Methods for rapid identification of pathogens in humans and animals

• US 8,071,309 B2
• Filed: 10/30/2007
• Issued: 12/06/2011
• Est. Priority Date: 12/06/2002
• 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, Crooke, Stanley T., Blyn, Lawrence, Ranken, Ray, Hall, Thomas A.
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): THOMAS, DAVID C
• Application Number:
  US11/930,002
• Publication Number:
  US 20090148829A1
• Time in Patent Office: 1,498 Days
• Field of Search: None
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 1/70 involving virus or bacteriophage immunoassay for viruses G01N33/56983
• CPC Class Codes: C12Q 1/701 Specific hybridization probes
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays
• CPC Class Codes: G01N 1/30 Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• CPC Class Codes: G01N 21/64 Fluorescence; Phosphorescence
• CPC Class Codes: G01N 23/00 Investigating or analysing materials by the use of wave or particle radiation, e.g. X-rays or neutrons, not covered by groups G01N3/00 – G01N17/00, G01N21/00 or G01N22/00
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/20 Polymerase chain reaction [PCR]; Primer or probe design; Probe optimisation

Abstract

The present invention provides methods of: identifying pathogens in biological samples from humans and animals, resolving a plurality of etiologic agents present in samples obtained from humans and animals, determining detailed genetic information about such pathogens or etiologic agents, and rapid detection and identification of bioagents from environmental, clinical or other samples.

Claims

1. A method of differentiating two or more bioagents, comprising:

a) contacting nucleic acid from two or more bioagents with two or more oligonucleotide primers that hybridize to sequence regions of said nucleic acid from said two or more bioagents that are conserved among different bioagents, wherein said conserved sequence regions flank a variable sequence region to produce two or more amplification products;

b) determining base compositions of said two or more amplification products wherein said base compositions of said two or more amplification products comprise identification of the number, but not the nucleic acid sequence order, of A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof in said two or more amplification products; and

c) differentiating said two or more bioagents by comparing said determined base compositions to a database comprising a plurality of calculated or measured base compositions of amplification products from a plurality of different bioagents obtained by amplification of at least one nucleic acid gene sequence from said plurality of different bioagents using said two or more oligonucleotide primers.

2. The method of claim 1, wherein at least one of said two or more bioagents is selected from the group of bioagents consisting of a viral bioagent, a bacterial bioagent, a fungal bioagent, a protozoal bioagent, a parasitic bioagent, a mammalian bioagent, and a human bioagent.

3. The method of claim 1, wherein at least one of said two or more bioagents is selected from the group of bioagents consisting of an evolving bioagent, a mutating bioagent, a recombinant bioagent, and an engineered bioagent.

4. The method of claim 1, wherein at least one of said two or more bioagents is not previously known to exist.

5. The method of claim 1, wherein said two or more bioagents are differentiated in a sample.

6. The method of claim 5, wherein said sample is selected from the group consisting of a body fluid sample, an environmental sample, a culture media sample, a manufacturing sample, and a forensic sample.

7. The method of claim 5, wherein said sample comprises two or more bioagents of different genus, and wherein said nucleic acid from said sample is contacted with two or more oligonucleotide primers configured to differentiate two or more bioagents of different genus.

8. The method of claim 5, wherein said sample comprises two or more bioagents of different species, and wherein said nucleic acid from said sample is contacted with two or more oligonucleotide primers configured to differentiate two or more bioagents of different species.

9. The method of claim 1, further comprising purifying said nucleic acid prior to production of said amplification product.

10. The method of claim 1, further comprising modifying said nucleic acid prior to production of said two or more amplification products.

11. The method of claim 1, wherein said nucleic acid is selected from the group consisting of genomic DNA, RNA, DNA that is complementary to RNA, DNA that is synthesized from RNA, double-stranded DNA, single stranded DNA, DNA that is the product of amplification, DNA that is fragmented, nuclear DNA, mitochondrial DNA, cytoplasmic DNA and extracellular DNA.

12. The method of claim 1, wherein said two or more oligonucleotide primers are about 15-35 nucleobases in length, and wherein said two or more oligonucleotide primers comprise at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% sequence identify with said conserved sequence regions.

13. The method of claim 1, wherein at least one of said two or more amplification products is 45 to 200 nucleobases in length.

14. The method of claim 1, wherein a non-templated T residue on the 5′

-end of at least one of said two or more oligionucleotide primers is removed.

15. The method of claim 1, wherein at least one of said two or more oligonucleotide primers comprises a non-templated T residue on the 5′

-end.

16. The method of claim 1, wherein at least one of said two or more oligonucleotide primers comprises at least one molecular mass modifying tag.

17. The method of claim 1, wherein at least one of said two or more oligonucleotide primers comprises at least one modified nucleobase.

18. The method of claim 17, wherein said modified nucleobase is 5-propynyluracil or 5-propynylcytosine.

19. The method of claim 17, wherein said modified nucleobase is a mass modified nucleobase.

20. The method of claim 19, wherein said mass modified nucleobase is 5-Iodo-C.

21. The method of claim 17, wherein said modified nucleobase is a universal nucleobase.

22. The method of claim 21, wherein said universal nucleobase is inosine.

23. The method of claim 1, wherein said two or more bioagents are differentiated at the genus, species, sub-species, strain, sub-type, or nucleotide polymorphism levels.

24. The method of claim 1, wherein said two or more oligonucleotide primers comprise multiple oligonucleotide primer sets configured for differentiation of diverse bioagents.

