterial identification.
  • assays were performed by multiplex PCR (i.e. using several pairs of primers in a single PCR reaction) to (i) reach an ubiquity of 100% for the specific target pathogen or (ii) to detect simultaneously several species of bacterial pathogens.
  • Multiplex PCR assays could also be used to (i) detect simultaneously several bacterial species or, alternatively, (ii) to simultaneously identify the bacterial pathogen and detect specific antibiotic resistance genes either directly from a clinical specimen or from a bacterial colony.
  • amplicon detection methods should be adapted to differentiate the various amplicons produced.
  • Standard agarose gel electrophoresis could be used because it discriminates the amplicons based on their sizes.
  • Another useful strategy for this purpose would be detection using a variety of fluorochromes emitting at different wavelengths which are each coupled with a specific oligonucleotide linked to a fluorescence quencher which is degraded during amplification to release the fluorochrome (e.g. TaqManTM, Perkin Elmer).
  • Detection of amplification Products The person skilled in the art will appreciate that alternatives other than standard agarose gel electrophoresis (Example 7) may be used for the revelation of amplification products. Such methods may be based on the detection of fluorescence after amplification (e.g. AmplisensorTM, Biotronics; TaqManTM) or other labels such as biotin (SHARP SignalTM system, Digene Diagnostics). These methods are quantitative and easily automated.
  • One of the amplification primers or an internal oligonucleotide probe specific to the amplicon(s) derived from the species-specific fragment probes is coupled with the fluorochrome or with any other label. Methods based on the detection of fluorescence are particularly suitable for diagnostic tests since they are rapid and flexible as fluorochromes emitting different wavelengths are available (Perkin Elmer).
  • Species-specific, universal and antibiotic resistance gene amplification primers can be used in other rapid amplification procedures such as the ligase chain reaction (LCR), transcription-based amplification systems (TAS), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) and branched DNA (bDNA) or any other methods to increase the sensitivity of the test.
  • Amplifications can be performed from an isolated bacterial colony or directly from clinical specimens. The scope of this invention is therefore not limited to the use of PCR but rather includes the use of any procedures to specifically identify bacterial DNA and which may be used to increase rapidity and sensitivity of the tests.
  • test kit would contain sets of probes specific for each bacterium as well as a set of universal probes.
  • the kit is provided in the form of test components, consisting of the set of universal probes labeled with non-radioactive labels as well as labeled specific probes for the detection of each bacterium of interest in specific clinical samples.
  • the kit will also include test reagents necessary to perform the pre-hybridization, hybridization, washing steps and hybrid detection. Finally, test components for the detection of known antibiotic resistance genes (or derivatives therefrom) will be included.
  • the kit will include standard samples to be used as negative and positive controls for each hybridization test.
  • kits Components to be included in the kits will be adapted to each specimen type and to detect pathogens commonly encountered in that type of specimen. Reagents for the universal detection of bacteria will also be included. Based on the sites of infection, the following kits for the specific detection of pathogens may be developed:
  • a kit for the universal detection of bacterial pathogens from most clinical specimens which contains sets of probes specific for highly conserved regions of the bacterial genomes.
  • a kit for the detection of bacterial pathogens retrieved from urine samples which contains eight specific test components (sets of probes for the detection of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Staphylococcus aureus and Staphylococcus epidermidis ).
  • a kit for the detection of respiratory pathogens which contains seven specific test components (sets of probes for detecting Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus ).
  • a kit for the detection of pathogens retrieved from blood samples which contains eleven specific test components (sets of probes for the detection of Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Staphylococcus epidermidis ).
  • a kit for the detection of pathogens causing meningitis which contains four specific test components (sets of probes for the detection of Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli and Pseudomonas aeruginosa ).
  • a kit for the detection of clinically important antibiotic resistance genes which contains sets of probes for the specific detection of at least one of the 19 following genes associated with bacterial resistance: bla tem , bla rob , bla shv , aadB, aacC1, aacC2, aacC3, aacA4, mecA, vanA, vanH, vanX, satA, aacA-aphD, vat, vga, msrA, sul and int.
  • kits adapted for the detection of pathogens from skin, abdominal wound or any other clinically relevant kits will be developed.
  • test kits contain all reagents and controls to perform DNA amplification assays. Diagnostic kits will be adapted for amplification by PCR (or other amplification methods) performed directly either from clinical specimens or from a bacterial colony. Components required for universal bacterial detection, bacterial identification and antibiotic resistance genes detection will be included.
  • Amplification assays could be performed either in tubes or in microtitration plates having multiple wells.
  • the wells will be coated with the specific amplification primers and control DNAs and the detection of amplification products will be automated.
  • Reagents and amplification primers for universal bacterial detection will be included in kits for tests performed directly from clinical specimens.
  • Components required for bacterial identification and antibiotic resistance gene detection will be included in kits for testing directly from colonies as well as in kits for testing directly from clinical specimens.
  • kits will be adapted for use with each type of specimen as described in example 13 for hybridization-based diagnostic kits.
  • probes and amplification primers described in this invention for bacterial detection and identification is not limited to clinical microbiology applications. In fact, we feel that other sectors could also benefit from these new technologies. For example, these tests could be used by industries for quality control of food, water, pharmaceutical products or other products requiring microbiological control. These tests could also be applied to detect and identify bacteria in biological samples from organisms other than humans (e.g. other primates, mammals, farm animals and live stocks). These diagnostic tools could also be very useful for research purposes including clinical trials and epidemiological studies.
  • Bacterial Species used for testing the specificity of DNA fragment probes, oligonucleotides probes and PCR primers Number Number of of Bacterial species strains Bacterial species strains Gram negative: tested Gram negative: tested Proteus mirabilis 5 Streptococcus pneumoniae 7 Klebsiella pneumoniae 5 Streptococcus salivarius 2 Pseudomonas aeruginosa 5 Streptococcus viridans 2 Escherichia coli 5 Streptococcus pyogenes 2 Moraxella catarrhalis 5 Staphylococcus aureus 2 Proteus vulgaris 2 Staphylococcus 2 epidermidis Morganella morganii 2 Staphylococcus 5 saprophyticus Enterobater cloacae 2 Micrococcus species 2 Providencia stuartii 1 Corynebacterium species 2 Providencia spp.
  • Streptococcus group B Enterobacter 2 Staphylococcus simulans 2 agglomerans Providencia rettgeri 2 Staphylococcus 1 ludgunesis Neisseria mucosa 1 Staphylococcus capitis 2 Providencia 1 Staphylococcus 2 alcalifaciens haemolyticus Providencia 1 Staphylococcus hominis 2 rustigianii Burkholderia cepacia 2 Enterococcus faecalis 2 Enterobacter aerogenes 2 Enterococcus faecium 1 Stenotrophomonas 2 Staphylococcus warneri 1 maltophilia Pseudomonas 1 Enterococcus durans 1 fluorescens Comamonas acidovorans 2 Streptococcus bovis 1 Pseudomonas putida 2 Diphteriods 2 Haemophilus 5 Lactobacillus 1 influenzae acid
  • faecalis 1 e (38–39) 200 71/80 + + 2 e (40–41) 121 79/80 + + 1 + 2 — 80/80 + + K. pneumoniae 1 (67–68) 198 76/80 + + 2 (61–62) 143 67/80 + + 3 h (135–136) 148 78/80 + N.T. i 4 (137–138) 116 69/80 + N.T. 1 + 2 + 3 — 80/80 + N.T. P. mirabilis 1 (74–75) 167 73/80 + N.T. 2 (133–134) 123 8080 + N.T. P.
  • aeruginosa 1 e (83–84) 139 79/80 + N.T. 2 e (85–86) 223 80/80 + N.T. S. saprophyticus 1 (98–99) 126 79/80 + + 2 (139–140) 190 80/80 + N.T. M. catarrhalis 1 (112–113) 157 79/80 + N.T. 2 (118–119) 118 80/80 + N.T. 3 (160–119) 137 80/80 + N.T. H. influenzae 1 e (154–155) 217 80/80 + N.T. S. pneumoniae 1 e (156–157) 134 80/80 + N.T.
  • Primer pairs with no “e” are derived from our species-specific fragments. f For S. pyogenes , primer pair #1 is specific for Group A Streptococci (GAS). Primer pair #2 is specific for GAS-producing exotoxin A gene (SpeA) g Ubiquity tested on 195 isolates from 23 species representative of bacterial pathogens commonly encountered in clinical specimens. h Optimizations are in progress to eliminate non-specific amplification observed with some bacterial species other than the target species. i N.T.: not tested.
  • ANNEX I Annex I Specific and ubiquitous oligonucleotide probes for hybridization Originating DNA fragment SEQ ID SEQ ID Nucleotide NO: Nucleotide Sequence NO: position Bacterial species: Escherichia coli 44 5′-CAC CCG CTT GCG TGG CAA GCT GCC C 5 a 213-237 45 5′-CGT TTG TGG ATT CCA GTT CCA TCC G 5 a 489-513 48 5′-TGA AGC ACT GGC CGA AAT GCT GCG T 6 a 759-783 49 5′-GAT GTA CAG GAT TCG TTG AAG GCT T 6 a 898-922 50 5′-TAG CGA AGG CGT AGC AGA AAC TAA C 7 a 1264-1288 51 5′-GCA ACC CGA ACT CAA CGC CGG ATT T 7 a 1227-1251 52 5′-ATA CAC AAG GGT CGC ATC TGC GGC C 7 a
  • ANNEX II Specific and ubiquitous primers for DNA amplification Originating DNA fragment SEQ ID SEQ ID Nucleotide NO: Nucleotide Sequence NO: position Bacterial species: Escherichia coli 42 5′-GCT TTC CAG CGT CAT ATT G 4 177-195 43 b 5′-GAT CTC GAC AAA ATG GTG A 4 260-278 46 5′-TCA CCC GCT TGC GTG GC 5 a 212-228 47 b 5′-GGA ACT GGA ATC CAC AAA C 5 a 490-508 55 5′-GCA ACC CGA ACT CAA CGC C 7 a 1227-1245 56 b 5′-GCA GAT GCG ACC CTT GTG T 7 a 1315-1333 131 5′-CAG GAG TAC GGT GAT TTT TA 3 60-79 132 b 5′-ATT TCT GGT TTG GTC ATA CA 3 174-193 Bacterial species
  • ANNEX III ANNEX III Selection of Universal Probes by Alignment of the Sequences of Bacterial 16S and 23S Ribosomal RNA Genes Reverse strand of TGGACGG AGCCGCCTA GGTGGGAT SEQ ID NO:122 1251 1300 Streptococcus TGAGGTAACC TTTTGGAGCC AGCCGCCTAA GGTGGGATAG ATGANNGGGG salivarius Proteus vulgaris TAGCTTAACC TTCGGGAGGG CGCTTACCAC TTTGTGATTC ATGACTGGGG Pseudomonas aeruginosa TAGTCTAACC GCAAGGGGGA CGGTTACCAC GGAGTGATTC ATGACTGGGG Neiserria gonorrhoeae TAGGGTAACC GCAAGGAGTC CGCTTACCAC GGTATGCTTC ATGACTGGGG Streptococcus lactis TTGCCTAACC GCAAGGAGGG CGCTTCCTAA GGTAAGACCG AT

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Abstract

The present invention relates to DNA-based methods for universal bacterial detection, for specific detection of the common bacterial pathogens Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, 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. With these methods, bacteria can be detected (universal primers and/or probes) and identified (species-specific primers and/or probes) directly from the clinical specimens or from an isolated bacterial colony. Bacteria are further evaluated for their putative susceptibility to antibiotics by resistance gene detection (antibiotic resistance gene specific primers and/or probes). Diagnostic kits for the detection of the presence, for the bacterial identification of the above-mentioned bacterial species and for the detection of antibiotic resistance genes are also claimed. These kits for the rapid (one hour or less) and accurate diagnosis of bacterial infections and antibiotic resistance will gradually replace conventional methods currently used in clinical microbiology laboratories for routine diagnosis. They should provide tools to clinicians to help prescribe promptly optimal treatments when necessary. Consequently, these tests should contribute to saving human lives, rationalizing treatment, reducing the development of antibiotic resistance and avoid unnecessary hospitalizations.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a continuation of U.S. patent application Ser. No. 10/121,120 to Bergeron et al., entitled “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,” filed Apr. 11, 2002, which is a continuation of U.S. patent application Ser. No. 09/452,599, filed Dec. 1, 1999, now abandoned, which is a continuation of U.S. patent application Ser. No. 08/526,840, filed Sep. 11, 1995, now U.S. Pat. No. 6,001,564, which is a continuation-in-part of U.S. patent application Ser. No. 08/304,732, filed Sep. 12, 1994, now abandoned.
