Methods for making oligonucleotide probes for the detection and/or quantitation of non-viral organisms

• US 6,150,517 A
• Filed: 05/30/1995
• Issued: 11/21/2000
• Est. Priority Date: 11/24/1986
• Status: Expired due to Term

• Current Assignee: Gen-Probe
• Original Assignee: Gen-Probe
• Inventors: Hogan, James John, McDonough, Sherrol Hoffa, Kop, Jo Ann, Smith, Richard Dana
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/454,063
• Time in Patent Office: 2,002 Days
• Field of Search: 435/6, 435/810, 436/501, 536/23.1, 536/24.1, 536/24.3-24.33, 536/25.3, 935/77, 935/78
• US Class Current: 536/25.3
• 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. A method for making a probe for use in a nucleic acid hybridization assay which comprises constructing a nucleotide polymer that is sufficiently complementary to hybridize to a region of rRNA, or the encoding DNA, selected to distinguish one or more non-viral target species from at least one non-viral non-target species, said nucleotide polymer comprising a target-complementary sequence obtained by:

a) aligning rRNA, or the encoding DNA, sequences of said one or more target species and said at least one non-target species to identify a variable region; and

b) designing said target-complementary sequence of said nucleotide polymer by substantially maximizing the complementarity of said nucleotide polymer to said variable region present in said one or more target species while substantially minimizing the complementarity of said nucleotide polymer to said variable region present in rRNA, or the encoding DNA, sequences of said at least one non-target species, such that a duplex formed between said nucleotide polymer and said one or more target species has a higher T_m than a duplex formed between said nucleotide polymer and said at least one non-target species,

wherein said variable region is located in an rRNA sequence, or a DNA sequence encoding for said rRNA sequence, corresponding to a target region selected from the group consisting of;

bases 125-150 of E. coli 16S rRNA or the encoding DNA;

bases 175-210 of E. coli 16S rRNA or the encoding DNA;

bases 185-225 of E. coli 16S rRNA or the encoding DNA;

bases 190-230 of E. coli 16S rRNA or the encoding DNA;

bases 600-635 of E. coli 16S rRNA or the encoding DNA;

bases 630-675 of E. coli 16S rRNA or the encoding DNA;

bases 820-860 of E. coli 16S rRNA or the encoding DNA;

bases 825-860 of E. coli 16S rRNA or the encoding DNA;

bases 830-870 of E. coli 16S rRNA or the encoding DNA;

bases 975-1020 of E. coli 16S rRNA or the encoding DNA;

bases 980-1010 of E. coli 16S rRNA or the encoding DNA;

bases 980-1015 of E. coli 16S rRNA or the encoding DNA;

bases 995-1030 of E. coli 16S rRNA or the encoding DNA;

bases 1025-1060 of E. coli 16S rRNA or the encoding DNA;

bases 1255-1290 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;

provided that if said target region corresponds to bases 125-150 of E. coli 16S rRNA or the encoding DNA, then said target species is Neisseria gonorrhoeae,

if said target region corresponds to bases 175-210 of E. coli 16S rRNA or the encoding DNA, then said target species is Chlamydia trachomatis,

if said target region corresponds to bases 185-225 of E. coli 16S rRNA or the encoding DNA, then said target species is either Mycobacterium avium, Mycobacterium intracellulare, or a Mycobacterium tuberculosis complex organism,

if said target region corresponds to bases 190-230 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,

if said target region corresponds to bases 600-635 of E. coli 16S rRNA or the encoding DNA, then said target species is Chlamydia trachomatis,

if said target region corresponds to bases 630-675 of E. coli 16S rRNA or the encoding DNA, then said target species is a Legionella organism,

if said target region corresponds to bases 820-860 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,

if said target region corresponds to bases 825-860 of E. coli 16S rRNA or the encoding DNA, then said target species is a Group D Streptococcus organism selected from the group consisting of Streptococcus faecium, Streptococcus faecalis and Streptococcus avium,

if said target region corresponds to bases 830-870 of E. coli 16S rRNA or the encoding DNA, then said target species is Chlamydia trachomatis,

if said target region corresponds to bases 975-1020 of E. coli 16S rRNA or the encoding DNA, then said target species is a Legionella organism,

if said target region corresponds to bases 980-1010 of E. coli 16S rRNA or the encoding DNA, then said target species is a Campylobacter organism,

if said target region corresponds to bases 980-1015 of E. coli 16S rRNA or the encoding DNA, then said target species is Neisseria gonorrhoeae,

if said target region corresponds to bases 995-1030 of E. coli 16S rRNA or the encoding DNA, then said target species is Escherichia coli,

if said target region corresponds to bases 1025-1060 of E. coli 16S rRNA or the encoding DNA, then said target species is a Mycobacterium organism, and

if said target region corresponds to bases 1255-1290 of E. coli 16S or the encoding DNA, then said target species is Mycoplasma pneumoniae.

2. The method of claim 1 wherein the percent similarity in the rRNA sequences of said at least one nontarget species and said one or more target species is between 90 and 99%.

