Fingerprinting bacterial strains using repetitive DNA sequence amplification

• US 5,691,136 A
• Filed: 08/24/1993
• Issued: 11/25/1997
• Est. Priority Date: 10/23/1991
• Status: Expired due to Term

• Current Assignee: Baylor College of Medicine (Baylor College)
• Original Assignee: Baylor College of Medicine (Baylor College)
• Inventors: Versalovic, James, Koeuth, Thearith, Lupski, James R.
• Primary Examiner(s): Campbell, Eggerton A.
• Application Number:
  US08/111,077
• Time in Patent Office: 1,554 Days
• Field of Search: 435/6, 435/91.2, 435/885, 935/77, 935/78
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Oligonucleotide primers and methods for identifying strains of bacteria by genomic fingerprinting are described. The methods are applicable to a variety of samples. The testing procedure includes amplifying the bacterial DNA in the sample to be tested by adding a pair of outwardly-directed primers to the sample. The primers are capable of hybridizing to repetitive DNA sequences in the bacterial DNA and extending outwardly from one hybridizable repetitive sequence to another hybridizable repetitive sequence. After amplification the extension products are separated by size and the specific strain of bacteria is determined by measuring the pattern of sized extension products. The procedure to identify strains of bacteria by fingerprinting has a variety of uses including: identifying bacteria in infections, agriculture and horticulture plots, bioremediation, food monitoring, production monitoring and quality assurance and quality control.

Claims

1. A method for identifying a species of bacteria in a sample, comprising the steps of:

amplifying DNA between interspersed, non-coding BOX repetitive sequences in a sample containing said bacteria by

adding an outwardly-directed BOX primer pair to said sample,

hybridizing primers of said primer pair to the interspersed, non-coding BOX repetitive DNA sequences in the bacterial DNA and

extending said primers outwardly from one hybridizable interspersed, non-coding, BOX repetitive sequence to another hybridizable interspersed, non-coding BOX repetitive sequence;

separating the extension products generated in the amplification step by size; and

determining the specific species of bacteria by measuring the pattern of sized extension products.

2. The method of claim 1, wherein the primers are between about 10 to 29 nucleotides in length.

3. The method of claim 1 wherein the primers are 15 to 25 nucleotides in length.

4. The method of claim 1 wherein the pair of primers is selected from the group consisting of SEQ. ID. NOs. 61, 62 and 63.

5. The method of claim 1, wherein the DNA is extracted from said bacteria prior to adding the primers.

6. The method of claim 1, wherein the separating step includes gel electrophoresis of the extension products.

7. The method of claim 1, wherein the separating step includes gel electrophoresis and ethidium bromide staining of the extension products.

8. The method of claim 1 wherein the primers are labelled and the determining step includes measuring the pattern of labelling.

9. The method of claim 1 wherein the separating step includes chromatography of the extension products.

10. The method of claim 1, wherein the primers are labelled.

11. The method of claim 1, wherein the primers are labelled with a fluorescer.

12. The method of claim 1, wherein the sample contains a plurality of species of bacteria and wherein each specific species of bacteria is distinguished by comparing the size pattern of extension products to a library of known fingerprints to determine the identity of each specific species of bacteria in the sample.

28. The method of claim 1, wherein the sample contains a plurality of species of bacteria and wherein each specific species of bacteria is isolated prior to the amplification step.

30. The method of claims 12 or 28, wherein said sample is selected from the group consisting of blood, urine, spinal fluid, tissue, vaginal swab, stool, amniotic fluid, and buccal mouthwash.

31. The method of claim 30, wherein said sample is from a human subject.

32. The method of claim 30, wherein said sample is from an animal subject.

33. The method of claim 12 or 28, wherein said sample is an agriculture sample.

34. The method of claims 12 or 28, wherein said sample is food.

35. The method of claims 12 or 28, where in said sample is an environmental sample.

37. The method of claim 1 for diagnosing bacterial disease wherein said sample is collected from a subject suspected of having a bacterial disease.

38. The method of claim 37, wherein the subject is a human.

39. The method of claim 37, wherein the subject is an animal.

40. The method of claim 37, wherein the subject is a plant.

41. The method of claim 1 for monitoring bacterial contamination in an environment wherein the sample is collected from an environmental source suspected of being contaminated.

42. The method of claim 41, wherein the environmental source is a liquid.

43. The method of claim 41, wherein the environmental source is sludge.

44. The method of claim 41, wherein the environmental source is sewage.

45. The method of claim 41, wherein the environmental source is a treatment plant.

46. The method of claim 41, wherein the environmental source is soil.

47. The method of claim 1 for monitoring bacterial contamination of food wherein the sample is collected from food suspected of being contaminated.