    • REFERENCE TO SEQUENCE LISTING
  • The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled GENOM.046CP1CC3.TXT, created Aug. 20, 2007, which is 115 KB in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
    • BACKGROUND OF THE INVENTION Classical Identification of Bacteria
  • Bacteria are classically identified by their ability to utilize different substrates as a source of carbon and nitrogen through the use of biochemical tests such as the API20E™ system. Susceptibility testing of Gram negative bacilli has progressed to microdilution tests. Although the API and the microdilution systems are cost-effective, at least two days are required to obtain preliminary results due to the necessity of two successive overnight incubations to isolate and identify the bacteria from the specimen. Some faster detection methods with sophisticated and expensive apparatus have been developed. For example, the fastest identification system, the autoSCAN-Walk-Away System™ identifies both Gram negative and Gram positive from isolated bacterial colonies in 2 hours and susceptibility patterns to antibiotics in only 7 hours. However, this system has an unacceptable margin of error, especially with bacterial species other than Enterobacteriaceae (York et al., 1992. J. Clin. Microbiol. 30:2903-2910). Nevertheless, even this fastest method requires primary isolation of the bacteria as a pure culture, a process which takes at least 18 hours if there is a pure culture or 2 to 3 days if there is a mixed culture.
    • Urine Specimens
  • A large proportion (40-50%) of specimens received in routine diagnostic microbiology laboratories for bacterial identification are urine specimens (Pezzlo, 1988, Clin. Microbiol. Rev. 1:268-280). Urinary tract infections (UTI) are extremely common and affect up to 20% of women and account for extensive morbidity and increased mortality among hospitalized patients (Johnson and Stamm, 1989; Ann. Intern. Med. 111:906-917). UTI are usually of bacterial etiology and require antimicrobial therapy. The Gram negative bacillus Escherichia coli is by far the most prevalent urinary pathogen and accounts for 50 to 60% of UTI (Pezzlo, 1988, op. cit.). The prevalence for bacterial pathogens isolated from urine specimens observed recently at the “Centre Hospitalier de l'Universit Laval (CHUL)” is given in Tables 1 and 2.
  • Conventional pathogen identification in urine specimens. The search for pathogens in urine specimens is so preponderant in the routine microbiology laboratory that a myriad of tests have been developed. The gold standard is still the classical semi-quantitative plate culture method in which a calibrated loop of urine is streaked on plates and incubated for 18-24 hours. Colonies are then counted to determine the total number of colony forming units (CFU) per liter of urine. A bacterial UTI is normally associated with a bacterial count of .gtoreq.10.sup.7 CFU/L in urine. However, infections with less than 10.sup.7 CFU/L in urine are possible, particularly in patients with a high incidence of diseases or those catheterized (Stark and Maki, 1984, N. Engl. J. Med. 311:560-564). Importantly, close to 80% of urine specimens tested are considered negative (<10.sup.7 CFU/L; Table 3).
  • Accurate and rapid urine screening methods for bacterial pathogens would allow a faster identification of negative results and a more efficient clinical investigation of the patient. Several rapid identification methods (Uriscreen™, UTIscreen™, Flash Track™ DNA probes and others) were recently compared to slower standard biochemical methods which are based on culture of the bacterial pathogens. Although much faster, these rapid tests showed low sensitivities and specificities as well as a high number of false negative and false positive results (Koening et al., 1992. J. Clin. Microbiol. 30:342-345; Pezzlo et al., 1992. J. Clin. Microbiol. 30:640-684).
  • Urine specimens found positive by culture are further characterized using standard biochemical tests to identify the bacterial pathogen and are also tested for susceptibility to antibiotics.
    • Any Clinical Specimens
  • As with urine specimen which was used here as an example, our probes and amplification primers are also applicable to any other clinical specimens. The DNA-based tests proposed in this invention are superior to standard methods currently used for routine diagnosis in terms of rapidity and accuracy. While a high percentage of urine specimens are negative, in many other clinical specimens more than 95% of cultures are negative (Table 4). These data further support the use of universal probes to screen out the negative clinical specimens. Clinical specimens from organisms other than humans (e.g. other primates, mammals, farm animals or live stocks) may also be used.
    • Towards the Development of Rapid DNA-Based Diagnostic
  • A rapid diagnostic test should have a significant impact on the management of infections. For the identification of pathogens and antibiotic resistance genes in clinical samples, DNA probe and DNA amplification technologies offer several advantages over conventional methods. There is no need for subculturing, hence the organism can be detected directly in clinical samples thereby reducing the costs and time associated with isolation of pathogens. DNA-based technologies have proven to be extremely useful for specific applications in the clinical microbiology laboratory. For example, kits for the detection of fastidious organisms based on the use of hybridization probes or DNA amplification for the direct detection of pathogens in clinical specimens are commercially available (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).
  • The present invention is an advantageous alternative to the conventional culture identification methods used in hospital clinical microbiology laboratories and in private clinics for routine diagnosis. Besides being much faster, DNA-based diagnostic tests are more accurate than standard biochemical tests presently used for diagnosis because the bacterial genotype (e.g. DNA level) is more stable than the bacterial phenotype (e.g. biochemical properties). The originality of this invention is that genomic DNA fragments (size of at least 100 base pairs) specific for 12 species of commonly encountered bacterial pathogens were selected from genomic libraries or from data banks. Amplification primers or oligonucleotide probes (both less than 100 nucleotides in length) which are both derived from the sequence of species-specific DNA fragments identified by hybridization from genomic libraries or from selected data bank sequences are used as a basis to develop diagnostic tests. Oligonucleotide primers and probes for the detection of commonly encountered and clinically important bacterial resistance genes are also included. For example, Annexes I and II present a list of suitable oligonucleotide probes and PCR primers which were all derived from the species-specific DNA fragments selected from genomic libraries or from data bank sequences. It is clear to the individual skilled in the art that oligonucleotide sequences appropriate for the specific detection of the above bacterial species other than those listed in Annexes 1 and 2 may be derived from the species-specific fragments or from the selected data bank sequences. For example, the oligonucleotides may be shorter or longer than the ones we have chosen and may be selected anywhere else in the identified species-specific sequences or selected data bank sequences. Alternatively, the oligonucleotides may be designed for use in amplification methods other than PCR. Consequently, the core of this invention is the identification of species-specific genomic DNA fragments from bacterial genomic DNA libraries and the selection of genomic DNA fragments from data bank sequences which are used as a source of species-specific and ubiquitous oligonucleotides. Although the selection of oligonucleotides suitable for diagnostic purposes from the sequence of the species-specific fragments or from the selected data bank sequences requires much effort it is quite possible for the individual skilled in the art to derive from our fragments or selected data bank sequences suitable oligonucleotides which are different from the ones we have selected and tested as examples (Annexes I and II).
  • Others have developed DNA-based tests for the detection and identification of some of the bacterial pathogens for which we have identified species-specific sequences (PCT patent application Serial No. WO 93/03186). However, their strategy was based on the amplification of the highly conserved 16S rRNA gene followed by hybridization with internal species-specific oligonucleotides. The strategy from this invention is much simpler and more rapid because it allows the direct amplification of species-specific targets using oligonucleotides derived from the species-specific bacterial genomic DNA fragments.
  • Since a high percentage of clinical specimens are negative, oligonucleotide primers and probes were selected from the highly conserved 16S or 23S rRNA genes to detect all bacterial pathogens possibly encountered in clinical specimens in order to determine whether a clinical specimen is infected or not. This strategy allows rapid screening out of the numerous negative clinical specimens submitted for bacteriological testing.
  • We are also developing other DNA-based tests, to be performed simultaneously with bacterial identification, to determine rapidly the putative bacterial susceptibility to antibiotics by targeting commonly encountered and clinically relevant bacterial resistance genes. Although the sequences from the selected antibiotic resistance genes are available and have been used to develop DNA-based tests for their detection (Ehrlich and Greenberg, 1994. PCR-based Diagnostics in Infectious Diseases, Blackwell Scientific Publications, Boston, Mass.; Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.), our approach is innovative as it represents major improvements over current “gold standard” diagnostic methods based on culture of the bacteria because it allows the rapid identification of the presence of a specific bacterial pathogen and evaluation of its susceptibility to antibiotics directly from the clinical specimens within one hour.
  • We believe that the rapid and simple diagnostic tests not based on cultivation of the bacteria that we are developing will gradually replace the slow conventional bacterial identification methods presently used in hospital clinical microbiology laboratories and in private clinics. In our opinion, these rapid DNA-based diagnostic tests for severe and common bacterial pathogens and antibiotic resistance will (i) save lives by optimizing treatment, (ii) diminish antibiotic resistance by reducing the use of broad spectrum antibiotics and (iii) decrease overall health costs by preventing or shortening hospitalizations.
    • SUMMARY OF THE INVENTION
  • In accordance with the present invention, there is provided sequence from genomic DNA fragments (size of at least 100 base pairs and all described in the sequence listing) selected either by hybridization from genomic libraries or from data banks and which are specific for the detection of commonly encountered bacterial pathogens (i.e. Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Staphylococcus saprophyticus, Streptococcus pyogenes, Haemophilus influenzae and Moraxella catarrhalis) in clinical specimens. These bacterial species are associated with approximately 90% of urinary tract infections and with a high percentage of other severe infections including septicemia, meningitis, pneumonia, intraabdominal infections, skin infections and many other severe respiratory tract infections. Overall, the above bacterial species may account for up to 80% of bacterial pathogens isolated in routine microbiology laboratories.
  • Synthetic oligonucleotides for hybridization (probes) or DNA amplification (primers) were derived from the above species-specific DNA fragments (ranging in sizes from 0.25 to 5.0 kilobase pairs (kbp)) or from selected data bank sequences (GenBank and EMBL). Bacterial species for which some of the oligonucleotide probes and amplification primers were derived from selected data bank sequences are Escherichia coli, Enterococcus faecalis, Streptococcus pyogenes and Pseudomonas aeruginosa. The person skilled in the art understands that the important innovation in this invention is the identification of the species-specific DNA fragments selected either from bacterial genomic libraries by hybridization or from data bank sequences. The selection of oligonucleotides from these fragments suitable for diagnostic purposes is also innovative. Specific and ubiquitous oligonucleotides different from the ones tested in the practice are considered as embodiments of the present invention.
