Method for Rapid Detection and Identification of Bioagents

• US 20090148836A1
• Filed: 10/30/2007
• Published: 06/11/2009
• Est. Priority Date: 03/02/2001
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Hofstadler, Steven A., Sampath, Rangarajan, Griffey, Richard H., McNeil, John, Ecker, David J.
• Granted Patent: US 8,017,743 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

1-34. -34. (canceled)

35. A method of identifying an unknown bioagent, comprising:

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

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

c) determining the identity of said unknown bioagent by comparing the determined base composition to a database of base compositions from a plurality of different known bioagents.

36. The method of claim 35, wherein said unknown bioagent 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.

37. The method of claim 35, wherein said unknown bioagent is selected from the group of bioagents consisting of an evolving bioagent, a mutating bioagent, a recombinant bioagent, and an engineered bioagent.

38. The method of claim 35, wherein said unknown bioagent is not previously known to exist.

39. The method of claim 35, wherein said unknown bioagent is identified in a sample.

40. The method of claim 39, 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.

41. The method of claim 39, wherein said sample comprises at least two bioagents of different genus, and wherein said nucleic acid from said sample is contacted with two or more oligonucleotide primers configured to identify two or more bioagents of different genus.

42. The method of claim 39, wherein said sample comprises at least two bioagents of different species, and wherein said nucleic acid from said sample is contacted with two or more oligonucleotide primers configured to identify two or more bioagents of different species.

43. The method of claim 35, further comprising purifying said nucleic acid prior to production of said amplification product.

44. The method of claim 35, further comprising modifying said nucleic acid prior to production of said amplification product.

45. The method of claim 35, 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.

46. The method of claim 35, 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.

47. The method of claim 35, wherein said amplification product is 45 to 200 nucleobases in length.

48. The method of claim 35, wherein a non-templated T residue on the 5′

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

49. The method of claim 35, wherein at least one of said two or more oligonucleotide primers comprises a non-templated T residue on the 5′

-end.

50. The method of claim 35, wherein at least one of said two or more oligonucleotide primers comprises at least one molecular mass modifying tag.

51. The method of claim 35, wherein at least one of said two or more oligonucleotide primers comprises at least one modified nucleobase.

52. The method of claim 51, wherein said modified nucleobase is 5-propynyluracil or 5-propynylcytosine.

53. The method of claim 51, wherein said modified nucleobase is a mass modified nucleobase.

54. The method of claim 53, wherein said mass modified nucleobase is 5-Iodo-C.

55. The method of claim 51, wherein said modified nucleobase is a universal nucleobase.

56. The method of claim 55, wherein said universal nucleobase is inosine.

57. The method of claim 35, wherein said unknown bioagent is identified at the genus, species, sub-species, strain, sub-type, or nucleotide polymorphism levels.

58. The method of claim 35, wherein said two or more oligonucleotide primers comprise multiple oligonucleotide primer sets configured for identification of diverse bioagents.

59. The method of claim 35, wherein said conserved sequence regions are within a gene.

60. The method of claim 35, wherein said conserved sequence regions are within a coding region of a gene.

61. The method of claim 35, wherein said conserved sequence regions are within a regulatory region of a gene.

62. The method of claim 35, wherein said variable sequence region is within a gene.

63. The method of claim 35, wherein said variable sequence region is within a coding region of a gene.

64. The method of claim 35, wherein said variable sequence region is within a regulatory region of a gene.

65. The method of claim 35, wherein said conserved sequence regions are about 10 to 100 nucleobases in length.

66. The method of claim 35, wherein said variable sequence region is about 10 to 200 nucleobases in length.

67. The method of claim 35, wherein said amplification product is produced by polymerase chain reaction.

68. The method of claim 35, further comprising purifying said amplification product.

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

70. The method of claim 69, wherein said mass spectrometry is ESI mass spectrometry.

71. The method of claim 35, wherein said base composition of said amplification product comprises identification of the number of A residues, C residues, T residues, G residues, U residues, analogs thereof and/or mass tag residues thereof in said amplification product.

72. The method of claim 35, wherein said base composition is determined without sequencing said amplification product.

73. The method of claim 35, wherein said comparing comprises identifying a match between said determined base composition and at least one entry within said database of base compositions from a plurality of different bioagents.

74. The method of claim 35, wherein said identifying said bioagent requires two or more oligonucleotide primer pairs.

75. The method of claim 35, 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 bioagent, classifies a major classification of said bioagent, or differentiates between subgroups of known and unknown bioagents.

76. The method of claim 35, wherein said database of base compositions comprises base composition information for at least 3 different bioagents.

77. The method of claim 35, wherein said database of base compositions comprises base composition information for at least 4 different bioagents.

78. The method of claim 35, wherein said database of base compositions comprises base composition information for at least 8 different bioagents.

79. The method of claim 35, wherein said database of base compositions comprises base composition information for at least 19 different bioagents.