48. The method of claim 47, wherein the food is infant formula.

49. The method of claim 47, wherein the food is sea food.

50. The method of claim 47, wherein the food is fresh produce.

51. The method of claim 47, wherein the food is processed food.

52. The method of claim 1 for monitoring a bacterial population at a bioremediation site wherein the sample is collected from said site.

53. The method of claim 52, wherein the sample is soil.

54. The method of claim 52, wherein the sample is liquid.

55. The method of claim 52, wherein the sample is sludge.

56. The method of claim 52, wherein the sample is from the bacteria which is to be added to the site.

57. The method of claim 1 for monitoring a horticulture sample wherein the sample is collected from a horticulture source to be tested.

58. The method of claim 1 for monitoring an agriculture sample wherein the sample is collected from an agriculture source to be tested.

59. The method of claim 1 for monitoring bacterial additions to an agricultural environment wherein the sample is collected from an agriculture source to be tested.

60. The method of claim 59, wherein the sample is a liquid.

61. The method of claim 59, wherein the sample is soil.

62. The method of claim 59, wherein the sample is from a plant.

63. The method of claim 59, wherein the sample is from an animal.

64. The method of claim 1 for monitoring manufacturing processes for bacteria wherein the sample is collected from the process to be tested.

65. The method of claim 64, wherein the sample is selected from the group consisting of drug manufacturing processes, fermentation processes, microorganism-aided synthesis processes, chemical manufacturing process and food manufacturing processes.

66. The method of claim 1 for quality assurance/quality control of laboratory tests involving microbiological assays wherein the sample is collected from the bacterial stock to be tested.

67. The method of claim 1 for tracing outbreaks of bacterial infections, wherein the sample is collected from an organism to be tested.

71. The method of claim 1, wherein the blood sample is tested for bacterial contamination and the sample is stored blood or blood used for transfusions.

72. The method of claim 71, wherein the sample is tested for the specific bacterial species Yersinia enterocolitica.

13. A method for differentiating a strain in the same species of bacteria, comprising the steps of:

amplifying DNA between interspersed, non-coding BOX repetitive sequences in a sample containing said bacteria by adding a pair of outwardly-directed BOX primers to said sample, said primers hybridizing to BOX repetitive DNA sequences in the bacterial DNA and extending outwardly from one hybridizable BOX repetitive sequence to another hybridizable BOX repetitive sequence;

separating the extension products generated in the amplification step by size; and

determining the specific strain of bacteria by measuring the pattern of sized extension products.

14. The method of claim 13, wherein the pair of primers is selected from the group consisting of SEQ. ID. NOS. 61, 62 and 63.

15. The method of claim 13, wherein the primers are between about 10 to 29 nucleotides.

16. The method of claim 13, wherein the primers are 15 to 25 nucleotides.

17. The method of claim 13, wherein the DNA is extracted from the bacteria prior to adding the primers.

18. The method of claim 13, wherein the separating step includes gel electrophoresis of the extension products.

19. The method of claim 18, wherein the extension products are stained with ethidium bromide.

20. The method of claim 13, wherein the primers are labelled and the determining step includes measuring the pattern of labelling.

21. The method of claim 13, wherein the separation step includes chromatography of the extension products.

22. The method of claim 21, wherein the primers are labelled.

23. The method of claim 21, wherein the label is a fluorescer.

24. The method of claim 13, wherein the sample contains a plurality of strains of bacteria, and wherein each specific strain of bacteria is distinguished by comparing the size pattern of extension products to a library of known fingerprints to determine the identity of each specific strain of bacteria in the sample.

25. The method of claim 13, for identifying a strain of the same species of bacteria, wherein primers of the primer pair are the same and the primers hybridize to the interspersed, non-coding BOX repetitive sequence in either strand of the DNA.

29. The method of claim 13, wherein the sample contains a plurality of strains of bacteria and wherein each specific strain of bacteria is isolated prior to the amplification step.

36. The method of claims 13 or 28, wherein said sample is a horticulture sample.

74. The method of claim 24 or 29 wherein the sample is selected from the group consisting of blood, urine, spinal fluid, tissue, vaginal swab, stool, amniotic fluid, and buccal mouthwash.