  • The development of hybridization (with either fragment or oligonucleotide probes) or of DNA amplification protocols for the detection of pathogens from clinical specimens renders possible a very rapid bacterial identification. This will greatly reduce the time currently required for the identification of pathogens in the clinical laboratory since these technologies can be applied for bacterial detection and identification directly from clinical specimens with minimum pretreatment of any biological specimens to release bacterial DNA. In addition to being 100% specific, probes and amplification primers allow identification of the bacterial species directly from clinical specimens or, alternatively, from an isolated colony. DNA amplification assays have the added advantages of being faster and more sensitive than hybridization assays, since they allow rapid and exponential in vitro replication of the target segment of DNA from the bacterial genome. Universal probes and amplification primers selected from the 16S or 23S rRNA genes highly conserved among bacteria, which permit the detection of any bacterial pathogens, will serve as a procedure to screen out the numerous negative clinical specimens received in diagnostic laboratories. The use of oligonucleotide probes or primers complementary to characterized bacterial genes encoding resistance to antibiotics to identify commonly encountered and clinically important resistance genes is also under the scope of this invention.
    • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Development of Species-Specific DNA Probes
  • DNA fragment probes were developed for the following bacterial species: Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus mirabilis, Streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Haemophilus influenzae and Moraxella catarrhalis. (For Enterococcus faecalis and Streptococcus pyogenes, oligonucleotide sequences were exclusively derived from selected data bank sequences). These species-specific fragments were selected from bacterial genomic libraries by hybridization to DNA from a variety of Gram positive and Gram negative bacterial species (Table 5).
  • The chromosomal DNA from each bacterial species for which probes were seeked was isolated using standard methods. DNA was digested with a frequently cutting restriction enzyme such as Sau3AI and then ligated into the bacterial plasmid vector pGEM3Zf (Promega) linearized by appropriate restriction endonuclease digestion. Recombinant plasmids were then used to transform competent E. coli strain DH5α thereby yielding a genomic library. The plasmid content of the transformed bacterial cells was analyzed using standard methods. DNA fragments of target bacteria ranging in size from 0.25 to 5.0 kilobase pairs (kbp) were cut out from the vector by digestion of the recombinant plasmid with various restriction endonucleases. The insert was separated from the vector by agarose gel electrophoresis and purified in low melting point agarose gels. Each of the purified fragments of bacterial genomic DNA was then used as a probe for specificity tests.
  • For each given species, the gel-purified restriction fragments of unknown coding potential were labeled with the radioactive nucleotide α32P(dATP) which was incorporated into the DNA fragment by the random priming labeling reaction. Non-radioactive modified nucleotides could also be incorporated into the DNA by this method to serve as a label.
  • Each DNA fragment probe (i.e. a segment of bacterial genomic DNA of at least 100 bp in length cut out from clones randomly selected from the genomic library) was then tested for its specificity by hybridization to DNAs from a variety of bacterial species (Table 5). The double-stranded labeled DNA probe was heat-denatured to yield labeled single-stranded DNA which could then hybridize to any single-stranded target DNA fixed onto a solid support or in solution. The target DNAs consisted of total cellular DNA from an array of bacterial species found in clinical samples (Table 5). Each target DNA was released from the bacterial cells and denatured by conventional methods and then irreversibly fixed onto a solid support (e.g. nylon or nitrocellulose membranes) or free in solution. The fixed single-stranded target DNAs were then hybridized with the single-stranded probe. Pre-hybridization, hybridization and post-hybridization conditions were as follows: (i) Pre-hybridization; in 1 M NaCl+10% dextran sulfate+1% SDS (sodium dodecyl sulfate)+1 .mu.g/ml salmon sperm DNA at 650.degree. C. for 15 min. (ii) Hybridization; in fresh pre-hybridization solution containing the labeled probe at 650.degree. C. overnight. (iii) Post-hybridization; washes twice in 3.times.SSC containing 1% SDS (1.times.SSC is 0.15M NaCl, 0.015M NaCitrate) and twice in 0.1.times.SSC containing 0.1% SDS; all washes were at 650.degree. C. for 15 min. Autoradiography of washed filters allowed the detection of selectively hybridized probes. Hybridization of the probe to a specific target DNA indicated a high degree of similarity between the nucleotide sequence of these two DNAs. Species-specific DNA fragments selected from various bacterial genomic libraries ranging in size from 0.25 to 5.0 kbp were isolated for 10 common bacterial pathogens (Table 6) based on hybridization to chromosomal DNAs from a variety of bacteria performed as described above. All of the bacterial species tested (66 species listed in Table 5) were likely to be pathogens associated with common infections or potential contaminants which can be isolated from clinical specimens. A DNA fragment probe was considered specific only when it hybridized solely to the pathogen from which it was isolated. DNA fragment probes found to be specific were subsequently tested for their ubiquity (i.e. ubiquitous probes recognized most isolates of the target species) by hybridization to bacterial DNAs from approximately 10 to 80 clinical isolates of the species of interest (Table 6). The DNAs were denatured, fixed onto nylon membranes and hybridized as described above.
    • Sequencing of the Species-Specific Fragment Probes
  • The nucleotide sequence of the totality or of a portion of the species-specific DNA fragments isolated (Table 6) was determined using the dideoxynucleotide termination sequencing method which was performed using Sequenase™ (USB Biochemicals) or T7 DNA polymerase (Pharmacia). These nucleotide sequences are shown in the sequence listing. Alternatively, sequences selected from data banks (GenBank and EMBL) were used as sources of oligonucleotides for diagnostic purposes for Escherichia coli, Enterococcus faecalis, Streptococcus pyogenes and Pseudomonas aeruginosa. For this strategy, an array of suitable oligonucleotide primers or probes derived from a variety of genomic DNA fragments (size of more than 100 bp) selected from data banks was tested for their specificity and ubiquity in PCR and hybridization assays as described later. It is important to note that the data bank sequences were selected based on their potential of being species-specific according to available sequence information. Only data bank sequences from which species-specific oligonucleotides could be derived are included in this invention.
  • Oligonucleotide probes and amplification primers derived from species-specific fragments selected from the genomic libraries or from data bank sequences were synthesized using an automated DNA synthesizer (Millipore). Prior to synthesis, all oligonucleotides (probes for hybridization and primers for DNA amplification) were evaluated for their suitability for hybridization or DNA amplification by polymerase chain reaction (PCR) by computer analysis using standard programs (e.g. Genetics Computer Group (GCG) and Oligo™ 4.0 (National Biosciences)). The potential suitability of the PCR primer pairs was also evaluated prior to the synthesis by verifying the absence of unwanted features such as long stretches of one nucleotide, a high proportion of G or C residues at the 3′ end and a 3′-terminal T residue (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.).
  • Hybridization with Oligonucleotide Probes
  • In hybridization experiments, oligonucleotides (size less than 100 nucleotides) have some advantages over DNA fragment probes for the detection of bacteria such as ease of preparation in large quantities, consistency in results from batch to batch and chemical stability. Briefly, for the hybridizations, oligonucleotides were 5′ end-labeled with the radionucleotide γ32P(ATP) using T4 polynucleotide kinase (Pharmacia). The unincorporated radionucleotide was removed by passing the labeled single-stranded oligonucleotide through a Sephadex G50 column. Alternatively, oligonucleotides were labeled with biotin, either enzymatically at their 3′ ends or incorporated directly during synthesis at their 5′ ends, or with digoxigenin. It will be appreciated by the person skilled in the art that labeling means other than the three above labels may be used.
  • The target DNA was denatured, fixed onto a solid support and hybridized as previously described for the DNA fragment probes. Conditions for pre-hybridization and hybridization were as described earlier. Post-hybridization washing conditions were as follows: twice in 3×SSC containing 1% SDS, twice in 2×SSC containing 1% SDS and twice in 1×SSC containing 1% SDS (all of these washes were at 65° C. for 15 min), and a final wash in 0.1×SSC containing 1% SDS at 25° C. for 15 min. For probes labeled with radioactive labels the detection of hybrids was by autoradiography as described earlier. For non-radioactive labels detection may be calorimetric or by chemiluminescence.
  • The oligonucleotide probes may be derived from either strand of the duplex DNA. The probes may consist of the bases A, G, C, or T or analogs. The probes may be of any suitable length and may be selected anywhere within the species-specific genomic DNA fragments selected from the genomic libraries or from data bank sequences.
    • DNA Amplification
  • For DNA amplification by the widely used PCR (polymerase chain reaction) method, primer pairs were derived either from the sequenced species-specific DNA fragments or from data bank sequences or, alternatively, were shortened versions of oligonucleotide probes. Prior to synthesis, the potential primer pairs were analyzed by using the program Oligo™4.0 (National Biosciences) to verify that they are likely candidates for PCR amplifications.
  • During DNA amplification by PCR, two oligonucleotide primers binding respectively to each strand of the denatured double-stranded target DNA from the bacterial genome are used to amplify exponentially in vitro the target DNA by successive thermal cycles allowing denaturation of the DNA, annealing of the primers and synthesis of new targets at each cycle (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). Briefly, the PCR protocols were as follows. Clinical specimens or bacterial colonies were added directly to the 50 μL PCR reaction mixtures containing 50 mM KCl, 10 mM Tris-HCl pH 8.3, 2.5 mM MgCl2, 0.4 μM of each of the two primers, 200 μM of each of the four dNTPs and 1.25 Units of Taq DNA polymerase (Perkin Elmer). PCR reactions were then subjected to thermal cycling (3 min at 95° C. followed by 30 cycles of 1 second at 95° C. and 1 second at 55° C.) using a Perkin Elmer 480™ thermal cycler and subsequently analyzed by standard ethidium bromide-stained agarose gel electrophoresis. It is clear that other methods for the detection of specific amplification products, which may be faster and more practical for routine diagnosis, may be used. Such methods may be based on the detection of fluorescence after amplification (e.g. TaqMan™ system from Perkin Elmer or Amplisensor™ from Biotronics) or liquid hybridization with an oligonucleotide probe binding to internal sequences of the specific amplification product. These novel probes can be generated from our species-specific fragment probes. Methods based on the detection of fluorescence are particularly promising for utilization in routine diagnosis as they are, very rapid and quantitative and can be automated.
  • To assure PCR efficiency, glycerol or dimethyl sulfoxide (DMSO) or other related solvents, can be used to increase the sensitivity of the PCR and to overcome problems associated with the amplification of target with a high GC content or with strong secondary structures. The concentration ranges for glycerol and DMSO are 5-15% (v/v) and 3-10% (v.backslash.v), respectively. For the PCR reaction mixture, the concentration ranges for the amplification primers and the MgCl2 are 0.1-1.0 μM and 1.5-3.5 mM, respectively. Modifications of the standard PCR protocol using external and nested primers (i.e. nested PCR) or using more than one primer pair (i.e. multiplex PCR) may also be used (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). For more details about the PCR protocols and amplicon detection methods see examples 7 and 8.
  • The person skilled in the art of DNA amplification knows the existence of other rapid amplification procedures such as ligase chain reaction (LCR), transcription-based amplification systems (TAS), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) and branched DNA (bDNA) (Persing et al, 1993. Diagnostic Molecular Microbiology: Principles and Applications, American Society for Microbiology, Washington, D.C.). The scope of this invention is not limited to the use of amplification by PCR, but rather includes the use of any rapid nucleic acid amplification methods or any other procedures which may be used to increase rapidity and sensitivity of the tests. Any oligonucleotides suitable for the amplification of nucleic acid by approaches other than PCR and derived from the species-specific fragments and from selected antibiotic resistance gene sequences included in this document are also under the scope of this invention.
