Oligonucleotide probes for the detection and/or quantitation of non-viral organisms

• US 7,138,516 B1
• Filed: 05/30/1995
• Issued: 11/21/2006
• Est. Priority Date: 11/24/1986
• Status: Expired due to Fees

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Smith, Richard Dana, Hogan, James John, McDonough, Sherrol Hoffa, Kop, Jo Ann
• Primary Examiner(s): Moran, Marjorie A.
• Application Number:
  US08/454,064
• Time in Patent Office: 4,193 Days
• Field of Search: 435/6, 435/810, 436/501, 536/23.1, 536/24.1, 536 243- 2433, 935/77, 935/78
• US Class Current: 536/24.3
• CPC Class Codes: C12Q 1/6888 for detection or identification of organisms
• CPC Class Codes: C12Q 1/689 for bacteria
• 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

A method for preparing probes, as well as several probes for use in qualitative or quantitative hybridization assays are disclosed. The method comprises constructing an oligonucleotide that is sufficiently complementary to hybridize to a region of rRNA selected to be unique to a non-viral organism or group of non-viral organisms sought to be detected, said region of rRNA being selected by comparing one or more variable region rRNA sequences of said non-viral organism or group of non-viral organisms with one or more variable region rRNA sequences from one or more non-viral organisms sought to be distinguished. Hybridization assay probes for Mycobacterium avium, Mycobacterium intracellulare, the Mycobacterium tuberculosis-complex bacteria, Mycoplasma pneumoniae, Legionella, Salmonella, Chlamydia trachomatis, Campylobacter, Proteus mirabilis, Enterococcus, Enterobacter cloacae, E. coli, Pseudomonas group I, Neisseria gonorrhoeae, bacteria, and fungi also are disclosed.

Claims

1. An oligonucleotide probe able to distinguish between nucleic acid from a target species and nucleic acid from a non-target species belonging to the same non-viral genus, made by a process comprising the following steps:

a) identifying a variable region present in nucleic acid from said target species and nucleic acid from said non-target species, wherein said variable region is present in an rRNA sequence, or a DNA sequence encoding for said rRNA sequence, in a location corresponding to a target region selected from the group consisting of;

bases 60–

100 of E. coli 16S rRNA or the encoding DNA;

bases 120–

150 of E. coli 16S rRNA or the encoding DNA;

bases 170–

230 of E. coli 16S rRNA or the encoding DNA;

bases 405–

480 of E. coli 16S rRNA or the encoding DNA;

bases 600–

670 of E. coli 16S rRNA or the encoding DNA;

bases 820–

860 of E. coli 16S rRNA or the encoding DNA;

bases 980–

1050 of E. coli 16S rRNA or the encoding DNA;

bases 270–

390 of E. coli 23S rRNA or the encoding DNA;

bases 535–

560 of E. coli 23S rRNA or the encoding DNA;

bases 1150–

1200 of E. coli 23S rRNA or the encoding DNA;

bases 1440–

1600 of E. coli 23S rRNA or the encoding DNA;

bases 1710–

1750 of E. coli 23S rRNA or the encoding DNA; and

bases 2190–

2330 of E. coli 23S rRNA or the encoding DNA;

b) substantially maximizing complementarity of a target-complementary nucleotide sequence which is present in said oligonucleotide probe to said variable region present in nucleic acid from said target species, while substantially minimizing complementarity of said nucleotide sequence to said variable region present in nucleic acid from said non-target species, such that a duplex formed between said oligonucleotide probe and nucleic acid from said target species has a higher T_m than a duplex formed between said oligonucleotide probe and nucleic acid from said non-target species; and

c) producing said oligonucleotide probe to comprise said target-complementary sequence, wherein under high stringency hybridization assay conditions said oligonucleotide probe hybridizes to nucleic acid from said target species to form a detectable probe;

target duplex, but does not hybridize to nucleic acid from said non-target species to form a detectable probe;

non-target duplex,

provided that said oligonucleotide probe does not distinguish Enterobacter cloacae rRNA or the encoding DNA present in a location corresponding to bases 270–

