TARGETED WHOLE GENOME AMPLIFICATION METHOD FOR IDENTIFICATION OF PATHOGENS

• US 20100035232A1
• Filed: 09/14/2007
• Published: 02/11/2010
• Est. Priority Date: 09/14/2006
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: Ibis Biosciences Incorporated
• Inventors: Eshoo, Mark W., Ecker, David J.
• Granted Patent: US 9,149,473 B2
• Time in Patent Office: Days
• US Class Current: 435/5
• CPC Class Codes: A61K 31/4741 condensed with ring systems having oxygen as a ring hetero atom, e.g. tubocuraran derivatives, noscapine, bicuculline
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• 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 2563/167 Mass label
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: H01J 49/0027 Methods for using particle spectrometers
• CPC Class Codes: H01J 49/40 Time-of-flight spectrometers H01J49/36 takes precedence
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The methods disclosed herein relate to methods and compositions for amplifying nucleic acid sequences, more specifically, from nucleic acid sequences of pathogens by targeted whole genome amplification.

Claims

1. A method comprising:

amplifying at least one pathogen genome from a sample suspected of comprising at least one pathogen genome and at least one background genome using a plurality of targeted whole genome amplification primers, thereby elevating the quantity of nucleic acid representing said at least one pathogen genome relative to the quantity of nucleic acid representing said at least one background genome, wherein said plurality of targeted whole genome amplification primers is selected by;

i. identifying at least one pathogen genome;

ii. identifying at least one background genome;

iii. identifying a plurality of genome sequence segments having unique sequences within said pathogen genome sequence;

iv. determining frequency of occurrence of members of said plurality of genome sequence segments within said pathogen genome sequence and determining frequency of occurrence of said plurality of genome sequence segments within said background genome sequences;

v. calculating a selectivity ratio for said members by dividing said frequency of occurrence within said pathogen genome sequence by said frequency of occurrence of said plurality of genome sequence segments within said background genome sequences;

vi. selecting a selectivity ratio threshold value, thereby defining a first sub-set of said plurality of genome sequence segments having selectivity ratios equal to or greater than said selectivity ratio threshold value;

vii. determining the lengths of pathogen genome sequence occurring between genome sequence segments of said first sub-set;

viii. selecting a second sub-set of genome sequence segments from said first sub-set wherein members of said second sub-set have a mean separation distance of less than a selected length of nucleobases; and

ix. selecting targeted whole genome amplification primers that hybridize to members of said second sub-set of genome sequence segments such that, under whole genome amplification conditions, said at least one pathogen genome is amplified selectively over said at least one background genomes.

2. The method of claim 1 further comprising the step of producing one or more amplification products representing bioagent identifying amplicons from said amplified pathogen genome using one or more primer pairs.

3. The method of claim 2 further comprising the step of measuring molecular masses of said amplification products by mass spectrometry.

4. The method of claim 3 wherein said mass spectrometry is electrospray time-of-flight mass spectrometry.

5. The method of claim 3 further comprising the step of comparing said molecular masses with a database comprising molecular masses of bioagent identifying amplicons of pathogens produced with said primer pairs, thereby identifying said pathogen in said sample.

6. The method of claim 3 further comprising the step of calculating base compositions of said amplification products from said molecular masses.

7. The method of claim 6 further comprising the step of comparing said base compositions with a database comprising base compositions of bioagent identifying amplicons of pathogens produced with said primer pairs, thereby identifying said pathogen in said sample.

8. The method of claim 2 wherein said amplification products are generated using a plurality of primer pairs that define bioagent identifying amplicons.

9. The method of claim 8 wherein said plurality of primer pairs are used in a multiplex reaction to generate a plurality of amplification products.

10. The method of claim 8 wherein said plurality of primer pairs comprises at least two primer pairs from the group consisting of primer pair numbers:

346 (SEQ ID NOs;

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604), 354 (SEQ ID NOs;

597;

605), 358 (SEQ ID NOs;

598;

606), 359 (SEQ ID NOs;

599;

607), 3346 (SEQ ID NOs;

616;

631), 449 (SEQ ID NOs;

600;

608), 3350 (SEQ ID NOs;

614;

629), 2249 (SEQ ID NOs;

601;

609), 3361 (SEQ ID NOs;

620;

635), and 3360 (SEQ ID NOs;

612;

627).