    • Specificity and Ubiquity Tests for Oligonucleotide Probes and Primers
  • The specificity of oligonucleotide probes, derived either from the sequenced species-specific fragments or from data bank sequences, was tested by hybridization to DNAs from the array of bacterial species listed in Table 5 as previously described. Oligonucleotides found to be specific were subsequently tested for their ubiquity by hybridization to bacterial DNAs from approximately 80 isolates of the target species as described for fragment probes. Probes were considered ubiquitous when they hybridized specifically with the DNA from at least 80% of the isolates. Results for specificity and ubiquity tests with the oligonucleotide probes are summarized in Table 6. The specificity and ubiquity of the amplification primer pairs were tested directly from cultures (see example 7) of the same bacterial strains. For specificity and ubiquity tests, PCR assays were performed directly from bacterial colonies of approximately 80 isolates of the target species. Results are summarized in Table 7. All specific and ubiquitous oligonucleotide probes and amplification primers for each of the 12 bacterial species investigated are listed in Annexes I and II, respectively. Divergence in the sequenced DNA fragments can occur and, insofar as the divergence of these sequences or a part thereof does not affect the specificity of the probes or amplification primers, variant bacterial DNA is under the scope of this invention.
    • Universal Bacterial Detection
  • In the routine microbiology laboratory a high percentage of clinical specimens sent for bacterial identification is negative (Table 4). For example, over a 2 year period, around 80% of urine specimens received by the laboratory at the “Centre Hospitalier de l'Université Laval (CHUL)” were negative (i.e. <107 CFU/L) (Table 3). Testing clinical samples with universal probes or universal amplification primers to detect the presence of bacteria prior to specific identification and screen out the numerous negative specimens is thus useful as it saves costs and may rapidly orient the clinical management of the patients. Several oligonucleotides and amplification primers were therefore synthesized from highly conserved portions of bacterial 16S or 23S ribosomal RNA gene sequences available in data banks (Annexes III and IV). In hybridization tests, a pool of seven oligonucleotides (Annex I; Table 6) hybridized strongly to DNA from all bacterial species listed in Table 5. This pool of universal probes labeled with radionucleotides or with any other modified nucleotides is consequently very useful for detection of bacteria in urine samples with a sensitivity range of ≧107 CFU/L. These probes can also be applied for bacterial detection in other clinical samples.
  • Amplification primers also derived from the sequence of highly conserved ribosomal RNA genes were used as an alternative strategy for universal bacterial detection directly from clinical specimens (Annex IV; Table 7). The DNA amplification strategy was developed to increase the sensitivity and the rapidity of the test. This amplification test was ubiquitous since it specifically amplified DNA from 23 different bacterial species encountered in clinical specimens.
  • Well-conserved bacterial genes other than ribosomal RNA genes could also be good candidates for universal bacterial detection directly from clinical specimens. Such genes may be associated with processes essential for bacterial survival (e.g. protein synthesis, DNA synthesis, cell division or DNA repair) and could therefore be highly conserved during evolution. We are working on these candidate genes to develop new rapid tests for the universal detection of bacteria directly from clinical specimens.
    • Antibiotic Resistance Genes
  • Antimicrobial resistance complicates treatment and often leads to therapeutic failures. Furthermore, overuse of antibiotics inevitably leads to the emergence of bacterial resistance. Our goal is to provide the clinicians, within one hour, the needed information to prescribe optimal treatments. Besides the rapid identification of negative clinical specimens with DNA-based tests for universal bacterial detection and the identification of the presence of a specific pathogen in the positive specimens with DNA-based tests for specific bacterial detection, the clinicians also need timely information about the ability of the bacterial pathogen to resist antibiotic treatments. We feel that the most efficient strategy to evaluate rapidly bacterial resistance to antimicrobials is to detect directly from the clinical specimens the most common and important antibiotic resistance genes (i.e. DNA-based tests for the detection of antibiotic resistance genes). Since the sequence from the most important and common bacterial antibiotic resistance genes are available from data banks, our strategy is to use the sequence from a portion or from the entire gene to design specific oligonucleotides which will be used as a basis for the development of rapid DNA-based tests. The sequence from the bacterial antibiotic resistance genes selected on the basis of their clinical relevance (i.e. high incidence and importance) is given in the sequence listing. Table 8 summarizes some characteristics of the selected antibiotic resistance genes.
    • EXAMPLES
  • The following examples are intended to be illustrative of the various methods and compounds of the invention.
    • Example 1
  • Isolation and cloning of fragments. Genomic DNAs from Escherichia coli strain ATCC 25922, Klebsiella pneumoniae strain CK2, Pseudomonas aeruginosa strain ATCC 27853, Proteus mirabilis strain ATCC 35657, Streptococcus pneumoniae strain ATCC 27336, Staphylococcus aureus strain ATCC 25923, Staphylococcus epidermidis strain ATCC 12228, Staphylococcus saprophyticus strain ATCC 15305, Haemophilus influenzae reference strain Rd and Moraxella catarrhalis strain ATCC 53879 were prepared using standard procedures. It is understood that the bacterial genomic DNA may have been isolated from strains other than the ones mentioned above. (For Enterococcus faecalis and Streptococcus pyogenes oligonucleotide sequences were derived exclusively from data banks). Each DNA was digested with a restriction enzyme which frequently cuts DNA such as Sau3AI. The resulting DNA fragments were ligated into a plasmid vector (pGEM3Zf) to create recombinant plasmids and transformed into competent E. coli cells (DH5α). It is understood that the vectors and corresponding competent cells should not be limited to the ones herein above specifically exemplified. The objective of obtaining recombinant plasmids and transformed cells is to provide an easily reproducible source of DNA fragments useful as probes. Therefore, insofar as the inserted fragments are specific and selective for the target bacterial DNA, any recombinant plasmids and corresponding transformed host cells are under the scope of this invention. The plasmid content of the transformed bacterial cells was analyzed using standard methods. DNA fragments from target bacteria ranging in size from 0.25 to 5.0 kbp were cut out from the vector by digestion of the recombinant plasmid with various restriction endonucleases. The insert was separated from the vector by agarose gel electrophoresis and purified in a low melting point agarose gel. Each of the purified fragments was then used for specificity tests.
  • Labeling of DNA fragment probes. The label used was α32P(dATP), a radioactive nucleotide which can be incorporated enzymatically into a double-stranded DNA molecule. The fragment of interest is first denatured by heating at 95° C. for 5 min, then a mixture of random primers is allowed to anneal to the strands of the fragments. These primers, once annealed, provide a starting point for synthesis of DNA. DNA polymerase, usually the Klenow fragment, is provided along with the four nucleotides, one of which is radioactive. When the reaction is terminated, the mixture of new DNA molecules is once again denatured to provide radioactive single-stranded DNA molecules (i.e. the probe). As mentioned earlier, other modified nucleotides may be used to label the probes.
  • Specificity and ubiquity tests for the DNA fragment probes. Species-specific DNA fragments ranging in size from 0.25 to 5.0 kbp were isolated for 10 common bacterial pathogens (Table 6) based on hybridization to chromosomal DNAs from a variety of bacteria. Samples of whole cell DNA for each bacterial strain listed in Table 5 were transferred onto a nylon membrane using a dot blot apparatus, washed and denatured before being irreversibly fixed. Hybridization conditions were as described earlier. A DNA fragment probe was considered specific only when it hybridized solely to the pathogen from which it was isolated. Labeled DNA fragments hybridizing specifically only to target bacterial species (i.e. specific) were then tested for their ubiquity by hybridization to DNAs from approximately 10 to 80 isolates of the species of interest as described earlier. The conditions for pre-hybridization, hybridization and post-hybridization washes were as described earlier. After autoradiography (or other detection means appropriate for the non-radioactive label used), the specificity of each individual probe can be determined. Each probe found to be specific (i.e. hybridizing only to the DNA from the bacterial species from which it was isolated) and ubiquitous (i.e. hybridizing to most isolates of the target species) was kept for further experimentations.
    • Example 2
  • Same as example 1 except that testing of the strains is by colony hybridization. The bacterial strains were inoculated onto a nylon membrane placed on nutrient agar. The membranes were incubated at 37° C. for two hours and then bacterial lysis and DNA denaturation were carried out according to standard procedures. DNA hybridization was performed as described earlier.
    • Example 3
  • Same as example 1 except that bacteria were detected directly from clinical samples. Any biological samples were loaded directly onto a dot blot apparatus and cells were lysed in situ for bacterial detection. Blood samples should be heparizined in order to avoid coagulation interfering with their convenient loading on a dot blot apparatus.
    • Example 4
  • Nucleotide sequencing of DNA fragments. The nucleotide sequence of the totality or a portion of each fragment found to be specific and ubiquitous (Example 1) was determined using the dideoxynucleotide termination sequencing method (Sanger et al., 1977, Proc. Natl. Acad. Sci. USA. 74:5463-5467). These DNA sequences are shown in the sequence listing. Oligonucleotide probes and amplification primers were selected from these nucleotide sequences, or alternatively, from selected data banks sequences and were then synthesized on an automated Biosearch synthesizer (Millipore™) using phosphoramidite chemistry.
  • Labeling of oligonucleotides. Each oligonucleotide was 5′ end-labeled with γ32P-ATP by the T4 polynucleotide kinase (Pharmacia) as described earlier. The label could also be non-radioactive.
  • Specificity test for oligonucleotide probes. All labeled oligonucleotide probes were tested for their specificity by hybridization to DNAs from a variety of Gram positive and Gram negative bacterial species as described earlier. Species-specific probes were those hybridizing only to DNA from the bacterial species from which it was isolated. Oligonucleotide probes found to be specific were submitted to ubiquity tests as follows.
  • Ubiquity test for oligonucleotide probes. Specific oligonucleotide probes were then used in ubiquity tests with approximately 80 strains of the target species. Chromosomal DNAs from the isolates were transferred onto nylon membranes and hybridized with labeled oligonucleotide probes as described for specificity tests. The batteries of approximately 80 isolates constructed for each target species contain reference ATCC strains as well as a variety of clinical isolates obtained from various sources. Ubiquitous probes were those hybridizing to at least 80% of DNAs from the battery of clinical isolates of the target species. Examples of specific and ubiquitous oligonucleotide probes are listed in Annex I.
    • Example 5
  • Same as example 4 except that a pool of specific oligonucleotide probes is used for bacterial identification (i) to increase sensitivity and assure 100% ubiquity or (ii) to identify simultaneously more than one bacterial species. Bacterial identification could be done from isolated colonies or directly from clinical specimens
    • Example 6
  • PCR amplification. The technique of PCR was used to increase sensitivity and rapidity of the tests. The PCR primers used were often shorter derivatives of the extensive sets of oligonucleotides previously developed for hybridization assays (Table 6). The sets of primers were tested in PCR assays performed directly from a bacterial colony or from a bacterial suspension (see Example 7) to determine their specificity and ubiquity (Table 7). Examples of specific and ubiquitous PCR primer pairs are listed in annex II.
  • Specificity and ubiquity tests for amplification primers. The specificity of all selected PCR primer pairs was tested against the battery of Gram negative and Gram positive bacteria used to test the oligonucleotide probes (Table 5). Primer pairs found specific for each species were then tested for their ubiquity to ensure that each set of primers could amplify at least 80% of DNAs from a battery of approximately 80 isolates of the target species. The batteries of isolates constructed for each species contain reference ATCC strains and various clinical isolates representative of the clinical diversity for each species.
