Method for rapid detection and identification of bioagents

• US 20030124556A1
• Filed: 05/24/2002
• Published: 07/03/2003
• Est. Priority Date: 03/02/2001
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: David J Ecker, John Mcneil, Rangarajan Sampath, Richard Griffey, Steven Hofstadler
• Inventors: Sampath, Rangarajan, McNeil, John, Ecker, David J., Hofstadler, Steven, Griffey, Richard
• Granted Patent: US 7,108,974 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 an unknown bioagent comprising:

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

(b) amplifying said variable nucleic acid sequence to produce an amplification product;

(c) determining the molecular mass of said amplification product; and

(d) comparing said molecular mass to one or more molecular masses of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies said unknown bioagent.

2. The method of claim 1, wherein said sequences to which said at least one pair of oligonucleotide primers hybridize are highly conserved.

3. The method of claim 1, wherein said amplifying step comprises polymerase chain reaction.

4. The method of claim 1, wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.

5. The method of claim 1, wherein said bioagent is a bacterium, virus, cell or spore.

6. The method of claim 1, wherein said nucleic acid is ribosomal RNA.

7. The method of claim 1, wherein said nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.

8. The method of claim 1, wherein said amplification product is ionized prior to molecular mass determination.

9. The method of claim 1, further comprising the step of isolating nucleic acid from said bioagent prior to contacting said nucleic acid with said at least one pair of oligonucleotide primers.

10. The method of claim 1, further comprising the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more molecular mass amplification product obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d).

11. The method of claim 1, wherein said one or more molecular masses are contained in a database of molecular masses.

12. The method of claim 1, wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.

13. The method of claim 1, wherein said molecular mass is determined by mass spectrometry.

14. The method of claim 11, wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF and triple quadrupole.

15. The method of claim 1, further comprising performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.

16. The method of claim 1, wherein said oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within said primer, wherein said base analog binds with increased affinity to its complement compared to the native base.

17. The method of claim 16, wherein said primer comprises a universal base at position 3 of each triplet within said primer.

18. The method of claim 16, wherein said base analog is selected from the group consisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.

19. The method of claim 16, wherein said universal base is selected from the group consisting of inosine, guanidine uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, and 1-(2-deoxy-β

-D-ribofuranosyl)-imidazole-4-carboxamide.

20. A method of identifying an unknown bioagent comprising:

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

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

determining the base composition of said amplification product; and

comparing said base composition to one or more base compositions of amplification products obtained by performing steps (a)-(c) on a plurality of known organisms, wherein a match identifies said unknown bioagent.

21. The method of claim 20, wherein said sequences to which said at least one pair of oligonucleotide primers hybridize are highly conserved.

22. The method of claim 20, wherein said amplifying step comprises polymerase chain reaction.

23. The method of claim 20, wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.

24. The method of claim 20, wherein said bioagent is a bacterium, virus, cell or spore.

25. The method of claim 20, wherein said nucleic acid is ribosomal RNA.

26. The method of claim 20, wherein said nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.

27. The method of claim 20, wherein said amplification product is ionized prior to base composition determination.

28. The method of claim 20, further comprising the step of isolating nucleic acid from said bioagent prior to contacting said nucleic acid with said at least one pair of oligonucleotide primers.

29. The method of claim 20, further comprising the step of performing steps (a)-(d) using a different oligonucleotide primer pair and comparing the results to one or more base compositions of amplification product obtained by performing steps (a)-(c) on a different plurality of known organisms from those in step (d).

30. The method of claim 20, wherein said one or more base composition signatures are contained in a database of base composition signatures.

31. The method of claim 20, wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.

32. The method of claim 20, wherein said base composition signature is determined by mass spectrometry.

33. The method of claim 32, wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), q-TOF and triple quadrupole.

34. The method of claim 20, further comprising performing step (b) in the presence of an analog of adenine, thymidine, guanosine or cytidine having a different molecular weight than adenosine, thymidine, guanosine or cytidine.

35. The method of claim 20, wherein said oligonucleotide primer comprises a base analog at positions 1 and 2 of each triplet within said primer, wherein said base analog binds with increased affinity to its complement compared to the native base.

36. The method of claim 35, wherein said primer comprises a universal base at position 3 of each triplet within said primer.

37. The method of claim 35, wherein said base analog is selected from the group consisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.

38. The method of claim 36, wherein said universal base is selected from the group consisting of inosine, guanidine uridine, 5-nitroindole, 3-nitropyrrole, dP, dK, and 1-(2-deoxy-β

-D-ribofuranosyl)-imidazole-4-carboxamide.

39. A method for detecting a single nucleotide polymorphism in an individual, comprising the steps of:

isolating nucleic acid from said individual;

contacting said nucleic acid with oligonucleotide primers which hybridize to regions of said nucleic acid which flank a region comprising said potential polymorphism;

amplifying said region to produce an amplification product;

determining the molecular mass of said amplification product;

comparing said molecular mass to the molecular mass of said region in an individual known to have said polymorphism, wherein if said molecular masses are the same then said individual has said polymorphism.

40. The method of claim 39, wherein said polymorphism is associated with a disease.

41. The method of claim 39, wherein said polymorphism is a blood group antigen.

42. The method of claim 39, wherein said amplification step is the polymerase chain reaction.

43. The method of claim 39, wherein said amplification step is ligase chain reaction or strand displacement amplification.

44. The method of claim 39, wherein said amplification product is ionized prior to mass determination.

45. The method of claim 39, wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.

46. The method of claim 39, wherein said primers hybridize to conserved sequences.

47. The method of claim 39, wherein said molecular mass is determined by mass spectrometry.

48. The method of claim 47, wherein said mass spectrometry is selected from the group consisting of Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS), ion trap, quadrupole, magnetic sector, time of flight (TOF), Q-TOF and triple quadrupole.