390 of E. coli 23S rRNA or the encoding DNA from Enterobacter aerogenes nucleic acid present in a location corresponding to bases 270–

390 of E. coli 23S rRNA or the encoding DNA, and

further provided that said oligonucleotide probe does not distinguish Pseudomonas denitrificans rRNA or the encoding DNA present in a location corresponding to bases 270–

390 of E. coli 23S rRNA or the encoding DNA from Pseudomonas pickettii nucleic acid present in a location corresponding to bases 270–

390 of E. coli 23S rRNA or the encoding DNA.

2. The oligonucleotide probe of claim 1, wherein said non-target species is selected based on it being the species most closely related phylogenetically to said target species.

3. The oligonucleotide probe of claim 1, wherein said target region is either bases 60–

100 of E. coli 16S rRNA or the encoding DNA.

4. The oligonucleotide probe of claim 3, wherein said target region is bases 60–

100 of E. coli 16S rRNA.

5. The oligonucleotide probe of claim 1, wherein said target region is either bases 120–

150 of E. coli 16S rRNA or the encoding DNA.

6. The oligonucleotide probe of claim 5, wherein said target region is bases 120–

150 of E. coli 16S rRNA.

7. The oligonucleotide probe of claim 1, wherein said target region is either bases 170–

230 of E. coli 16S rRNA or the encoding DNA.

8. The oligonucleotide probe of claim 7, wherein said target region is bases 170–

230 of E. coli 16S rRNA.

9. The oligonucleotide probe of claim 1, wherein said target region is either bases 405–

480 of E. coli 16S rRNA or the encoding DNA.

10. The oligonucleotide probe of claim 9, wherein said target region is bases 405–

480 of E. coli 16S rRNA.

11. The oligonucleotide probe of claim 1, wherein said target region is either bases 600–

670 of E. coli 16S rRNA or the encoding DNA.

12. The oligonucleotide probe of claim 11, wherein said target region is bases 600–

670 of E. coli 16S rRNA.

13. The oligonucleotide probe of claim 1, wherein said target region is either bases 820–

860 of E. coli 16S rRNA or the encoding DNA.

14. The oligonucleotide probe of claim 13, wherein said target region is bases 820–

860 of E. coli 16S rRNA.

15. The oligonucleotide probe of claim 1, wherein said target region is either bases 980–

1050 of E. coli 16S rRNA or the encoding DNA.

16. The oligonucleotide probe of claim 15, wherein said target region is bases 980–

1050 of E. coli 16S rRNA.

17. The oligonucleotide probe of claim 1, wherein said target region is either bases 270–

390 of E. coli 23S rRNA or the encoding DNA.

18. The oligonucleotide probe of claim 17, wherein said target region is bases 270–

390 of E. coli 23S rRNA.

19. The oligonucleotide probe of claim 1, wherein said target region is either bases 535–

560 of E. coli 23S rRNA or the encoding DNA.

20. The oligonucleotide probe of claim 19, wherein said target region is bases 535–

560 of E. coli 23S rRNA.

21. The oligonucleotide probe of claim 1, wherein said target region is either bases 1150–

1200 of E. coli 23S rRNA or the encoding DNA.

22. The oligonucleotide probe of claim 21, wherein said target region is bases 1150–

1200 of E. coli 23S rRNA.

23. The oligonucleotide probe of claim 1, wherein said target region is either bases 1440–

1600 of E. coli 23S rRNA or the encoding DNA.

24. The oligonucleotide probe of claim 23, wherein said target region is bases 1440–

1600 of E. coli 23S rRNA.

25. The oligonucleotide probe of claim 1, wherein said target region is either bases 1710–

1750 of E. coli 23S rRNA or the encoding DNA.

26. The oligonucleotide probe of claim 25, wherein said target region is bases 1710–

1750 of E. coli 23S rRNA.

27. The oligonucleotide probe of claim 1, wherein said target region is either bases 2190–

2330 of E. coli 23S rRNA or the encoding DNA.