3. The method of claim 2 wherein said at least one nontarget species is selected based on it being known to be the species most closely related phylogenetically to said one or more target species.

4. The method of claim 1 comprising the further step of verifying that said probe does not cross hybridize to said at least one nontarget species under high stringency hybridization assay conditions to form a detectable probe:

non-target duplex, wherein under said high stringency hybridization assay conditions said probe forms a detectable probe;

target duplex with nucleic acid from said one or more target species.

7. The method of any one of claims 1, 3 or 4, wherein said target region corresponds to either:

bases 125-150 of E. coli 16S rRNA

bases 175-210 of E. coli 16S rRNA;

bases 185-225 of E. coli 16S rRNA;

bases 190-230 of E. coli 16S rRNA;

bases 600-635 of E. coli 16S rRNA;

bases 630-675 of E. coli 16S rRNA;

bases 820-860 of E. coli 16S rRNA;

bases 825-860 of E. coli 16S rRNA;

bases 830-870 of E. coli 16S rRNA;

bases 975-1020 of E. coli 16S rRNA;

bases 980-1010 of E. coli 16S rRNA;

bases 980-1015 of E. coli 16S rRNA;

bases 995-1030 of E. coli 16S rRNA;

bases 1025-1060 of E. coli 16S rRNA;

bases 1255-1290 of E. coli 16S rRNA;

bases 270-390 of E. coli 23S rRNA;

bases 535-560 of E. coli 23S rRNA;

bases 1150-1200 of E. coli 23S rRNA;

bases 1440-1600 of E. coli 23S rRNA;

bases 1710-1750 of E. coli 23S rRNA;

or

bases 2190-2330 of E. coli 23S rRNA.

48. The method of claim 2, wherein said target region corresponds to bases 125-150 of E. coli 16S rRNA or the encoding DNA.

49. The method of claim 2, wherein said target region corresponds to bases 125-150 of E. coli 16S rRNA.

50. The method of claim 2, wherein said target region corresponds to either bases 175-210 of E. coli 16S rRNA or the encoding DNA bases 185-225 of E. coli 16S rRNA or the encoding DNA, or bases 190-230 of E. coli 16S rRNA or the encoding DNA.

51. The method of claim 2, wherein said target region corresponds to either bases 175-210 of E. coli 16S rRNA, bases 185-225 of E. coli 16S rRNA or bases 190-230 of E. coli 16S rRNA.

52. The method of claim 2, wherein said target region corresponds either to bases 600-635 of E. coli 16S rRNA or the encoding DNA, or bases 630-675 of E. coli 16S rRNA or the encoding DNA.

53. The method of claim 2, wherein said target region corresponds to bases 600-635 of E. coli 16S rRNA, or bases 630-675 of E. coli 16S rRNA.

54. The method of claim 2, wherein said target region corresponds to either bases 820-860 of E. coli 16S rRNA or the encoding DNA, bases 825-860 of E. coli 16S rRNA or the encoding DNA, or bases 830-870 of E. coli 16rRNA or the encoding DNA.

55. The method of claim 2, wherein said target region corresponds to either bases 820-860 of E. coli 16S rRNA, bases 825-860 of E. coli 16S rRNA, or bases 830-870 of E. coli 16S rRNA.

56. The method of claim 2, wherein said target region corresponds to either bases 975-1020 of E. coli 16S rRNA or the encoding DNA, bases 980-1010 of E. coli 16 S rRNA or the encoding DNA, bases 980-1015 of E. coli 16S rRNA or the encoding DNA, bases 995-1030 of E. coli 16S rRNA or the encoding DNA, or bases 1025-1060 of E. coli 16S rRNA or the encoding DNA.

57. The method of claim 2, wherein said target region corresponds to either bases 975-1020 of E. coli 16S rRNA, bases 989-1010 of E. coli 16S rRNA, bases 980-1015 of E. coli 16S rRNA, bases 995-1030 of E. coli 16S rRNA, or bases 1025-1060 of E. coli 16S rRNA.

58. The method of claim 2, wherein said target region corresponds to bases 1255-1290 of E. coli 16S rRNA or the encoding DNA.

59. The method of claim 2, wherein said target region corresponds to bases 1255-1290 of E. coli 16S rRNA.

60. The method of claim 2, wherein said target region corresponds to bases 270-390 of E. coli 23S rRNA or the encoding DNA.

61. The method of claim 2, wherein said target region corresponds to bases 270-390 of E. coli 23S rRNA.

62. The method of claim 2, wherein said target region corresponds to bases 535-560 of E. coli 23S rRNA or the encoding DNA.

63. The method of claim 2, wherein said target region corresponds to bases 535-560 of E. coli 23S rRNA.

64. The method of claim 2, wherein said target region corresponds to bases 1150-1200 of E. coli 23S rRNA or the encoding DNA.

65. The method of claim 2, wherein said target region corresponds to bases 1150-1200 of E. coli 23S rRNA.

66. The method of claim 2, wherein said target region corresponds to bases 1440-1600 of E. coli 23S rRNA or the encoding DNA.