11. The method of claim 8 wherein said plurality of primer pairs comprises primer pair numbers:

346 (SEQ ID NOs;

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604), 3346 (SEQ ID NOs;

616;

631).

12. The method of claim 8 wherein said plurality of primer pairs comprises primer pair numbers:

346 (SEQ ID NOs;

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604), and 3361 (SEQ ID NOs;

620;

635).

13. The method of claim 8 wherein said plurality of primer pairs comprises primer pair numbers 346 (SEQ ID NOs:

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604) and at least one of the primer pairs selected from the group consisting of 354 358 (SEQ ID NOs;

598;

606), 359 (SEQ ID NOs;

599;

607), 3346 (SEQ ID NOs;

616;

631), 449 (SEQ ID NOs;

600;

608), 3350 (SEQ ID NOs;

614;

629), 3361 (SEQ ID NOs;

620;

635), and 3360 (SEQ ID NOs;

612;

627).

14. The method of claim 1 wherein a high processivity polymerase enzyme is used at said amplification step.

15. The method of claim 14 wherein said high processivity polymerase enzyme is a recombinant polymerase enzyme.

16. The method of claim 14 wherein said high processivity polymerase enzyme is a genetically engineered polymerase enzyme.

17. The method of claim 14 wherein said high processivity polymerase enzyme is phi29.

18. The method of claim 1, wherein said sample comprises human whole blood.

19. The method of claim 18 further comprising the step of extracting total nucleic acid from said sample before carrying out said amplifying step.

20. The method of claim 1 wherein said sample comprises human buffy coat.

21. The method of claim 20 further comprising the step of extracting total nucleic acid from said sample before carrying out said amplifying step.

22. The method of claim 1 wherein said sample comprises human serum.

23. The method of claim 22 further comprising the step of extracting total nucleic acid from said sample before carrying out said amplifying step.

24. The method of claim 1 wherein said sample comprises human hepatic cells.

25. The method of claim 24 further comprising the step of extracting total nucleic acid from sample before carrying out said amplifying step.

26. The method of claim 1 wherein said sample comprises sputum.

27. The method of claim 26 further comprising the step of extracting total nucleic acid from sample before carrying out said amplifying step.

28. The method of claim 1 wherein said sample comprises urine.

29. The method of claim 28 further comprising the step of extracting total nucleic acid from sample before carrying out said amplifying step.

30. The method of claim 1 wherein said sample comprises biopsy tissue.

31. The method of claim 30 further comprising the step of extracting total nucleic acid from sample before carrying out said amplifying step

32. The method of claim 1 wherein said at least one pathogen is a bacterium.

33. The method of claim 32 wherein said bacterium is selected from the group consisting of:

Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Enterobacter cloacae, Enterobacter aerogenes, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Enterococcus faecium, Enterococcus faecalis, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata, Mycobacterium tuberculosis, and Aspergillus fumigatus.

34. The method of claim 1 wherein said at least one background genome comprises a human nucleic acid.

35. The method of claim 5 wherein said identifying step indicates the presence of bacterial sepsis in a human patient.

36. The method of claim 5 wherein said identifying step indicates the presence of bacteremia in a human patient.

37. The method of claim 1 wherein said pathogen is a virus.

38. The method of claim 37 wherein said virus is HIV.

39. The method of claim 37 wherein said virus is HCV.

40. The method of claim 37 wherein said virus is influenza virus.

41. A diagnostic kit for performing the method of claim 1.

42. A diagnostic kit comprising a high processivity polymerase enzyme and a plurality of purified targeted whole genome amplification primers.