  • Standard precautions to avoid false positive PCR results should be taken. Methods to inactivate PCR amplification products such as the inactivation by uracil-N-glycosylase may be used to control PCR carryover.
    • Example 7
  • Amplification directly from a bacterial colony or suspension. PCR assays were performed either directly from a bacterial colony or from a bacterial suspension, the latter being adjusted to a standard McFarland 0.5 (corresponds to 1.5 times.10.sup.8 bacteria/mL). In the case of direct amplification from a colony, a portion of the colony was transferred directly to a 50 μL PCR reaction mixture (containing 50 mM KCl, 10 mM Tris pH 8.3, 2.5 mM MgCl2, 0.4 μM of each of the two primers, 200 μM of each of the four dNTPs and 1.25 Unit of Taq DNA polymerase (Perkin Elmer)) using a plastic rod. For the bacterial suspension, 4 μL of the cell suspension was added to 46 μL of the same PCR reaction mixture. For both strategies, the reaction mixture was overlaid with 50 μL of mineral oil and PCR amplifications were carried out using an initial denaturation step of 3 min. at 95° C. followed by 30 cycles consisting of a 1 second denaturation step at 95° C. and of a 1 second annealing step at 55° C. in a Perkin Elmer 480™ thermal cycler. PCR amplification products were then analyzed by standard agarose gel (2%) electrophoresis. Amplification products were visualized in agarose gels containing 2.5 μg/mL of ethidium bromide under UV at 254 mm. The entire PCR assay can be completed in approximately one hour.
  • Alternatively, amplification from bacterial cultures was performed as described above but using a “hot start” protocol. In that case, an initial reaction mixture containing the target DNA, primers and dNTPs was heated at 85° C. prior to the addition of the other components of the PCR reaction mixture. The final concentration of all reagents was as described above. Subsequently, the PCR reactions were submitted to thermal cycling and analysis as described above.
    • Example 8
  • Amplification directly from clinical specimens. For amplification from urine specimens, 4 .mu.L of undiluted or diluted (1:10) urine was added directly to 46 μL of the above PCR reaction mixture and amplified as described earlier.
  • To improve bacterial cell lysis and eliminate the PCR inhibitory effects of clinical specimens, samples were routinely diluted in lysis buffer containing detergent(s). Subsequently, the lysate was added directly to the PCR reaction mixture. Heat treatments of the lysates, prior to DNA amplification, using the thermocycler or a microwave oven could also be performed to increase the efficiency of cell lysis.
  • Our strategy is to develop rapid and simple protocols to eliminate PCR inhibitory effects of clinical specimens and lyse bacterial cells to perform DNA amplification directly from a variety of biological samples. PCR has the advantage of being compatible with crude DNA preparations. For example, blood, cerebrospinal fluid and sera may be used directly in PCR assays after a brief heat treatment. We intend to use such rapid and simple strategies to develop fast protocols for DNA amplification from a variety of clinical specimens.
    • Example 9
  • Detection of antibiotic resistance genes. The presence of specific antibiotic resistance genes which are frequently encountered and clinically relevant is identified using the PCR amplification or hybridization protocols described in previous sections. Specific oligonucleotides used as a basis for the DNA-based tests are selected from the antibiotic resistance gene sequences. These tests can be performed either directly from clinical specimens or from a bacterial colony and should complement diagnostic tests for specific bacterial identification.
    • Example 10
  • Same as examples 7 and 8 except that assays were performed by multiplex PCR (i.e. using several pairs of primers in a single PCR reaction) to (i) reach an ubiquity of 100% for the specific target pathogen or (ii) to detect simultaneously several species of bacterial pathogens.
  • For example, the detection of Escherichia coli requires three pairs of PCR primers to assure a ubiquity of 100%. Therefore, a multiplex PCR assay (using the “hot-start” protocol (Example 7)) with those three primer pairs was developed. This strategy was also used for the other bacterial pathogens for which more than one primer pair was required to reach a ubiquity of 100%.
  • Multiplex PCR assays could also be used to (i) detect simultaneously several bacterial species or, alternatively, (ii) to simultaneously identify the bacterial pathogen and detect specific antibiotic resistance genes either directly from a clinical specimen or from a bacterial colony.
  • For these applications, amplicon detection methods should be adapted to differentiate the various amplicons produced. Standard agarose gel electrophoresis could be used because it discriminates the amplicons based on their sizes. Another useful strategy for this purpose would be detection using a variety of fluorochromes emitting at different wavelengths which are each coupled with a specific oligonucleotide linked to a fluorescence quencher which is degraded during amplification to release the fluorochrome (e.g. TaqMan™, Perkin Elmer).
    • Example 11
  • Detection of amplification Products. The person skilled in the art will appreciate that alternatives other than standard agarose gel electrophoresis (Example 7) may be used for the revelation of amplification products. Such methods may be based on the detection of fluorescence after amplification (e.g. Amplisensor™, Biotronics; TaqMan™) or other labels such as biotin (SHARP Signal™ system, Digene Diagnostics). These methods are quantitative and easily automated. One of the amplification primers or an internal oligonucleotide probe specific to the amplicon(s) derived from the species-specific fragment probes is coupled with the fluorochrome or with any other label. Methods based on the detection of fluorescence are particularly suitable for diagnostic tests since they are rapid and flexible as fluorochromes emitting different wavelengths are available (Perkin Elmer).
    • Example 12
  • Species-specific, universal and antibiotic resistance gene amplification primers can be used in other rapid amplification procedures such as the ligase chain reaction (LCR), transcription-based amplification systems (TAS), self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) and branched DNA (bDNA) or any other methods to increase the sensitivity of the test. Amplifications can be performed from an isolated bacterial colony or directly from clinical specimens. The scope of this invention is therefore not limited to the use of PCR but rather includes the use of any procedures to specifically identify bacterial DNA and which may be used to increase rapidity and sensitivity of the tests.
    • Example 13
  • A test kit would contain sets of probes specific for each bacterium as well as a set of universal probes. The kit is provided in the form of test components, consisting of the set of universal probes labeled with non-radioactive labels as well as labeled specific probes for the detection of each bacterium of interest in specific clinical samples. The kit will also include test reagents necessary to perform the pre-hybridization, hybridization, washing steps and hybrid detection. Finally, test components for the detection of known antibiotic resistance genes (or derivatives therefrom) will be included. Of course, the kit will include standard samples to be used as negative and positive controls for each hybridization test.
  • Components to be included in the kits will be adapted to each specimen type and to detect pathogens commonly encountered in that type of specimen. Reagents for the universal detection of bacteria will also be included. Based on the sites of infection, the following kits for the specific detection of pathogens may be developed:
  • A kit for the universal detection of bacterial pathogens from most clinical specimens which contains sets of probes specific for highly conserved regions of the bacterial genomes.
  • A kit for the detection of bacterial pathogens retrieved from urine samples, which contains eight specific test components (sets of probes for the detection of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas aeruginosa, Staphylococcus saprophyticus, Staphylococcus aureus and Staphylococcus epidermidis).
  • A kit for the detection of respiratory pathogens which contains seven specific test components (sets of probes for detecting Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Streptococcus pyogenes and Staphylococcus aureus).
  • A kit for the detection of pathogens retrieved from blood samples, which contains eleven specific test components (sets of probes for the detection of Streptococcus pneumoniae, Moraxella catarrhalis, Haemophilus influenzae, Proteus mirabilis, Klebsiella pneumoniae, Pseudomonas aeruginosa, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Streptococcus pyogenes and Staphylococcus epidermidis).
  • A kit for the detection of pathogens causing meningitis, which contains four specific test components (sets of probes for the detection of Haemophilus influenzae, Streptococcus pneumoniae, Escherichia coli and Pseudomonas aeruginosa).
  • A kit for the detection of clinically important antibiotic resistance genes which contains sets of probes for the specific detection of at least one of the 19 following genes associated with bacterial resistance: blatem, blarob, blashv, aadB, aacC1, aacC2, aacC3, aacA4, mecA, vanA, vanH, vanX, satA, aacA-aphD, vat, vga, msrA, sul and int.
  • Other kits adapted for the detection of pathogens from skin, abdominal wound or any other clinically relevant kits will be developed.
    • Example 14
  • Same as example 13 except that the test kits contain all reagents and controls to perform DNA amplification assays. Diagnostic kits will be adapted for amplification by PCR (or other amplification methods) performed directly either from clinical specimens or from a bacterial colony. Components required for universal bacterial detection, bacterial identification and antibiotic resistance genes detection will be included.
  • Amplification assays could be performed either in tubes or in microtitration plates having multiple wells. For assays in plates, the wells will be coated with the specific amplification primers and control DNAs and the detection of amplification products will be automated. Reagents and amplification primers for universal bacterial detection will be included in kits for tests performed directly from clinical specimens. Components required for bacterial identification and antibiotic resistance gene detection will be included in kits for testing directly from colonies as well as in kits for testing directly from clinical specimens.
  • The kits will be adapted for use with each type of specimen as described in example 13 for hybridization-based diagnostic kits.
    • Example 15
  • It is understood that the use of the probes and amplification primers described in this invention for bacterial detection and identification is not limited to clinical microbiology applications. In fact, we feel that other sectors could also benefit from these new technologies. For example, these tests could be used by industries for quality control of food, water, pharmaceutical products or other products requiring microbiological control. These tests could also be applied to detect and identify bacteria in biological samples from organisms other than humans (e.g. other primates, mammals, farm animals and live stocks). These diagnostic tools could also be very useful for research purposes including clinical trials and epidemiological studies.
  • TABLE 1
    Distribution of urinary isolates from positive urine samples
    (≧107CFU/L) at the Centre Hospitalier de l'Université Laval
    (CHUL) for the 1992-1994 period
    % of isolates
    November April July January
    1992 1993 1993 1994
    Organisms n = 267a n = 265 n = 238 n = 281
    Escherichia coli 53.2 51.7 53.8 54.1
    Enterococcus faecalis 13.8 12.4 11.7 11.4
    Klebsiella pneumoniae 6.4 6.4 5.5 5.3
    Staphylococcus epidermidis 7.1 7.9 3.0 6.4
    Proteus mirabilis 2.6 3.4 3.8 2.5
    Pseudomonas aeruginosa 3.7 3.0 5.0 2.9
    Staphylococcus 3.0 1.9 5.4 1.4
    saprophyticus
    Othersb 10.2 13.3 11.8 16.0
    an = total number of isolates for the indicated month
    bSee Table 2
  • TABLE 2
    Distribution of uncommona urinary isolates from positive urine samples
    (≧107CFU/L) at the Centre Hospitalier de l'Université Laval
    (CHUL) for the 1992-1994 period
    % of isolates
    November April July January
    Organisms 1992 1993 1993 1994
    Staphylococcus aureus 0.4 1.1 1.3 1.4
    Staphylococcus spp. 2.2 4.9 1.7 6.0
    Micrococcus spp. 0.0 0.0 0.4 0.7
    Enterococcus faecium 0.4 0.4 1.3 1.4
    Citrobacter spp. 1.4 0.8 0.4 0.7
    Enterobacter spp. 1.5 1.1 1.3 1.4
    Klebsiella oxytoca 1.1 1.5 2.5 1.8
    Serratia spp. 0.8 0.0 0.5 0.0
    Proteus spp. 0.4 0.4 0.0 1.1
    Moganella and 0.4 0.8 0.4 0.0
    Providencia
    Hafania alvei 0.8 0.0 0.0 0.0
    NFBb 0.0 0.4 1.3 1.1
    (Stenotrophomonas,
    Acinetobacter
    Candida spp. 0.8 1.9 0.7 0.4
    aUncommon urinary isolates are those identified as “Others” in Table 1.