75. The method of claim 74, wherein the sample is from a human subject.

76. The method of claim 74, wherein the test sample is from an animal subject.

77. The method of claims 24 or 29, wherein the test sample is an agriculture sample.

78. The method of claims 24 or 29, wherein the test sample is food.

79. The method of claims 24 or 29, where in the test sample is an environmental sample.

80. The method of claims 24 or 29, wherein the test sample is a horticulture sample.

81. The method of claim 74 for diagnosing bacterial disease wherein the sample is collected from a subject suspected of having a bacterial disease.

82. The method of claim 81, wherein the subject is a human.

83. The method of claim 81, wherein the subject is an animal.

84. The method of claim 81, wherein the subject is a plant.

85. The method of claim 13 for monitoring bacterial contamination in an environment wherein the sample is collected from an environmental source suspected of being contaminated.

86. The method of claim 85, wherein the environmental source is a liquid.

87. The method of claim 85, wherein the environmental source is sludge.

88. The method of claim 85, wherein the environmental source is sewage.

89. The method of claim 85, wherein the environmental source is a treatment plant.

90. The method of claim 85, wherein the environmental source is soil.

91. The method of claim 13 for monitoring bacterial contamination of food wherein the sample is collected from food suspected of being contaminated.

92. The method of claim 91, wherein the food is infant formula.

93. The method of claim 91, wherein the food is sea food.

94. The method of claim 91, wherein the food is fresh produce.

95. The method of claim 91, wherein the food is processed food.

96. The method of claim 13 for monitoring a bacterial population at a bioremediation site wherein the sample is collected from said site.

97. The method of claim 96, wherein the sample is soil.

98. The method of claim 96, wherein the sample is liquid.

99. The method of claim 96, wherein the sample is sludge.

100. The method of claim 96, wherein the sample is from the bacteria which is to be added to the site.

101. The method of claim 13 for monitoring a horticulture sample wherein the sample is collected from a horticulture source to be tested.

102. The method of claim 13 for monitoring an agriculture sample wherein the sample is collected from an agriculture source to be tested.

103. The method of claim 13 for monitoring bacterial additions to an agricultural environment wherein the sample is collected from an agriculture source to be tested.

104. The method of claim 103, wherein the sample is a liquid.

105. The method of claim 103, wherein the sample is soil.

106. The method of claim 103, wherein the sample is from a plant.

107. The method of claim 103, wherein the sample is from an animal.

108. The method of claim 13 for monitoring manufacturing processes for bacteria wherein the sample is collected from the process to be tested.

109. The method of claim 108, wherein the sample is selected from the group consisting of drug manufacturing processes, fermentation processes, microorganism-aided synthesis processes, chemical manufacturing process and food manufacturing processes.

110. The method of claim 13 for quality assurance/quality control of laboratory tests involving microbiological assays wherein the sample is collected from the bacterial stock to be tested.

111. The method of claim 13 for tracing outbreaks of bacterial infections, wherein the sample is collected from an organism to be tested.

112. The method of claim 13, wherein a blood sample is tested for bacterial contamination and the sample is taken from stored blood or blood used for transfusions.

113. The method of claim 112, wherein the sample is tested for the specific bacterial species Yersinia enterocolitica.

26. A kit for determining the identity of a strain of bacteria, comprising a container having outwardly-directed PCR primer pairs to interspersed, non-coding, BOX repetitive sequences in said bacteria.

27. The kit of claim 26, wherein the PCR primer pairs are selected from the group consisting of SEQ. ID. NOS. 61, 62, 63 and any combination thereof.

68. A kit for determining the identity of a species of bacteria, comprising a container having outwardly-directed PCR primer pairs to interspersed, non-coding, BOX repetitive sequences in said bacteria.

69. The kit of claim 68, wherein the PCR primer pairs are selected from the group consisting of SEQ. ID. NOS. 61, 62, 63 and any combination thereof.

70. As a composition of matter the DNA oligonucleotide selected from the group consisting of SEQ. ID. NOS. 61, 62 and 63.

73. A method for identifying a species of bacteria, comprising the steps of:

amplifying DNA between interspersed, non-coding repetitive sequences in a sample containing said bacteria by adding to said sample a BOX primer selected from the group consisting of SEQ. ID. NOs. 61, 62 and 63, wherein said primer hybridizes to the interspersed, non-coding repetitive sequence in either of the complementary strands of the DNA and wherein the hybridized primer extends from one interspersed, non-coding repetitive sequence across non-repetitive DNA to another interspersed, non-coding repetitive sequence and wherein said extension product hybridizes to the primer for generations of further extension products;

separating the extension products by size; and

determining the specific strain of bacteria by measuring the pattern of sized extension products.