43. The kit of claim 42 further comprising at least one primer pair that defines a bioagent identifying amplicon.

44. The kit of claim 43 wherein said plurality of primer pairs comprises at least two primer pairs from the group consisting of primer pair numbers:

346 (SEQ ID NOs;

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604), 354 (SEQ ID NOs;

597;

605), 358 (SEQ ID NOs;

598;

606), 359 (SEQ ID NOs;

599;

607), 3346 (SEQ ID NOs;

616;

631), 449 (SEQ ID NOs;

600;

608), 3350 (SEQ ID NOs;

614;

629), 2249 (SEQ ID NOs;

601;

609), 3361 (SEQ ID NOs;

620;

635), and 3360 (SEQ ID NOs;

612;

627).

45. The kit of claim 43 wherein said plurality of primer pairs comprises primer pair numbers:

346 (SEQ ID NOs;

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604), 3346 (SEQ ID NOs;

616;

631).

46. The kit of claim 43 wherein said plurality of primer pairs comprises primer pair numbers:

346 (SEQ ID NOs;

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604), and 3361 (SEQ ID NOs;

620;

635).

47. The kit of claim 43 wherein said plurality of primer pairs comprises primer pair numbers 346 (SEQ ID NOs:

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604) and at least one of the primer pairs selected from the group consisting of 354 358 (SEQ ID NOs;

598;

606), 359 (SEQ ID NOs;

599;

607), 3346 (SEQ ID NOs;

616;

631), 449 (SEQ ID NOs;

600;

608), 3350 (SEQ ID NOs;

614;

629), 3361 (SEQ ID NOs;

620;

635), and 3360 (SEQ ID NOs;

612;

627).

48. The kit of claim 43 wherein said high processivity enzyme is phi29.

49. A method comprising the steps of:

a. extracting nucleic acids from a sample; and

b. mixing said nucleic acids with a plurality of targeted whole genome amplification primers, a high processivity polymerase enzyme to produce an amplification mixture, wherein said plurality of targeted whole genome amplification primers is selected by;

i. identifying at least one target genome suspected of being present in said sample;

ii. identifying at least one background genome suspected of being present in said sample;

iii. identifying a plurality of genome sequence segments having unique sequences within said target genome sequence;

iv. determining frequency of occurrence of members of said plurality of genome sequence segments within said target genome sequence and within said background genome sequences;

v. calculating a selectivity ratio for said members by dividing said frequency of occurrence within said target genome by said frequency of occurrence of said plurality of genome sequence segments within said background genome sequences;

vi. selecting a selectivity ratio threshold value, thereby defining a first sub-set of said plurality of genome sequence segments having selectivity ratios equal to or greater than said selectivity ratio threshold value;

vii. determining the lengths of target genome sequence occurring between genome sequence segments of said first sub-set;

viii. selecting a second sub-set of genome sequence segments from said first sub-set wherein members of said second sub-set have a mean separation of less than a selected length of nucleobases; and

ix. selecting targeted whole genome amplification primers that hybridize to members of said second sub-set of genome sequence segments such that said at least one target genome is amplified selectively over said at least one background genome.

50. The method of claim 49 further comprising the step of amplifying one or more of said extracted nucleic acids in said mixture of step b.

51. The method of claim 49 wherein said amplifying step is a targeted whole genome amplification reaction.

52. The method of claim 51 further comprising the step of performing a second amplification step using at least one primer pair that defines a bioagent identifying amplicon to obtain at least a second amplification product.

53. The method of claim 52 further comprising the step of measuring the molecular mass of said second amplification product by mass spectrometry.

54. The method of claim 53 wherein said mass spectrometry is electrospray time-of-flight mass spectrometry.

55. The method of claim 52 further comprising the step of comparing said molecular mass with a database comprising molecular masses of bioagent identifying amplicons of pathogens produced with said primer pairs, thereby identifying said pathogen in said sample.

56. The method of claim 53 further comprising the step of calculating a base composition of said amplification products from said molecular mass.

57. The method of claim 56 further comprising the step of comparing said base compositions with a database comprising base compositions of bioagent identifying amplicons of pathogens produced with said primer pairs, thereby identifying said pathogen in said sample.

58. The method of claim 52 wherein said second amplification step comprises obtaining a plurality of amplification products generated using a plurality of primer pairs that define bioagent identifying amplicons.

59. The method of claim 58 wherein said plurality of primer pairs is used in one or more multiplex reactions to generate a plurality of amplification products.

