Method for rapid detection and identification of bioagents

• US 20060275788A1
• Filed: 01/13/2006
• Published: 12/07/2006
• Est. Priority Date: 03/02/2001
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Sampath, Rangarajan, McNeil, John, Ecker, David J., Hofstadler, Steven, Griffey, Richard
• Granted Patent: US 7,741,036 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

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

Claims

1. A method of identifying a bacterium comprising:

selecting at least one pair of oligonucleotide primers which are not specific for a particular bacterial genus, wherein one member of said pair of primers hybridizes to a first conserved region of nucleic acid encoding ribosomal RNA and the other member of said pair of primers hybridizes to a second conserved region of nucleic acid encoding ribosomal RNA wherein said first and second conserved regions flank a variable nucleic acid region which varies among bacteria;

amplifying nucleic acid from said bacterium with said pair of oligonucleotide primers to produce an amplification product;

determining the molecular mass of said amplification product by mass spectrometry; and

comparing said molecular mass to calculated or measured molecular masses of amplification products of known bacteria produced by using said pair of oligonucleotide primers, thereby identifying said bacterium.

2. The method of claim 1 wherein said amplification product is between about 46 to 166 nucleobases in length.

3. The method of claim 2 wherein each member of said pair of oligonucleotide primers hybridize to nucleic acid encoding ribosomal RNA of one hundred or more bacterial species and produce said amplification products of known bacteria.

4. The method of claim 1 wherein each member of said pair of oligonucleotide primers can hybridize to nucleic acid encoding ribosomal RNA of one hundred or more bacterial species and produce said amplification products of known bacteria.

5. The method of claim 1 wherein the level of identification of said bacterium is the genus level.

6. The method of claim 1 wherein the level of identification of said bacterium is the species level.

7. The method of claim 1 wherein the level of identification of said bacterium is the strain level.

8. The method of claim 1 wherein said bacterium is a member of the genus Acinetobacter, Aeromonas, Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio, or Yersinia.

9. A method of identifying a bacterium comprising:

selecting at least one pair of oligonucleotide primers which are not specific for a particular bacterial genus, wherein one member of said pair of primers hybridizes to a first conserved region of nucleic acid encoding a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion and the other member of said pair of primers hybridizes to a second conserved region of nucleic acid encoding a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion wherein said first and second conserved regions flank a variable nucleic acid region which varies among bacteria;

amplifying nucleic acid from said bacterium with said pair of oligonucleotide primers to produce an amplification product;

determining the molecular mass of said amplification product by mass spectrometry; and

comparing said molecular mass to calculated or measured molecular masses of amplification products of known bacteria produced by using said pair of oligonucleotide primers, thereby identifying said bacterium.

10. The method of claim 9 wherein said amplification product is between about 46 to 166 nucleobases in length.

11. The method of claim 10 wherein each member of said pair of oligonucleotide primers hybridize to nucleic acid encoding a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion of one hundred or more bacterial species and produce said amplification products of known bacteria.

12. The method of claim 9 wherein each member of said pair of oligonucleotide primers can hybridize to nucleic acid encoding a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion of one hundred or more bacterial species and produce said amplification products of known bacteria.

13. The method of claim 9 wherein the level of identification of said bacterium is the genus level.

14. The method of claim 9 wherein the level of identification of said bacterium is the species level.

15. The method of claim 9 wherein the level of identification of said bacterium is the strain level.

16. The method of claim 9 wherein said protein is DNA polymerase, RNA polymerase, elongation factor Tu, heat shock protein groEL, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase, or elongation factor G.

17. The method of claim 9 wherein said bacterium is a member of the genus Acinetobacter, Aeromonas, Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio, or Yersinia.

18. A method of identifying a bacterium comprising:

selecting at least one pair of oligonucleotide primers which are not specific for a particular bacterial genus, wherein one member of said pair of primers hybridizes to a first conserved region of nucleic acid encoding ribosomal RNA and the other member of said pair of primers hybridizes to a second conserved region of nucleic acid encoding ribosomal RNA wherein said first and second conserved regions flank a variable nucleic acid region which varies among bacteria;

amplifying nucleic acid from said bacterium with said pair of oligonucleotide primers to produce an amplification product;

determining the molecular mass of said amplification product by mass spectrometry;

calculating the base composition of said amplification product from said molecular mass; and

comparing said base composition to calculated or measured base compositions of amplification products of known bacteria produced by using said pair of oligonucleotide primers, thereby identifying said bacterium.

19. The method of claim 18 wherein said amplification product is between about 46 to 166 nucleobases in length.

20. The method of claim 19 wherein each member of said pair of oligonucleotide primers hybridize to nucleic acid encoding ribosomal RNA of one hundred or more bacterial species and produce said amplification products of known bacteria.

21. The method of claim 18 wherein each member of said pair of oligonucleotide primers can hybridize to nucleic acid encoding ribosomal RNA of one hundred or more bacterial species and produce said amplification products of known bacteria.

22. The method of claim 18 wherein the level of identification of said bacterium is the genus level.

23. The method of claim 18 wherein the level of identification of said bacterium is the species level.

24. The method of claim 18 wherein the level of identification of said bacterium is the strain level.

25. The method of claim 18 wherein said bacterium is a member of the genus Acinetobacter, Aeromonas, Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio, or Yersinia.

26. A method of identifying a bacterium comprising:

selecting at least one pair of oligonucleotide primers which are not specific for a particular bacterial genus, wherein one member of said pair of primers hybridizes to a first conserved region of nucleic acid encoding a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion and the other member of said pair of primers hybridizes to a second conserved region of nucleic acid encoding a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion wherein said first and second conserved regions flank a variable nucleic acid region which varies among bacteria;

amplifying nucleic acid from said bacterium with said pair of oligonucleotide primers to produce an amplification product;

determining the molecular mass of said amplification product by mass spectrometry;

calculating the base composition of said amplification product from said molecular mass; and

comparing said base composition to calculated or measured base compositions of amplification products of known bacteria produced by using said pair of oligonucleotide primers, thereby identifying said bacterium.

27. The method of claim 26 wherein said amplification product is between about 46 to 166 nucleobases in length.

28. The method of claim 27 wherein each member of said pair of oligonucleotide primers hybridize to nucleic acid encoding a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion of one hundred or more bacterial species and produce said amplification products of known bacteria.

29. The method of claim 26 wherein each member of said pair of oligonucleotide primers can hybridize to nucleic acid encoding a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion of one hundred or more bacterial species and produce said amplification products of known bacteria.

30. The method of claim 26 wherein the level of identification of said bacterium is the genus level.

31. The method of claim 26 wherein the level of identification of said bacterium is the species level.

32. The method of claim 26 wherein the level of identification of said bacterium is the strain level.

33. The method of claim 26 wherein said protein is DNA polymerase, RNA polymerase, elongation factor Tu, heat shock protein groEL, phosphoglycerate kinase, NADH dehydrogenase, DNA ligase, DNA topoisomerase, or elongation factor G.

34. The method of claim 26 wherein said bacterium is a member of the genus Acinetobacter, Aeromonas, Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Chlamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Klebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio, or Yersinia.