Method for rapid detection and identification of bioagents

• US 8,265,878 B2
• Filed: 10/30/2007
• Issued: 09/11/2012
• Est. Priority Date: 03/02/2001
• Status: Expired due to Fees

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Ecker, David J., Griffey, Richard H., Sampath, Rangarajan, Hofstadler, Steven A., McNeil, John
• Primary Examiner(s): Clow, Lori A.
• Application Number:
  US11/930,017
• Publication Number:
  US 20090148837A1
• Time in Patent Office: 1,778 Days
• Field of Search: None
• US Class Current: 702/19
• 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 system, comprising:

a) a nucleic acid amplification component;

b) a base composition determination component comprising a processor wherein said base composition identifies the number of, but not the nucleic acid gene sequence order of, A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof; and

c) a base composition identification component comprising a processor and a database of base compositions from a plurality of bioagents wherein said base compositions identify the number of, but not the nucleic acid gene sequence order of, A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof;

said database comprising a plurality of calculated or measured base compositions of amplification products of bioagents obtained by amplification of at least one target sequence of a bioagent nucleic acid gene sequence using a primer pair that hybridizes to the at least one target sequence region of a plurality of bioagents said target sequence region comprising two conserved regions that are hybridizable with the primer pair and that flank a variable region that uniquely varies between a plurality of bioagents.

2. The system of claim 1, further comprising a nucleic acid purification component.

3. The system of claim 2, wherein said nucleic acid purification component comprises one or more buffer manipulations, one or more salt manipulations, one or more thermal manipulations, one or more pH manipulations, one or more mechanical manipulations, one or more centrifugation manipulations, or one or more magnetic manipulations.

4. The system of claim 1, wherein said nucleic acid amplification component comprises a thermocycler.

5. The system of claim 1, wherein said nucleic acid amplification component comprises one or more salts, one or more buffers, one or more purified oligonucleotide primers, one or more dNTPs, or one or more enzymes.

6. The system of claim 1, wherein said base composition identification component comprises a mass spectrometer.

7. The system of claim 6, wherein said mass spectrometer is an ESI mass spectrometer.

8. The system of claim 1, further comprising a computer program on a computer readable medium configured to direct at least one said processor to coordinate the operation of said nucleic acid component, said base composition determination component, and said base composition identification component.

9. The system of claim 1, wherein at least one said processor is configured to process mass spectrometry data to base composition data.

10. The system of claim 1, wherein said base composition identification component processor is configured to process base composition data to identify a bioagent.

11. The system of claim 1, wherein said database of base compositions comprises at least 10 base compositions.

12. The system of claim 1, wherein said database of base compositions comprises at least 20 base compositions.

13. The system of claim 1, wherein said database of base compositions comprises at least 30 base compositions.

14. The system of claim 1, wherein said database of base compositions comprises at least 40 base compositions.

15. The system of claim 1, wherein said database of base compositions comprises at least 50 base compositions.

16. The system of claim 1, wherein said database of base compositions comprises at least 60 base compositions.

17. The system of claim 1, wherein said database of base compositions comprises at least 70 base compositions.

18. The system of claim 1, wherein said database of base compositions comprises at least 80 base compositions.

19. The system of claim 1, wherein said database of base compositions comprises at least 90 base compositions.

20. The system of claim 1, wherein said database of base compositions comprises at least 100 base compositions.

21. The system of claim 1, wherein said database of base compositions comprises at least 500 base compositions.

22. The system of claim 1, wherein said database of base compositions comprises at least 1000 base compositions.

23. The system of claim 1, wherein said plurality of bioagents comprises bioagents that differ by genus, species, sub-species, strain, sub-type or nucleotide polymorphism.

24. The system of claim 1, wherein said plurality of bioagents comprises one or more viral bioagents, one or more bacterial bioagents, one or more fungal bioagents, one or more protozoal bioagents, one or more parasitic bioagents, one or more mammalian bioagents, or one or more human bioagents.