60. The method of claim 58 wherein said plurality of primer pairs comprises at least two primer pairs from the group consisting of primer pair numbers:

346 (SEQ ID NOs;

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604), 354 (SEQ ID NOs;

597;

605), 358 (SEQ ID NOs;

598;

606), 359 (SEQ ID NOs;

599;

607), 3346 (SEQ ID NOs;

616;

631), 449 (SEQ ID NOs;

600;

608), 3350 (SEQ ID NOs;

614;

629), 2249 (SEQ ID NOs;

601;

609), 3361 (SEQ ID NOs;

620;

635), and 3360 (SEQ ID NOs;

612;

627).

61. The method of claim 58 wherein said plurality of primer pairs comprises primer pair numbers:

346 (SEQ ID NOs;

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604), 3346 (SEQ ID NOs;

616;

631).

62. The method of claim 58 wherein said plurality of primer pairs comprises primer pair numbers:

346 (SEQ ID NOs;

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604), and 3361 (SEQ ID NOs;

620;

635).

63. The method of claim 58 wherein said plurality of primer pairs comprises primer pair numbers 346 (SEQ ID NOs:

594;

602), 348 (SEQ ID NOs;

595;

603), 349 (SEQ ID NOs;

596;

604) and at least one of the primer pairs selected from the group consisting of 354 358 (SEQ ID NOs;

598;

606), 359 (SEQ ID NOs;

599;

607), 3346 (SEQ ID NOs;

616;

631), 449 (SEQ ID NOs;

600;

608), 3350 (SEQ ID NOs;

614;

629), 3361 (SEQ ID NOs;

620;

635), and 3360 (SEQ ID NOs;

612;

627).

64. The method of claim 49 wherein said high processivity polymerase enzyme is a recombinant polymerase enzyme.

65. The method of claim 49 wherein said high processivity polymerase enzyme is a genetically engineered polymerase enzyme.

66. The method of claim 49 wherein said high processivity polymerase enzyme is phi29.

67. The method of claim 49, wherein said sample comprises human whole blood.

68. The method of claim 67 further comprising the step of extracting total nucleic acid from said sample before carrying out said amplifying step.

69. The method of claim 49 wherein said sample comprises human buffy coat.

70. The method of claim 69 further comprising the step of extracting total nucleic acid from said sample before carrying out said amplifying step.

71. The method of claim 49 wherein said sample comprises human serum.

72. The method of claim 71 further comprising the step of extracting total nucleic acid from said sample before carrying out said amplifying step.

73. The method of claim 49 wherein said sample comprises human hepatic cells.

74. The method of claim 73 further comprising the step of total nucleic acid from sample before carrying out said amplifying step.

75. The method of claim 49 wherein said sample comprises sputum.

76. The method of claim 75 further comprising the step of extracting total nucleic acid from sample before carrying out said amplifying step.

77. The method of claim 49 wherein said sample comprises urine.

78. The method of claim 77 further comprising the step of extracting total nucleic acid from sample before carrying out said amplifying step.

79. The method of claim 49 wherein said sample comprises biopsy tissue.

80. The method of claim 79 further comprising the step of extracting total nucleic acid from sample before carrying out said amplifying step

81. The method of claim 49 wherein said sample comprises a bacterium.

82. The method of claim 81 wherein said bacterium is selected from the group consisting of:

Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens, Enterobacter cloacae, Enterobacter aerogenes, Proteus mirabilis, Pseudomonas aeruginosa, Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus haemolyticus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus mitis, Enterococcus faecium, Enterococcus faecalis, Candida albicans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata and Aspergillus fumigatus.

83. The method of claim 49 wherein said at least one background genome comprises a human nucleic acid.

84. The method of claim 56 wherein said identifying step indicates the presence of bacterial sepsis in a human.

85. The method of claim 56 wherein said pathogen is a virus.

86. The method of claim 85 wherein said virus is HIV.

87. The method of claim 85 wherein said virus is HCV.

88. The method of claim 85 wherein said virus is influenza virus.

89. A diagnostic kit for performing the method of claim 49, said kit comprising a plurality of targeted whole genome amplification primers.