80. The method of claim 35, wherein said database of base compositions comprises base composition information for at least 30 different bioagents.

81. The method of claim 35, wherein said database of base compositions comprises at least 12 unique base compositions.

82. The method of claim 35, wherein said database of base compositions comprises at least 40 unique base compositions.

83. The method of claim 35, 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.

84. The method of claim 35, 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.

85. The method of claim 35, wherein said database of base compositions comprises base composition information for a bioagent from two or more orders in families selected from the group consisting of Smallpox virus, Arenavirus, Bunyaviruses, Mononegavirales, Picornaviruses, Astroviruses, Calciviruses, Nidovirales, Flaviviruses, and Togaviruses.

86. The method of claim 35, 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.

87. The method of claim 35, wherein said base compositions in said database are associated with bioagent identity.

88. The method of claim 35, wherein said base compositions in said database are associated with bioagent geographic origin.

89. The method of claim 35, wherein said comparing step is performed by a computer.

90. The method of claim 89, 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.

91. The method of claim 35, wherein said database of base compositions comprises at least 10 base compositions.

92. The method of claim 35, wherein said database of base compositions comprises at least 20 base compositions.

93. The method of claim 35, wherein said database of base compositions comprises at least 30 base compositions.

94. The method of claim 35, wherein said database of base compositions comprises at least 40 base compositions.

95. The method of claim 35, wherein said database of base compositions comprises at least 50 base compositions.

96. The method of claim 35, wherein said database of base compositions comprises at least 60 base compositions.

97. The method of claim 35, wherein said database of base compositions comprises at least 70 base compositions.

98. The method of claim 35, wherein said database of base compositions comprises at least 80 base compositions.

99. The method of claim 35, wherein said database of base compositions comprises at least 90 base compositions.

100. The method of claim 35, wherein said database of base compositions comprises at least 100 base compositions.

101. The method of claim 35, wherein said database of base compositions comprises at least 500 base compositions.

102. The method of claim 35, wherein said database of base compositions comprises at least 1000 base compositions.

103. The method of claim 35, wherein said database is stored on a computer.

104. The method of claim 103, wherein said computer is a local computer.

105. The method of claim 103, wherein said computer is a remote computer.

106. The method of claim 35, wherein said identifying said unknown bioagent comprises interrogation of said database with two or more different base compositions associated with said unknown bioagent.

107. The method of claim 35, wherein said determining the identity of said unknown bioagent comprises determining the identity of said unknown bioagent at the kingdom, phylum, class, order, family, genus, species, sub-species, strain, sub-type, or nucleotide polymorphism level.

108. A system, comprising:

a) a nucleic acid amplification component;

b) a base composition determination component; and

c) a base composition identification component comprising a database of base compositions from a plurality of bioagents and a processor that compares said base compositions from said plurality of known bioagents to a base composition of an unknown bioagent to determine the identity of said unknown bioagent.

109. The system of claim 108, further comprising a nucleic acid purification component.

110. The system of claim 109, 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.

111. The system of claim 108, wherein said nucleic acid amplification component comprises a thermocycler.

112. The system of claim 108 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.

113. The system of claim 108, wherein said base composition identification component comprises a mass spectrometer.

114. The system of claim 113, wherein said mass spectrometer is an ESI mass spectrometer.

115. The system of claim 108, wherein said base composition identification component comprises a processor.

116. The system of claim 115, further comprising a computer program on a computer readable medium configured to direct said processor to coordinate the operation of said nucleic acid component, said base composition determination component, and said base composition identification component.

117. The system of claim 115, wherein said processor is configured to process mass spectrometry data to base composition data.

118. The system of claim 115, wherein said processor is configured to process base composition data to identify a bioagent.

119. The system of claim 108, wherein said database of base compositions comprises at least 10 base compositions.

120. The system of claim 108, wherein said database of base compositions comprises at least 20 base compositions.

121. The system of claim 108, wherein said database of base compositions comprises at least 30 base compositions.

122. The system of claim 108, wherein said database of base compositions comprises at least 40 base compositions.

123. The system of claim 108, wherein said database of base compositions comprises at least 50 base compositions.

124. The system of claim 108, wherein said database of base compositions comprises at least 60 base compositions.

125. The system of claim 108, wherein said database of base compositions comprises at least 70 base compositions.

126. The system of claim 108, wherein said database of base compositions comprises at least 80 base compositions.

127. The system of claim 108, wherein said database of base compositions comprises at least 90 base compositions.

128. The system of claim 108, wherein said database of base compositions comprises at least 100 base compositions.

129. The system of claim 108, wherein said database of base compositions comprises at least 500 base compositions.

130. The system of claim 108, wherein said database of base compositions comprises at least 1000 base compositions.

131. The system of claim 108, wherein said plurality of bioagents comprises bioagents that differ by genus, species, sub-species, strain, sub-type or nucleotide polymorphism.

132. The system of claim 108, 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.