Method for the detection and/or identification of a microorganism

Abstract

The present invention relates generally to a method of identifying micro-organisms in a sample and in particular to the identification of prokaryotic micro-organisms. In some embodiments, the present invention relates to a method for the detection and/or identification of at least one microorganism or for the simultaneous detection and/or identification of several microorganisms in a test sample wherein the test sample is contacted with one or more probes derived from a tuf gene variable region.

1 - 29 . (canceled) 
     
     
         30 . A method for the simultaneous detection and/or identification of several microorganisms in a test sample comprising:
 (a) providing a test sample suspected of containing one or more target microorganisms;   (b) releasing, isolating and/or concentrating polynucleic acids from said microorganisms from said test sample, if present in the test sample, the or each polynucleic acid comprising at least a portion of a tuf gene variable region;   (c) amplifying at least a portion of said tuf gene variable region in said polynucleic acid with a primer pair consisting of SEQ ID NO. 394 and SEQ ID NO. 395 or with a primer pair consisting of SEQ ID NO. 396 and SEQ ID NO. 395 to produce an amplified product;   (d) contacting said amplified product with at least one probe or combination thereof selected from the group consisting of SEQ ID NOs:12 to 393 for a time and under conditions sufficient for hybridization to take place; and   (e) detecting any hybridization which has taken place in step (d);   
       wherein the presence or absence of hybridization is indicative of the presence or absence of said one or more microorganisms in said test sample, and wherein the one or more microorganisms are selected from the group consisting of  Acinetobacter, Bordetella, Burkholderia, Campylobacter, Chlamydophila, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Enterobacter, Escherichia, Fusobacterium, Haemophilus, Helicobacter  species,  Klebsiella, Legionella, Listeria, Moraxella Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Serratia, Staphylococcus, Stenotrophomonas, Streptococcus, Vibrio , and  Yersinia  species. 
     
     
         31 . The method of  claim 30 , wherein the tuf gene variable region in said polynucleic acid consists essentially of nucleotides 108-220, 393-591, 708-774, 792-816, and/or 885-945 of SEQ ID NOs.:1 to 11. 
     
     
         32 . The method of  claim 30 , wherein the one or more microorganisms are selected from the group consisting of  Acinetobacter baumanii, Bordetella pertussis, Burkholderia cepacia, Campylobacter jejuni, Chlamydophyla abortus, Chlamydia pneumoniae, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterobacter cloacae, Enterococcus faecalis, Escherichia coli, Fusobacterium neucleatum, Haemophilus influenzae, Helicobacter pylori, Helicobacter hepaticus, Klebsiella pneumoniae, Legionella longbeachiae, Legionella pneumophila, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitides, Pseudomonas aeruginosa, Staphylococcus aureus, Staphyloccoccus epidermidis, Staphylococcus saprophyticus, Stenotrophomonas maltophilia, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Streptococcus sanguinis, Serratia marcescens, Yersinia pestis , and  Yersinia pseudotuberculosis.    
     
     
         33 . The method of  claim 30 , wherein the test sample is a sputum sample. 
     
     
         34 . A microarray comprising at least one probe selected from the group consisting of SEQ ID NOs: 12 to 393 for use in simultaneous detection and/or identification of several microorganisms in a test sample, wherein the microorganisms are selected from the group consisting of  Acinetobacter, Bordetella, Burkholderia, Campylobacter, Chlamydophila, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Enterobacter, Escherichia, Fusobacterium, Haemophilus, Helicobacter  species,  Klebsiella, Legionella, Listeria, Moraxella Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Serratia, Staphylococcus, Stenotrophomonas, Streptococcus, Vibrio , and  Yersinia  species. 
     
     
         35 . A method for diagnosing a bacterial infection in a subject, said method comprising:
 a) obtaining polynucleic acids from a test sample taken from said subject suspected of containing one or more target microorganisms;   b) releasing, isolating and/or concentrating polynucleic acids from said microorganism from said test sample, if present in the test sample, the or each polynucleic acid comprising at least a portion of a tuf gene variable region;   c) amplifying at least a portion of said tuf gene variable region in said polynucleic acids with a primer pair consisting of SEQ ID NO. 394 and SEQ ID NO. 395 or with a primer pair consisting of SEQ ID NO. 396 and SEQ ID NO. 395 to produce an amplified product;   d) contacting said amplified product with a microarray according to  claim 34  for a time and under conditions sufficient to form one or more hybridization complexes; and   e) detecting any hybridization which has taken place in step d), wherein the presence of hybridization is indicative of the presence of bacterial infection, wherein the bacterial infection is caused by a microorganism selected from the group consisting of  Acinetobacter, Bordetella, Burkholderia, Campylobacter, Chlamydophila, Chlamydia, Clostridium, Corynebacterium, Enterococcus, Enterobacter, Escherichia, Fusobacterium, Haemophilus, Helicobacter  species,  Klebsiella, Legionella, Listeria, Moraxella Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Serratia, Staphylococcus, Stenotrophomonas, Streptococcus, Vibrio , and  Yersinia  species.   
     
     
         36 . A kit for the detection and/or identification of several microorganisms simultaneously in a test sample, comprising:
 (a) at least one of a first primer pair consisting of SEQ ID NO. 394 and SEQ ID NO. 395 or and a second primer pair consisting of SEQ ID NO. 396 and SEQ ID NO. 395 allowing amplification of at least a portion of a tuf gene variable region;   (b) the microarray of  claim 34 ;   (c) a buffer or components necessary to produce the buffer enabling a hybridization reaction between the probes contained in said microarray and polynucleic acid present in an amplified product;   (d) a solution or components necessary for producing the solution enabling washing of hybrids formed under appropriate wash conditions; and   (e) optionally, a means for detecting hybrids.