Method for rapid detection and identification of bioagents

• US 7,108,974 B2
• Filed: 05/24/2002
• Issued: 09/19/2006
• Est. Priority Date: 03/02/2001
• Status: Expired due to Term

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Sampath, Rangarajan, McNeil, John, Ecker, David J., Hofstadler, Steven, Griffey, Richard
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US10/156,608
• Publication Number:
  US 20030124556A1
• Time in Patent Office: 1,579 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 536/23.1
• US Class Current: 435/6.19
• 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 bacterial bioagent comprising:

contacting nucleic acid from the bioagent with at least one pair of primers which hybridize to flanking sequences of the nucleic acid, wherein the flanking sequences flank a variable nucleic acid sequence of the bioagent;

amplifying the variable nucleic acid sequence to produce an amplification product;

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

comparing the molecular mass of the amplification product to calculated or measured molecular masses of analogous amplification products of one or more known bacterial bioagents present in a database comprising 19 or more molecular masses, with the proviso that sequencing of the amplification product is not used to identify the bacterial bioagent.

3. The method of claim 1 or claim 2 wherein the amplifying step comprises the polymerase chain reaction.

4. The method of claim 1 or claim 2 wherein the nucleic acid encodes ribosomal RNA or a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion.

5. The method of claims 1 or 2 wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) or time of flight mass spectrometry (TOF-MS).

6. The method of claim 1 or claim 2 wherein the variable nucleic acid sequence exhibits no greater than about 5% identity among bacterial bioagents.

7. The method of claim 1 or claim 2 wherein the sequences to which the primers hybridize are separated by between about 60–

100 nucleotides.

8. The method of claim 1 or claim 2 wherein the flanking sequences are between about 80 and 100% identical among bacterial bioagents.

9. The method of claim 1 or claim 2 wherein the flanking sequences are greater than about 95% identical among bacterial bioagents.

21. The method of claims 1 or 2 wherein the bacterial bioagent is a member of the genus Acinetobacter, Aeromonas, Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chiamydia, Chiamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Kiebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Ricketsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio, or Yersinia.

22. The method of claims 1, 2 or 10 wherein the bacterial bioagent is identified at the species level.

23. The method of claims 1, 2 or 10 wherein the bacterial bioagent is identified at the strain level.

24. The method of claims 1, 2 or 10 wherein the bacterial bioagent is a biological warfare agent.

25. The method of claim 24 wherein the biological warfare agent is Bacillus anthracis, Yersinia pestis, Franciscella tularensis, Brucella suis, Brucella abortus, Brucella melitensis, Burkholderia mallei, Burkholderia pseudomalleii, Salmonella typhi, Rickettsia typhii, Rickettsia prowasekii, Coxiella burnetii, Rhodobacter capsulatus, Chlamydia pneumoniae, Escherichia coli, Shigella dysenteriae, Shigellaflexneri, Bacillus cereus, Clostridium botulinum, Coxiella burnetti, Pseudomonas aeruginosa, Legionella pneumophila, or Vibrio cholerae.

26. The method of claims 1, 2 or 10 wherein the pair of primers comprises at least one nucleotide analog.

27. The method of claim 26 wherein the nucleotide analog is inosine, uridine, 2,6-diaminopurine, propyne C, or propyne T.

28. The method of claims 1, 2 or 10 wherein a molecular mass-modifying tag is incorporated into the amplification product to limit the number of possible base compositions consistent with the mass of the amplification product.

2. A method of identifying a bacterial bioagent comprising:

contacting nucleic acid from the bioagent with at least one pair of primers which hybridize to flanking sequences of the nucleic acid, wherein the flanking sequences flank a variable nucleic acid sequence of the bioagent;

amplifying the variable nucleic acid sequence to produce an amplification product;

determining the base composition of the amplification product by mass spectrometry; and

comparing the base composition of the amplification product to calculated or measured base composition of analogous amplification products of one or more known bacterial bioagents present in a database comprising 19 or more base compositions, with the proviso that sequencing of the amplification product is not used to identify the bacterial bioagent.

10. A method of identifying a bacterial bioagent comprising:

contacting nucleic acid from the bacterial bioagent with at least one pair of primers which hybridize to flanking sequences of the nucleic acid, wherein each member of the pair of primers hybridizes to one hundred or more bacterial bioagents wherein the flanking sequences flank a variable nucleic acid sequence of the one hundred or more bacterial bioagents;

amplifying the variable nucleic acid sequence to produce an amplification product;

determining the molecular mass or base composition of the amplification product by mass spectrometry; and

comparing the molecular mass to calculated or measured molecular masses or base compositions of analogous amplification products of more than one known bacterial bioagents, thereby identifying the bacterial bioagent.

11. The method of claim 10 wherein the bacterial bioagent is a member of the genus Acinetobacter, Aeromonas, Bacillus, Bacteriodes, Bartonella, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chiamydia, Chiamydophila, Clostridium, Coxiella, Enterococcus, Escherichia, Francisella, Fusobacterium, Haemophilus, Helicobacter, Kiebsiella, Legionella, Leptospira, Listeria, Moraxella, Mycobacterium, Mycoplasma, Neisseria, Proteus, Pseudomonas, Rhodobacter, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptobacillus, Streptomyces, Treponema, Ureaplasma, Vibrio, or Yersinia.

12. The method of claim 10 wherein the amplifying step comprises the polymerase chain reaction.

13. The method of claim 10 wherein the nucleic acid encodes ribosomal RINA or a protein involved in translation, replication, recombination and repair, transcription, nucleotide metabolism, amino acid metabolism, lipid metabolism, energy generation, uptake or secretion.

14. The method of claim 10 wherein the mass spectrometry is Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) or time of flight mass spectrometry (TOF-MS).

15. The method of claim 10 wherein the molecular masses of the more than one known bacterial bioagents are contained in a database of molecular masses.

16. The method of claim 10 wherein the base compositions of the more than one known bacterial bioagents are contained in a database of base compositions.

17. The method of claim 10 wherein the variable nucleic acid sequence exhibits no greater than about 5% identity among bacterial bioagents.

18. The method of claim 10 wherein the sequences to which the primers hybridize are separated by between about 60–

100 nucleotides.

19. The method of claim 10 wherein the flanking sequences are between about 80 and 100% identical among bacterial bioagents.

20. The method of claim 10 wherein the flanking sequences are greater than about 95% identical among bacterial bioagents.

29. The method of claim 13 wherein the 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.