Methods for rapid detection and identification of bioagents in containers

• US 20040121314A1
• Filed: 12/18/2002
• Published: 06/24/2004
• Est. Priority Date: 12/06/2002
• Status: Abandoned Application

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Sampath, Rangarajan, Griffey, Richard H., McNeil, John, Ecker, David J., Hofstadler, Steven
• Application Number:
  US10/326,642
• Publication Number:
  US 20040121314A1
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer

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 in a container comprising:

a) contacting nucleic acid from said bioagent in said container 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 in said container.

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, parasite, fungi, 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 products 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. The method of claim 1 wherein said container is a package, box, envelope, mail tube, or railroad box car.

21. A method of identifying an unknown bioagent in a container comprising:

a) contacting nucleic acid from said bioagent in a container 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;

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

c) determining the base composition of said amplification product; and

d) 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 in said container.

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

23. The method of claim 21 wherein said amplifying step comprises polymerase chain reaction.

24. The method of claim 21 wherein said amplifying step comprises ligase chain reaction or strand displacement amplification.

25. The method of claim 21 wherein said bioagent is a bacterium, virus, fungi, parasite, cell or spore.

26. The method of claim 21 wherein said nucleic acid is ribosomal RNA.

27. The method of claim 21 wherein said nucleic acid encodes RNase P or an RNA-dependent RNA polymerase.

28. The method of claim 21 wherein said amplification product is ionized prior to base composition determination.

29. The method of claim 21 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.

30. The method of claim 21 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).

31. The method of claim 21 wherein said one or more base composition signatures are contained in a database of base composition signatures.

32. The method of claim 21 wherein said amplification product is ionized by electrospray ionization, matrix-assisted laser desorption or fast atom bombardment.

33. The method of claim 21 wherein said base composition signature is determined by mass spectrometry.

34. The method of claim 33 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.

35. The method of claim 21 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.

36. The method of claim 21 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.

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

38. The method of claim 36 wherein said base analog is selected from the group consisting of 2,6-diaminopurine, propyne T, propyne G, phenoxazines and G-clamp.

39. 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.

40. The method of claim 21 wherein said container is a package, box, envelope, mail tube, or railroad box car.

41. A method of determining the absence of a bioagent in a container sample comprising:

a) contacting said container sample suspected of containing nucleic acid encoding said bioagent with at least one pair of oligonucleotide primers which are capable of hybridizing to sequences of said nucleic acid, wherein said sequences flank a variable nucleic acid sequence of said bioagent;

b) treating said variable nucleic acid sequence under amplification conditions capable of producing an amplification product of said variable nucleic acid sequence;

c) performing spectroscopy to determine the molecular mass or base composition of all amplification products; and

d) comparing said molecular mass to one or more molecular masses of amplification products or 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 the lack of a match indicates that said bioagent is absent from said container sample.

42. The method of claim 41 further comprising determining the presence of a positive control.

43. The method of claim 42 wherein said positive control is a known bioagent or residual primer signal.