25. The method of claim 1, wherein said conserved sequence regions are within a gene.

26. The method of claim 1, wherein said conserved sequence regions are within a coding region of a gene.

27. The method of claim 1, wherein said conserved sequence regions are within a regulatory region of a gene.

28. The method of claim 1, wherein said variable sequence region is within a gene.

29. The method of claim 1, wherein said variable sequence region is within a coding region of a gene.

30. The method of claim 1, wherein said variable sequence region is within a regulatory region of a gene.

31. The method of claim 1, wherein said conserved sequence regions are about 10 to 100 nucleobases in length.

32. The method of claim 1, wherein said variable sequence region is about 10 to 200 nucleobases in length.

33. The method of claim 1, wherein said amplification product is produced by polymerase chain reaction.

34. The method of claim 1, further comprising purifying said amplification product.

35. The method of claim 1, wherein said base composition is determined by mass spectrometry.

36. The method of claim 35, wherein said mass spectrometry is ESI mass spectrometry.

37. The method of claim 1, wherein said comparing comprises identifying a match between said determined two or more base compositions and at least one entry within said database of base compositions from a plurality of different bioagents.

38. The method of claim 1, wherein said differentiating said two or more bioagents requires two or more oligonucleotide primer pairs.

39. The method of claim 1, wherein said database of base compositions from a plurality of different bioagents comprises base compositions of genus specific amplification products, family specific amplification products, species specific amplification products, strain specific amplification products, sub-type specific amplification products, or nucleotide polymorphism specific amplification products produced with said two or more oligonucleotide primers, wherein one or more matches between said determined base composition of said amplification product and one or more entries in said database identifies said two or more bioagents, classifies a major classification of said two or more bioagents, or differentiates between subgroups of known and unknown said two or more bioagents.

40. The method of claim 1, wherein said database of base compositions comprises base composition information for at least 3 different bioagents.

41. The method of claim 1, wherein said database of base compositions comprises base composition information for at least 4 different bioagents.

42. The method of claim 1, wherein said database of base compositions comprises base composition information for at least 8 different bioagents.

43. The method of claim 1, wherein said database of base compositions comprises base composition information for at least 19 different bioagents.

44. The method of claim 1, wherein said database of base compositions comprises base composition information for at least 30 different bioagents.

45. The method of claim 1, wherein said database of base compositions comprises at least 12 unique base compositions.

46. The method of claim 1, wherein said database of base compositions comprises at least 40 unique base compositions.

47. The method of claim 1, wherein said database of base compositions comprises base composition information for a bioagent from two or more genuses selected from the group consisting of Acinetobacter, Aeromonas, Bacillus, Bacteroides, 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.

48. The method of claim 1, wherein said database of base compositions comprises base composition information for a bioagent from each of the genuses of Acinetobacter, Aeromonas, Bacillus, Bacteroides, 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.

49. The method of claim 1, wherein said database of base compositions comprises base composition information for a bioagent from two or more orders or families selected from the group consisting of Smallpox virus, Arenavirus, Bunyaviruses, Mononegavirales, Picornaviruses, Astroviruses, Calciviruses, Nidovirales, Flaviviruses, and Togaviruses.

50. The method of claim 1, wherein said database of base compositions comprises base composition information for a bioagent from each of the orders or families of Smallpox virus, Arenavirus, Bunyaviruses, Mononegavirales, Picornaviruses, Astroviruses, Calciviruses, Nidovirales, Flaviviruses, and Togaviruses.

51. The method of claim 1, wherein said base compositions in said database are associated with bioagent identity.

52. The method of claim 1, wherein said base compositions in said database are associated with bioagent geographic origin.

53. The method of claim 1, wherein said comparing step is performed by a computer.

54. The method of claim 53, wherein said computer identifies a match between said determined base composition of said amplification product and one or more entries in database of base compositions from a plurality of different bioagents with a probability algorithm.

55. The method of claim 1, wherein said database is stored on a computer.

56. The method of claim 55, wherein said computer is a local computer.

57. The method of claim 55, wherein said computer is a remote computer.

58. The method of claim 1, wherein said differentiating said two or more bioagents comprises interrogation of said database with two or more different base compositions associated with said two or more bioagents.

59. The method of claim 1, wherein said database of base compositions comprises at least 10 base compositions.

60. The method of claim 1, wherein said database of base compositions comprises at least 20 base compositions.

61. The method of claim 1, wherein said database of base compositions comprises at least 30 base compositions.

62. The method of claim 1, wherein said database of base compositions comprises at least 40 base compositions.

63. The method of claim 1, wherein said database of base compositions comprises at least 50 base compositions.

64. The method of claim 1, wherein said database of base compositions comprises at least 60 base compositions.

65. The method of claim 1, wherein said database of base compositions comprises at least 70 base compositions.

66. The method of claim 1, wherein said database of base compositions comprises at least 80 base compositions.

67. The method of claim 1, wherein said database of base compositions comprises at least 90 base compositions.

68. The method of claim 1, wherein said database of base compositions comprises at least 100 base compositions.

69. The method of claim 1, wherein said database of base compositions comprises at least 500 base compositions.

70. The method of claim 1, wherein said database of base compositions comprises at least 1000 base compositions.

71. The method of claim 1, wherein said two or more bioagents comprises at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 500 or at least 1000 bioagents.

72. The method of claim 1, wherein said two or more bioagents are differentiated by kingdom, phylum, class, order, family, genus, species, sub-species, strain, sub-type, or nucleotide polymorphism.