28. The oligonucleotide probe of claim 27, wherein said target region is bases 2190–

2330 of E. coli 23S rRNA.

29. The oligonucleotide probe of any one of claims 2, 3–

12, 13–

16 and 17–

28, wherein said process further comprises the step of labeling said oligonucleotide probe with either an isotopic label or a non-isotopic label.

30. The oligonucleotide probe of any one of claims 2, 3–

12, 13–

16 and 17–

28, wherein said oligonucleotide probe is 10 to 100 nucleotides in length.

31. The oligonucleotide probe of claim 1, wherein said process further comprises the step of labeling said oligonucleotide probe with a non-isotopic label selected from the group consisting of fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, and haptens.

32. The oligonucleotide probe of claim 31, wherein said non-isotopic label is an acridinium ester.

33. An oligonucleotide probe able to distinguish between nucleic acid from two or more target species belonging to a first non-viral genus and nucleic acid from one or more non-target species belonging to a second non-viral genus, made by a process comprising the following steps:

a) identifying a variable region present in nucleic acid from each of said two or more target species and in nucleic acid from said one or more non-target species, wherein said variable region is present in an rRNA sequence, or a DNA sequence encoding for said rRNA sequence, in a location corresponding to a target region selected from the group consisting of;

bases 60–

100 of E. coli 16S rRNA or the encoding DNA;

bases 120–

150 of E. coli 16S rRNA or the encoding DNA;

bases 170–

230 of E. coli 16S rRNA or the encoding DNA;

bases 405–

480 of E. coli 16S rRNA or the encoding DNA;

bases 600–

670 of E. coli 16S rRNA or the encoding DNA;

bases 820–

860 of E. coli 16S rRNA or the encoding DNA;

bases 980–

1050 of E. coli 16S rRNA or the encoding DNA;

bases 270–

390 of E. coli 23S rRNA or the encoding DNA;

bases 535–

560 of E. coli 23S rRNA or the encoding DNA;

bases 1150–

1200 of E. coli 23S rRNA or the encoding DNA;

bases 1440–

1600 of E. coli 23S rRNA or the encoding DNA;

bases 1710–

1750 of E. coli 23S rRNA or the encoding DNA; and

bases 2190–

2330 of E. coli 23S rRNA or the encoding DNA;

b) substantially maximizing complementarity of a target-complementary nucleotide sequence which is present in said oligonucleotide probe to said variable region present in nucleic acid from each of said two or more target species, while substantially minimizing complementarity of said nucleotide sequence to said variable region present in nucleic acid from said one or more non-target species, such that a duplex formed between said oligonucleotide probe and nucleic acid from any of said two or more target species has a higher T_m than a duplex formed between said oligonucleotide probe and nucleic acid from any of said one or more non-target species; and

c) producing said oligonucleotide probe to comprise said target-complementary sequence, wherein under high stringency hybridization assay conditions said oligonucleotide probe hybridizes to nucleic acid from said two or more target species to form detectable probe;

target duplexes, but does not hybridize to nucleic acid from said one or more non-target species to form detectable probe;

non-target duplexes,

provided that said oligonucleotide probe does not distinguish Enterobacter cloacae rRNA or the encoding DNA present in a location corresponding to bases 270–

390 of E. coli 23S rRNA or the encoding DNA from Enterobacter aerogenes nucleic acid present in a location corresponding to bases 270–

390 of E. coli 23S rRNA or the encoding DNA, and

further provided that said oligonucleotide probe does not distinguish Pseudomonas denitrificans rRNA or the encoding DNA present in a location corresponding to bases 270–

390 of E. coli 23S rRNA or the encoding DNA from Pseudomonas pickettii nucleic acid present in a location corresponding to bases 270–

390 of E. coli 23S rRNA or the encoding DNA.

34. The oligonucleotide probe of claim 33, wherein said second genus is selected based on it being the phylogenetically most closely related genus to said first genus.

35. The oligonucleotide probe of claim 33, wherein said target region is either bases 60–

100 of E. coli 16S rRNA or the encoding DNA.