    bNFB: non-fermentative bacilli
  • TABLE 3
    Distribution of positivea (bacterial count ≧107CFU/L) and negative
    samples (bacterial count ≦107CFU/L) urine specimens tested at the
    Centre Hospitalier de l'Université Laval (CHUL) for the
    1992-1994 period
    Number of isolates
    November 1992 April 1993 July 1993 January 1994
    n = 267a n = 265 n = 238 n = 281
    received 53.2 51.7 53.8 54.1
    positive 13.8 12.4 11.7 11.4
    negative 6.4 6.4 5.5 5.3
    an = total number of isolates for the indicated month
  • TABLE 4
    Distribution of positive and negative clinical specimens
    tested in the Microbiology Laboratory of the CHUL
    No. of % of % of
    samples positive negative
    Clinical Specimensa tested specimens specimens
    Urine 17,981 19.4 80.6
    Haemoclulture/marrow 10.010 6.9 93.1
    Sputum 1,266 68.4 31.6
    Superficial pus 1,136 72.3 27.7
    Cerebrospinal fluid 553 1.0 99.0
    Synovial fluid-articular 523 2.7 97.3
    Bronch./Trach./Amyg/Throat 502 56.6 43.4
    Deep pus 473 56.8 43.2
    Ears 289 47.1 52.9
    Pleural and pericardial fluid 132 1.0 99.0
    Peritonial fluid 101 28.6 71.4
    aSpecimens tested from February 1994 to January 1995
  • TABLE 5
    Bacterial Species (66) used for testing the specificity of DNA
    fragment probes, oligonucleotides probes and PCR primers
    Number Number
    of of
    Bacterial species strains Bacterial species strains
    Gram negative: tested Gram negative: tested
    Proteus mirabilis 5 Streptococcus pneumoniae 7
    Klebsiella pneumoniae 5 Streptococcus salivarius 2
    Pseudomonas aeruginosa 5 Streptococcus viridans 2
    Escherichia coli 5 Streptococcus pyogenes 2
    Moraxella catarrhalis 5 Staphylococcus aureus 2
    Proteus vulgaris 2 Staphylococcus 2
    epidermidis
    Morganella morganii 2 Staphylococcus 5
    saprophyticus
    Enterobater cloacae 2 Micrococcus species 2
    Providencia stuartii 1 Corynebacterium species 2
    Providencia spp. 1 Streptococcus group B 2
    Enterobacter 2 Staphylococcus simulans 2
    agglomerans
    Providencia rettgeri 2 Staphylococcus 1
    ludgunesis
    Neisseria mucosa 1 Staphylococcus capitis 2
    Providencia 1 Staphylococcus 2
    alcalifaciens haemolyticus
    Providencia 1 Staphylococcus hominis 2
    rustigianii
    Burkholderia cepacia 2 Enterococcus faecalis 2
    Enterobacter aerogenes 2 Enterococcus faecium 1
    Stenotrophomonas 2 Staphylococcus warneri 1
    maltophilia
    Pseudomonas 1 Enterococcus durans 1
    fluorescens
    Comamonas acidovorans 2 Streptococcus bovis 1
    Pseudomonas putida 2 Diphteriods 2
    Haemophilus 5 Lactobacillus 1
    influenzae acidophilus
    Haemophilus 2
    parainfluenzae
    Bordetella pertussis 2
    Haemophilus 2
    parahaemolyticus
    Haemophilus aegyptius 2
    Kingella indologenes 1
    Moraxella atlantae 1
    Neisseria cavaie 1
    Neisseria subflava 1
    Moraxella urethralis 1
    Shigella sonnei 1
    Shigella flexneri 1
    Klebsiella oxytoca 2
    Serratia marcescens 2
    Salmonella 1
    typhimurium
    Yersinia 1
    enterocolitica
    Acinetobacter 1
    calcoaceticus
    Acinetobacter lwoffi 1
    Haftnia alvei 2
    Citrobacter diversus 1
    Citrobacter freundii 1
    Salmonella species 1
  • TABLE 6
    Species-specific DNA fragment and oligonucleotide
    probes for hybridization
    Number of Number of
    fragment probes oligonucleotide probes
    Spe- Ubi- Synthe- Spe- Ubi-
    Organisms Tested cific quitous sized cific quitous
    E. coli d — — — 20 12  9f
    E. coli 14 2  2e — — —
    K. pneumoniae d — — — 15 1 1
    K. pneumoniae 33 3 3 18 12 8
    P. mirabilis d — — — 3 3 2
    P. mirabilis 14 3  3e 15 8 7
    P. aeruginosa d — — — 26 13 9
    P. aeruginosa 6 2  2e 6 0 0
    S. saprophyticus 7 4 4 20 9 7
    H. influenzae d — — — 16 2 2
    H. influenzae 1 1 1 20 1 1
    S. pneumoniae d — — — 6 1 1
    M. catarrhalis 2 2 2 9 8 8
    S. epidermidis 62 1 1 — — —
    S. aureus 30 1 1 — — —
    Universal probesd — — — 7 —  7g
    a No DNA fragment or oligonucleotide probes were tested for E. faecalis and S. pyogenes.
    b Sizes of DNA fragments range from 0.25 to 5.0 kbp
    c A specific probe was considered ubiquitous when at least 80% of isolates of the target species (approximately 80 isolates) were recognized by each specific probe. When 2 or more probes are combined, 100% of the isolates are recognized.
    dThese sequences were selected from data banks.
    eUbiquity tested with approximately 10 isolates of the target species
    fA majority of probes (8/9) do not discriminate E. coli and Shigella spp.
    gUbiquity testes with a pool of the 7 probes detected all 66 bacterial species listed in Table 5.
  • TABLE 7
    PCR amplification for bacterial pathogens commonly encountered in
    urine, sputum, blood, cerebrospinal fluid and other specimens
    DNA amplification
    Primer pair Amplicon from from
    Organism #(SEQ ID NO:) size (bp) Ubiquityb coloniesc specimensd
    E. coli 1e (55–56) 107 75/80 + +
    2e (46–47) 297 77/80 + +
    3 (42–43) 102 78/80 + +
    4 (131–132) 134 73/80 + +
    1 + 2 + 3 + 4 — 80/80 + +
    E. faecalis 1e (38–39) 200 71/80 + +
    2e (40–41) 121 79/80 + +
    1 + 2 — 80/80 + +
    K. pneumoniae 1 (67–68) 198 76/80 + +
    2 (61–62) 143 67/80 + +
    3h (135–136) 148 78/80 +  N.T.i
    4 (137–138) 116 69/80 + N.T.
    1 + 2 + 3 — 80/80 + N.T.
    P. mirabilis 1 (74–75) 167 73/80 + N.T.
    2 (133–134) 123 8080 + N.T.
    P. aeruginosa 1e (83–84) 139 79/80 + N.T.
    2e (85–86) 223 80/80 + N.T.
    S. saprophyticus 1 (98–99) 126 79/80 + +
    2 (139–140) 190 80/80 + N.T.
    M. catarrhalis 1 (112–113) 157 79/80 + N.T.
    2 (118–119) 118 80/80 + N.T.
    3 (160–119) 137 80/80 + N.T.
    H. influenzae 1e (154–155) 217 80/80 + N.T.
    S. pneumoniae 1e (156–157) 134 80/80 + N.T.
    2e (158–159) 197 74/80 + N.T.
    3 (78–79) 175 67/80 + N.T.
    S. epidermidis 1 (147–148) 175 80/80 + N.T.
    2 (145–146) 125 80/80 + N.T.
    S. aureus 1 (152–153) 108 80/80 + N.T.
    2 (149–150) 151 80/80 + N.T.
    3 (149–151) 176 80/80 + N.T.
    S. pyogenes f 1e (141–142) 213 80/80 + N.T.
    2e (143–144) 157 24/24 + N.T.
    Universal 1e (126–127) 241  194/195g + N.T.
    a All primer pairs are specific in PCR assays since no amplification was observed with DNA from 66 different species of both Gram positive and Gram negative bacteria other than the species of interest.
    bThe ubiquity was normally tested on 80 strains of the species of interest. All retained primer pairs amplified at least 90% of the isolates. When combinations of primers were used, a ubiquity of 100% was reached.
    cFor all primer pairs and multiplex combinations, PCR amplifications directly performed from a bacterial colony were 100% species specific.
    dPCR assays performed directly from urine specimens.
    ePrimer pairs derived from data bank sequences. Primer pairs with no “e” are derived from our species-specific fragments.
    fFor S. pyogenes, primer pair #1 is specific for Group A Streptococci (GAS). Primer pair #2 is specific for GAS-producing exotoxin A gene (SpeA)
    gUbiquity tested on 195 isolates from 23 species representative of bacterial pathogens commonly encountered in clinical specimens.
    hOptimizations are in progress to eliminate non-specific amplification observed with some bacterial species other than the target species.
    iN.T.: not tested.
  • TABLE 8
    Selected antibiotic resistance genes for diagnostic purposes
    Genes Antibiotics Bacteriaa SEQ ID NO:
    (blatem) TEM-1 β-lactams Enterobacteriaceae, 161
    Pseudomonadaceae,
    Haemophilus,
    Neisseria
    (blarob) ROB-1 β-lactams Haemophilus, 162
    Pasteurella
    (blashv) SHV-1 β-lactams Klebsiella and other 163
    Enterobacteriaceae
    aadB, aacC1, Aminoglycosides Enterobacteriaceae, 164, 165,
    aacC2, aacC3, Pseudomonadaceae 166, 167,
    aacC4, aacA4 168
    mecA β-lactams Staphylococci 169
    vanH, vanA, Vancomycin Enterococci 170
    vanX
    satA Macrolides Enterococci 173
    aacA-aphD Aminoglycosides Enterococci, 174
    Staphylococci
    vat Macrolides Staphylococci 175
    vga Macrolides Staphylococci 176
    msrA Erythromycin Staphylococci 177
    Int and Sul β-lactams, Enterobacteriaceae 171, 172
    trimethoprim
    conserved aminoglycosides, Pseudomonadaecae
    sequences antiseptic,
    chloramphenicol
    aBacteria having high incidence for the specified antibiotic resistance genes. The presence in other bacteria is not excluded.
  • ANNEX I
    Annex I: Specific and ubiquitous oligonucleotide
    probes for hybridization
    Originating DNA
    fragment
    SEQ ID SEQ ID Nucleotide
    NO: Nucleotide Sequence NO: position
    Bacterial species: Escherichia coli
     44 5′-CAC CCG CTT GCG TGG CAA GCT GCC C  5a 213-237
     45 5′-CGT TTG TGG ATT CCA GTT CCA TCC G  5a 489-513
     48 5′-TGA AGC ACT GGC CGA AAT GCT GCG T  6a 759-783
     49 5′-GAT GTA CAG GAT TCG TTG AAG GCT T  6a 898-922
     50 5′-TAG CGA AGG CGT AGC AGA AAC TAA C  7a 1264-1288
     51 5′-GCA ACC CGA ACT CAA CGC CGG ATT T  7a 1227-1251
     52 5′-ATA CAC AAG GGT CGC ATC TGC GGC C  7a 1313-1337
     53 5′-TGC GTA TGC ATT GCA GAC CTT GTG GC  7a 111-136
     54 5′-GCT TTC ACT GGA TAT CGC GCT TGG G  7a 373-397
    Bacterial species: Proteus mirabilis
     70b 5′-TGG TTC ACT GAC TTT GCG ATG TTT C 12 23-47
     72 5′-TCG AGG ATG GCA TGC ACT AGA AAA T 12 53-77
     72b 5′-CGC TGA TTA GGT TTC GCT AAA ATC TTA TTA 12 80-109
     73 5′-TTG ATC CTC ATT TTA TTA ATC ACA TGA CCA 12 174-203
     76 5′-CCG CCT TTA GCA TTA ATT GGT GTT TAT AGT 13 246-275
     77 5′-CCT ATT GCA GAT ACC TTA AAT GTC TTG GGC 13 291-320
     80b 5′-TTG AGT GAT GAT TTC ACT GAC TCC C 14 18-42
     81 5′-GTG AGA CAG TGA TGG TGA GGA CAC A 15a 1185-1203
     82 5′-TGG TTG TCA TGC TGT TTG TGT GAA AAT 15a 1224-1230
    Bacterial species: Klebsiella pneumoniae
     57 5′-GTG GTG TCG TTC AGG GGT TTC AC  8 45-67
     58 5′-GCG ATA TTC ACA CCC TAC GCA GCC A  9 161-185
     59b 5′-GTC GAA AAT GCC GGA AGA GGT ATA CG  9 203-228
     60b 5′-ACT GAG CTG CAG ACC GGT AAA ACT CA  9 233-258
     63b 5′-CGT GAT GGA TAT TCT TAA CGA AGG GC 10 250-275
     64b 5′-ACC AAA CTG TTG AGC CGC CTG GA 10 201-223
     65 5′-GTG ATC GCC CCT CAT CTG CTA CT 10 77-99
     66 5′-CGC CCT TCG TTA AGA ATA TCC ATC AC 10 249-274
     69 5′-CAG GAA GAT GCT GCA CCG GTT GTT G 11a 296-320
    Bacterial species: Pseudomonas aeruginosa
     87 5′-AAT GCG GCT GTA CCT CGG CGC TGG T 18a 2985-3009
     88 5′-GGC GGA GGG CCA GTT GCA CCT GCC A 18a 2929-2953
     89 5′-AGC CCT GCT CCT CGG CAG CCT CTG C 18a 2821-2845
     90 5′-TGG CTT TTG CAA CCG CGT TCA GGT T 18a 1079-1103
     91 5′-GCG CCC GCG AGG GCA TGC TTC GAT G 19a 705-729
     92 5′-ACC TGG GCG CCA ACT ACA AGT TCT A 19a 668-692
     93 5′-GGC TAC GCT GCC GGG CTG CAG GCC G 19a 505-529
     94 5′-CCG ATC TAG ACC ATC GAG ATG GGC G 20a 1211-1235
     95 5′-GAG CGC GGC TAT GTG TTC GTC GGC T 20a 2111-2135
    Bacterial species: Streptococcus pneumoniae
    120 5′-TCT GTG CTA GAG ACT GCC CCA TTT C 30 423-447
    121 5′-CGA TGT CTT GAT TGA GCA GGG TTA T 31a 1198-1222
    Bacterial species: Staphylococcus saprophyticus
     96 5′-CGT TTT TAC CCT TAC CTT TTC GTA CTA CC 21 45-73
     97b 5′-TCA GGC AGA GGT AGT ACG AAA AGG TAA GGG 21 53-82
    100 5′-CAC CAA GTT TGA CAC GTG AAG ATT CAT 22  89-115
    101b 5′-ATG AGT GAA GCG GAG TCA GAT TAT GTG CAG 23 105-134
    102 5′-CGC TCA TTA CGT ACA GTG ACA ATC G 24 20-44
    103 5′-CTG GTT AGC TTG ACT CTT AAC AAT CTT GTC 24 61-90
    104b 5′-GAC GCG ATT GTC ACT GTA CGT AAT GAG CGA 24 19-48
    Bacterial species: Moraxella catarrhalis
    108 5′-GCC CCA AAA CAA TGA AAC ATA TGG T 28  81-105
    109 5′-CTG CAG ATT TTG GAA TCA TAT CGC C 28 126-130
    110 5′-TGG TTT GAC CAG TAT TTA ACG CCA T 28 165-189
    111 5′-CAA CGG CAC CTG ATG TAC CTT GTA C 28 232-256
    114 5′-TTA CAA CCT GCA CCA CAA GTC ATC A 29 97-121
    115 5′-GTA CAA ACA AGC CGT CAG CGA CTT A 29 139-163
    116 5′-CAA TCT GCG TGT GTG CGT TCA CT 29 178-200
    117 5′-GCT ACT TTG TCA GCT TTA GCC ATT CA 29 287312
    Bacterial species: Haemophilus influenzae
    105b 5′-GCG TCA GAA AAA GTA GGC GAA ATG AAA G 25 138-165
    106b 5′-AGC GGC TCT ATC TTG TAA TGA CAC A 26a 770-794
    107b 5′-GAA ACG TGA ACT CCC CTC TAT ATA A 27a 5184-5208
    Universal probesc
    122b 5′-ATC CCA CCT TAG GCG GCT GGC TCC A — —
    123 5′-ACG TCA AGT CAT CAT GGC CCT TAC GAG TAG G — —
    124b 5′-GTG TGA CGG GCG GTG TGT ACA AGG C — —
    125b 5′-GAG TTG CAG ACT CCA ATC CGG ACT ACG A — —
    128b 5′-CCC TAT ACA TCA CCT TGC GGT TTA GCA GAG AG — —
    129 5′-GGG GGG ACC ATC CTC CA  GGC TAA ATA C — —
    130b 5′-CGT CCA CTT TCG TGT TTG CAG AGT GCT GTG TT — —
    aSequence from data banks
    bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing.
  • ANNEX II
    ANNEX II: Specific and ubiquitous primers for
    DNA amplification
    Originating DNA
    fragment
    SEQ ID SEQ ID Nucleotide
    NO: Nucleotide Sequence NO: position
    Bacterial species: Escherichia coli
     42 5′-GCT TTC CAG CGT CAT ATT G  4 177-195
     43b 5′-GAT CTC GAC AAA ATG GTG A  4 260-278
     46 5′-TCA CCC GCT TGC GTG GC  5a 212-228
     47b 5′-GGA ACT GGA ATC CAC AAA C  5a 490-508
     55 5′-GCA ACC CGA ACT CAA CGC C  7a 1227-1245
     56b 5′-GCA GAT GCG ACC CTT GTG T  7a 1315-1333
    131 5′-CAG GAG TAC GGT GAT TTT TA  3 60-79
    132b 5′-ATT TCT GGT TTG GTC ATA CA  3 174-193
    Bacterial species: Enterococcus faecalis
     38 5′-GCA ATA CAG GGA AAA ATG TC  1a 69-88
     39b 5′-CTT CAT CAA ACA ATT AAC TC  1a 249-268
     40 5′-GAA CAG AAG AAG CCA AAA AA  2a 569-588
     41b 5′-GCA ATC CCA AAT AAT ACG GT  2a 670-689
    Bacterial species: Klebsiella pneumoniae
     61 5′-GAC AGT CAG TTC GTC AGC C  9 37-55
     62b 5′-CGT AGG GTG TGA ATA TCG C  9 161-179
     67 5′-TCG CCC CTC ATC TGC TAC T 10 81-99
     68b 5′-GAT CGT GAT GGA TAT TCT T 10 260-278
    135 5′-GCA GCG TGG TGT CGT TCA  8 40-57
    136b 5′-AGC TGG CAA CGG CTG GTC  8 170-187
    137 5′-ATT CAC ACC CTA CGC AGC CA  9 166-185
    138b 5′-ATC CGG CAG CAT CTC TTT GT  9 262-281
    Bacterial species: Proteus mirabilis
     74 5′-GAA ACA TCG CAA AGT CAG T 12 23-41
     75b 5′-ATA AAA TGA GGA TCA AGT TC 12 170-189
    133 5′-CGG GAG TCA GTG AAA TCA TC 14 17-36
    134b 5′-CTA AAA TCG CCA CAC CTC TT 14 120-139
    Bacterial species: Staphylococcus saprophyticus
     98 5′-CGT TTT TAC CCT TAC CTT TTC GTA CT 21 45-70
     99b 5′-ATC GAT CAT CAC ATT CCA TTT GTT TTT A 21 143-170
    139 5′-CTG GTT AGC TTG ACT CTT AAC AAT C 24 61-85
    140b 5′-TCT TAA CGA TAG AAT GGA GCA ACT G 24  26-250
    Bacterial species: Psuedomonas aeruginosa
     83 5′-CGA GCG GGT GGT GTT CAT C 16a 554-572
     84b 5′-CAA GTC GTG GTG GGA GGG A 16a 674-692
     85 5′-TCG CTG TTC ATC AAG ACC C 17a 1423-1441
     86b 5′-CCG AGA ACC AGA CTT CAT C 17a 1627-1645
    Bacterial species: Moraxella catarrhalis
    112 5′-GGC ACC TGA TGT ACC TTG 28 235-252
    113b 5′-AAC AGC TCA CAC GCA TT 28 375-391
    118 5′-TGT TTT GAG CTT TTT ATT TTT TGA 29 41-64
    119 5′-CGC TGA CGG CTT GTT TGT ACC A 29 137-158
    160 5′-GCT CAA ATC AGG GTC AGC 29 22-39
    119b 5′-CGC TGA CGG CTT GTT TGT ACG A 29 137-158
    Bacterial species: Staphylococcus epidermidis
    145 5′-ATC AAA AAG TTG GCG AAC CTT TTC A 36 21-45
    146 5′-CAA AAG AGC GTG GAG AAA AGT ATC A 36 121-145
    147 5′-TCT CTT TTA ATT TCA TCT TCA ATT CCA TAG 36 448-477
    148b 5′-AAA CAC AAT TAC AGT CTG GTT ATC CAT ATC 36 593-622
    Bacterial species: Staphylococcus aureus
    149b 5′-CTT CAT TTT ACG GTG ACT TCT TAG AAG ATT 37 409-438
    150 5′-TCA ACT GTA GCT TCT TTA TCC ATA CGT TGA 37 288-317
    149b 5′-CTT CAT TTT ACG GTG ACT TCT TAG AAG ATT 37 409-438
    151 5′-ATA TTT TAG CTT TTC AGT TTC TAT ATC AAC 37 263-292
    152 5′-AAT CTT TGT CGG TAC ACG ATA TTC TTC ACG 37  5-34
    153b 5′-CGT AAT GAG ATT TCA GTA GAT AAT ACA ACA 37  83-112
    Bacterial species: Haemophilus influenzae
    154 5′-TTT AAC GAT CCT TTT ACT CCT TTT G 27a 5074-5098
    155b 5′-ACT GCT GTT GTA AAG AGG TTA AAA T 27a 5266-5290
    Bacterial species: Streptococcus pneumoniae
     78 5′-AGT AAA ATG AAA TAA GAA CAG GAC AG 34 164-189
     79b 5′-AAA ACA GGA TAG GAG AAC GGG AAA A 34 314-338
    156 5′-ATT TGG TGA CGG GTG ACT TT 31a 1401-1420
    157b 5′-GCT GAG GAT TTG TTC TTC TT 31a 1515-1534
    158 5′-GAG CGG TTT CTA TGA TTG TA 35a 1342-1361
    159b 5′-ATC TTT CCT TTC TTG TTC TT 35a 1519-1538
    Bacterial species: Steptococcus pyogenes
    149 5′-TGA AAA TTC TTG TAA CAG GC 32a 286-305
    142b 5′-GGC CAC CAG CTT GCC CAA TA 32a 479-498
    143 5′-ATA TTT TCT TTA TGA GGG TG 33a 966-985
    144b 5′-ATC CTT AAA TAA AGT TGC CA 33a 1103-1122
    Universal primersc
    126 5′-GGA GGA AGG TGG GGA TGA CG — —
    127b 5′-ATG GTG TGA CGG GCG GTG TG — —
    asequence from data banks
    bThese sequences are from the opposite DNA strand of the sequences given in the Sequence listing.