36. The oligonucleotide probe of claim 35, wherein said target region is bases 60–

100 of E. coli 16S rRNA.

37. The oligonucleotide probe of claim 33, wherein said target region is either bases 120–

150 of E. coli 16S rRNA or the encoding DNA.

38. The oligonucleotide probe of claim 37, wherein said target region is bases 120–

150 of E. coli 16S rRNA.

39. The oligonucleotide probe of claim 33, wherein said target region is either bases 170–

230 of E. coli 16S rRNA or the encoding DNA.

40. The oligonucleotide probe of claim 39, wherein said target region is bases 170–

230 of E. coli 16S rRNA.

41. The oligonucleotide probe of claim 33, wherein said target region is either bases 405–

480 of E. coli 16S rRNA or the encoding DNA.

42. The oligonucleotide probe of claim 41, wherein said target region is bases 405–

480 of E. coli 16S rRNA.

43. The oligonucleotide probe of claim 33, wherein said target region is either bases 600–

670 of E. coli 16S rRNA or the encoding DNA.

44. The oligonucleotide probe of claim 43, wherein said target region is bases 600–

670 of E. coli 16S rRNA.

45. The oligonucleotide probe of claim 33, wherein said target region is either bases 820–

860 of E. coli 16S rRNA or the encoding DNA.

46. The oligonucleotide probe of claim 45, wherein said target region is bases 820–

860 of E. coli 16S rRNA.

47. The oligonucleotide probe of claim 33, wherein said target region is either bases 980–

1050 of E. coli 16S rRNA or the encoding DNA.

48. The oligonucleotide probe of claim 47, wherein said target region is bases 980–

1050 of E. coli 16S rRNA.

49. The oligonucleotide probe of claim 33, wherein said target region is either bases 270–

390 of E. coli 23S rRNA or the encoding DNA.

50. The oligonucleotide probe of claim 49, wherein said target region is bases 270–

390 of E. coli 23S rRNA.

51. The oligonucleotide probe of claim 33, wherein said target region is either bases 535–

560 of E. coli 23S rRNA or the encoding DNA.

52. The oligonucleotide probe of claim 51, wherein said target region is bases 535–

560 of E. coli 23S rRNA.

53. The oligonucleotide probe of claim 33, wherein said target region is either bases 1150–

1200 of E. coli 23S rRNA or the encoding DNA.

54. The oligonucleotide probe of claim 53, wherein said target region is bases 1150–

1200 of E. coli 23S rRNA.

55. The oligonucleotide probe of claim 33, wherein said target region is either bases 1440–

1600 of E. coli 23S rRNA or the encoding DNA.

56. The oligonucleotide probe of claim 55, wherein said target region is bases 1440–

1600 of E. coli 23S rRNA.

57. The oligonucleotide probe of claim 33, wherein said target region is either bases 1710–

1750 of E. coli 23S rRNA or the encoding DNA.

58. The oligonucleotide probe of claim 57, wherein said target region is bases 1710–

1750 of E. coli 23S rRNA.

59. The oligonucleotide probe of claim 33, wherein said target region is either bases 2190–

2330 of E. coli 23S rRNA or the encoding DNA.

60. The oligonucleotide probe of claim 59, wherein said target region is bases 2190–

2330 of E. coli 23S rRNA.

61. The oligonucleotide probe of any one of claims 34, 35–

44, 45–

48, and 49–

60, wherein said process further comprising the step of labeling said oligonucleotide probe with either an isotopic label or a non-isotopic label.

62. The oligonucleotide probe of any one of claims 34, 35–

44, 45–

48 and 49–

60, wherein said oligonucleotide probe is 10 to 100 nucleotides in length.

63. The oligonucleotide probe of claim 33, wherein said process further comprising the step of labeling said oligonucleotide probe with a non-isotopic label selected from the group consisting of fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, and haptens.

64. The oligonucleotide probe of claim 63, wherein said non-isotopic label is an acridinium ester.