  • ANNEX III
    ANNEX III
    Selection of Universal Probes by Alignment of the
    Sequences of Bacterial 16S and 23S Ribosomal RNA Genes
    Reverse strand of                 TGGACGG AGCCGCCTA GGTGGGAT
    SEQ ID NO:122
    1251                                              1300
    Streptococcus TGAGGTAACC TTTTGGAGCC AGCCGCCTAA GGTGGGATAG ATGANNGGGG
    salivarius
    Proteus vulgaris TAGCTTAACC TTCGGGAGGG CGCTTACCAC TTTGTGATTC ATGACTGGGG
    Pseudomonas aeruginosa TAGTCTAACC GCAAGGGGGA CGGTTACCAC GGAGTGATTC ATGACTGGGG
    Neiserria gonorrhoeae TAGGGTAACC GCAAGGAGTC CGCTTACCAC GGTATGCTTC ATGACTGGGG
    Streptococcus lactis TTGCCTAACC GCAAGGAGGG CGCTTCCTAA GGTAAGACCG ATGACNNGGG
    SEQ ID NO: 123            ACGTCAAGTC ATCATGGC CCTTACGAGT AGG
    1251                                              1300
    Haemophilus influenzae GGTNGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGAGT AGGGCTACAC
    Neiserria gonorrhoeae GGTGGGGATG ACGTCAAGTC ..CTCATGGC CCTTATGACC AGGGCTTCAC
    Pseudomonas cepacia GGTNGGGATG ACGTCAAGTC ..CTCATGGC CCTTATGGGT AGGGCTTCAC
    Serratia marcescens GGTGGGGATG ACGTCAAGTC ..CTCATGGC CCTTATGGGT AGGGCTTCAC
    Escherichia coli GGTGGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGACC AGGGCTACAC
    Proteus vulgaris GGTGGGGATG ACGTTAAGTC GTATCATGGC CCTTACGAGT AGGGCTACAC
    Pseudomonas aeruginosa GGTGGGGATG ACGTCAAGTC ..ATCATGGC CCTTACGGCN AGGGCTACAC
    Clostridium pefringens GGTGGGGATG ACGTNNAATC ..ATCATGCC CNTTATGTGT AGGGCTACAC
    Mycoplasma hominis GGTGGGGATG ACGTCAAATC ..ATCATGCC TCTTACGAGT GGGGCCACAC
    Helicobacter pylori GGTGGGGACG ACGTCAAGTC ..ATCATGGC CCTTACGCCT AGGGCTACAC
    Mycoplasma pneumoniae GGAAGGGATG ACGTCAAATC ..ATCATGCC CCTTATGTCT AGGGCTGCAA
    Reverse of the probe            GCCTTGTACA CACCGCCCGT CACAC
    SEQ ID NO:124
    1451                                   1490
    Escherichia coli ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG
    Neiserria ghonorrhoeae ACGTTCCCNG NNCTTGTACA CACCGCCCGT CACACCATGG
    Pseudomonas cepacia ACGTTCCCGG GTCTTGTACA CACNGCCCGT CACACCATGG
    Serratia marcescens ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG
    Proteus vulgaris ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG
    Haemophilus influenzae ACGTTCCCGG GCNTTGTACA CACCGCCCGT CACACCATGG
    Pseudomonas aeruginosa ACGTTCCCGG GCCTTGTACA CACCGCCCGT CACACCATGG
    Clostridium pefringens ACGTTCCCNG GTCTTGTACA CACCGCNCGT CACACCATGA
    Mycoplasma hominis ACGTTCTCGG GTCTTGTACA CACCGCCCGT CACACCATGG
    Helicobacter pylori ACGTTCCCGG GTCTTGTACT CACCGCCCGT CACACCATGG
    Mycoplasma pneumoniae ACGTTCTCGG GTCTTGTACA CACCGCCCGT CAAACTATGA
    Reverse strand of        TCG TAGTCCGGAT TGGAGTCTGC AACTC
    SEQ ID NO: 125
    1361                                   1400
    Escherichia coli AAGTGCGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC
    Neiserria ghonorrhoeae AAACCGATCG TAGTCCGGAT TGCACTCTGC AACTCGAGTG
    Pseudomonas cepacia AAACCGATCG TAGTCCGGAT TGCACTCTGC AACTCGAGTG
    Serratia marcescens AAGTATGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC
    Proteus vulgaris AAGTCTGTCG TAGTCCGGAT TGGAGTCTGC AACTCGACTC
    Haemophilus influenzae AAGTACGTCT AAGTCCGGAT TGGAGTCTGC AACTCGACTC
    Pseudomonas aeruginosa AAACCGATCG TAGTCCGGAT CGCAGTCTGC AACTCGACTG
    Clostridium pefringens AAACCAGTCT CAGTTCGGAT TGTAGGCTGA AACTCGCCTA
    Mycoplasma hominis AAGCCGATCT CAGTTCGGAT TGGAGTCTGC AATTCGACTC
    Helicobacter pylori ACACC..TCT CAGTTCGGAT TGTAGGCTGC AACTCGCCTG
    Mycoplasma pneumoniae AAGTTGGTCT CAGTTCGGAT TGAGGGCTGC AATTCGTCTT
    Reverse strand of
    SEQ ID NO: 128
    481                                                530
    Lactobacillus lactis AAACACAGCT CTCTGCTAAA CCGCAAGGTG ATGTATAGGG GGTGACGCCT
    Escherichia coli AAACACAGCA CTGTGCAAAC ACGAAAGTGG ACGTATACGG TGTGACGCCT
    Pseudomonas aeruginosa AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG TGTGACGCCT
    Pseudomonas cepacia AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAAGG TGTGACGCCT
    Bacillus AAACACAGGT CTCTGCGAAG TCGTAAGGCG ACGTATAGGG GCTGACACCT
    stearothermophilus
    Micrococcus luteus AAACACAGGT CCATGCGAAG TCGTAAGACG ATGTATATGG ACTGACTCCT
    SEQ ID NO: 129             GGGGGGACC ATCCTCCAAG GCTAAATAC
    1991                                              2040
    Escherichia coli TGTCTGAATG TGGGGGGACC ATCCTCCAAG GCTAAATACT CCTGACTGAC
    Pseudomonas aeruginosa TGTCTGAACA TGGGGGGACC ATCCTCCAAG GCTAAATACT ACTGACTGAC
    Pseudomonas cepacia TGTCTGAAGA TGGGGGGACC ATCCTCCAAG GCTAAATACT CGTGATCGAC
    Lactobacillus lactis AGTTTGAATC GCCCAGGACC ATCTCCCAAC CCTAAATACT CCTTAGTGAC
    Micrococcus luteus CGTGTGAATC TGCCAGGACC ACCTGGTAAG CCTGAATACT ACCTGTTGAC
    Reverse strand of             AACACAGCA CTCTGCAAAC ACGAAAGTGG ACG
    SEQ ID NO: 130
    1981                                              2030
    Pseudomonas aeruginosa TGTTTATTAA AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG
    Escherichia coli TGTTTATTAA AAACACAGCA CTGTGGAAAC ACGAAAGTGG ACGTATACGG
    Bacillus TGTTTAATAA AAACACAGCA CTCTGCAAAC ACGAAAGTGG ACGTATAGGG
    stearothermophilus
    Lactobacillus lactis TGTTTATCAA AAACACAGCT CTCTGCTAAA CCACAAGGTG ATGTATAGGG
    Micrococcus luteus TGTTTATCAA AAACACAGGT CCATGCGAAG TCGTAAGACG ATGTATATGG
    SEQ ID NO: 126    GGAGGAA GGTGGGGATG ACG
    Reverse strand of                                            CA CACCGCCCGT CACACCAT
    SEQ ID NO: 127
    Escherichia coli ACTGGAGGAA GGTGGGGATG ACGTCAAGTC...GCCTTGTACA CACCGCCCGT CACACCATGG
    Neiserria ghonorrhoeae GCCGGAGGAA GGTGGGGATG ACGTCAAGTC...NNCTTGTACA CACCGCCCGT CACACCATGG
    Pseudomonas cepacia ACCGGAGGAA GGTNGGGATG ACGTCAAGTC...GTCTTGTACA CACNGCCCGT CACACCATGG
    Serratia marcescens ACTGGAGGAA GGTGGGGATG ACGTCAAGTC...GCCTTGTACA CACCGCCCGT CACACCATGG
    Proteus vulgaris ACCGGAGGAA GGTGGGGATG ACGTTAAGTC...GCCTTGTACA CACCGCCCGT CACACCATGG
    Haemophilus influenzae ACTGGAGGAA GGTNGGGATG ACGTCAAGTC...GCNTTGTACA CACCGCCCGT CACACCATGG
    Legionella pneumophila ACCGGAGGAA GGCGGGGATG ACGTCAAGTC...GCCTTGTACA CACCGCCCGT CACACCATGG
    Pseudomonas aeruginosa ACCGGAGGAA GGTGGGGATG ACGTCAAGTC...GCCTTGTACA CACCGCCCGT CACACCATGG
    Clostridium perfringens CCAGGAGGAA GGTGGGGATG ACGTNNAATC...GTCTTGTACA CACCGCNCGT CACACCATGA
    Mycoplasma hominis CTGGGAGGAA GGTGGGGATG ACGTCAAATC...GTCTTGTACA CACCGCCCGT CACACCATGG
    Helicobacter pylori GGAGGAGGAA GGTGGGGACG ACGTCAAGTC...GTCTTGTACT CACCGCCCGT CACACCATGG
    Mycoplasma pneumoniae ATTGGAGGAA GGAAGGGATG ACGTCAAATC...GTCTTGTACA CACCGCCCGT CAAACTATGA

Claims (1)

1. A method to detect and identify the presence of Staphylococcus aureus and at least a second and a third target bacterial species in a sample by performing an assay, comprising:
simultaneously contacting a sample with a set of amplification primers comprising a plurality of at least a first, second, and third primer pair, wherein said first primer pair hybridizes solely to the nucleic acids of Staphylococcus aureus, and wherein said second and third primer pairs hybridize solely to target DNA of said second and third target bacterial species, respectively, and are ubiquitous to at least 80% to Staphylococcus aureus, and second and third target bacterial species, respectively, wherein the plurality of primer pairs are chosen to allow amplification under a single amplification protocol;
amplifying target nucleic acid from said sample under said single amplification protocol; and
detecting the presence or amount of amplified product(s) as an indication of the presence of Staphylococcus aureus and said second and third target bacterial species in said sample.
US11/842,092 1994-09-12 2007-08-20 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 Abandoned US20090047671A1 (en)

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US9452599A 1999-12-01 1999-12-01
US10/121,120 US20050042606A9 (en) 1994-09-12 2002-04-11 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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