67. The method of claim 2, wherein said target region corresponds to bases 1440-1600 of E. coli 23S rRNA.

68. The method of claim 2, wherein said target region corresponds to bases 1710-1750 of E. coli 23S rRNA or the encoding DNA.

69. The method of claim 2, wherein said target region corresponds to bases 1710-1750 of E. coli 23S rRNA.

70. The method of claim 2, wherein said target region corresponds to bases 2190-2330 of E. coli 23S rRNA or the encoding DNA.

71. The method of claim 2, wherein said target region corresponds to bases 2190-2330 of E. coli 23S rRNA.

5. A method for making a probe for use in a hybridization assay to distinguish two or more non-viral target species of a target group of non-viral organisms from one or more non-viral non-target species which comprises constructing an oligonucleotide that is sufficiently complementary to hybridize a variable region of rRNA, or the encoding DNA, present in said two or more target species, said oligonucleotide comprising a target-complementary sequence obtained by:

a) aligning rRNA, or the encoding DNA, sequences of said two or more target species with rRNA, or the encoding DNA, sequences of said one or more nontarget species, so as to reveal inter-species hypervariability present in said variable region; and

b) designing said target-complementary sequence of said oligonucleotide by substantially maximizing complementarity of said oligonucleotide to said variable region present in said two or more target species while substantially minimizing the complementarity of said oligonucleotide to said variable region present in said one or more non-target species, such that a duplex formed between said oligonucleotide and each of said two or more target species has a higher T_m than a duplex formed between said oligonucleotide and said one or more non-target species,

wherein said variable region is located in an rRNA sequence, or a DNA sequence encoding for said rRNA sequence, corresponding to a target region selected from the group consisting of;

bases 185-225 of E. coli 16S rRNA or the encoding DNA;

bases 630-675 of E. coli 16S rRNA or the encoding DNA;

bases 825-860 of E. coli 16S rRNA or the encoding DNA;

bases 975-1020 of E. coli 16S rRNA or the encoding DNA;

bases 980-1010 of E. coli 16S rRNA or the encoding DNA;

bases 1025-1060 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;

provided that if said target region corresponds to bases 185-225 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Mycobacterium tuberculosis complex organisms,

if said target region corresponds to bases 630-675 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Legionella organisms,

if said target region corresponds to bases 825-860 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are group D Streptococcus organisms selected from the group consisting of Streptococcus faecium, Streptococcus faecalis and Streptococcus avium,

if said target region corresponds to bases 975-1020 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Legionella organisms,

if said target region corresponds to bases 980-1010 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Campylobacter organisms, and

if said target region corresponds to bases 1025-1060 of E. coli 16S rRNA or the encoding DNA, then said two or more target species are Mycobacterium organisms.

6. The method of claim 5 comprising the further step of verifying that said probe does not cross hybridize to said one or more nontarget species under high stringency hybridization assay conditions to form a detectable probe:

non-target duplex, wherein under said high stringency hybridization assay conditions said probe forms a detectable probe;

target duplex with nucleic acid from each of said two or more target species.

8. The method of claim 5 wherein said one or more nontarget species is selected based on it belonging to the group of organisms most closely related phylogenetically to said target group of organisms.

9. The method of any one of claims 5, 6, or 8, wherein said target region corresponds to either:

bases 185-225 of E. coli 16S;

bases 630-675 of E. coli 16S rRNA;

bases 825-860 of E. coli 16S rRNA;

bases 975-1020 of E. coli 16S rRNA;

bases 980-1010 of E. coli 16S rRNA;

bases 1025-1060 of E. coli 16S rRNA;

bases 270-390 of E. coli 23S rRNA;

bases 535-560 of E. coli 23S rRNA;

bases 1150-1200 of E. coli 23S rRNA;

bases 1440-1600 of E. coli 23S rRNA;

bases 1710-1750 of E. coli 23S rRNA;

or

bases 2190-2330 of E. coli 23S rRNA.

72. The method of claim 5, wherein said target region corresponds to bases 185-225 of E. coli 16S rRNA or the encoding DNA.

73. The method of claim 5, wherein said target region corresponds to bases 185-225 of E. coli 16S rRNA.

74. The method of claims 5, wherein said target region corresponds to bases 630-675 of E. coli 16S rRNA or the encoding DNA.

75. The method of claims 5, wherein said target region corresponds to bases 630-675 of E. coli 16S rRNA.

76. The method of claim 5, wherein said target region corresponds to bases 825-860 of E. coli 16S rRNA or the encoding DNA.

77. The method of claim 5, wherein said target region corresponds to bases 825-860 of E. coli 16S rRNA.

78. The method of claim 5, wherein said target region corresponds to either bases 975-1020 of E. coli 16S rRNA or the encoding DNA, bases 980-1010 of E. coli 16S rRNA or the encoding DNA, or bases 1025-1060 of E. coli 16S rRNA or the encoding DNA.

79. The method of claim 5, wherein said target region corresponds to either bases 975-1020 of E. coli 16S rRNA, bases 980-1010 of E. coli 16S rRNA or bases 1025-1060 of E. coli 16S rRNA.

80. The method of claim 5, wherein said target region corresponds to bases 270-390 of E. coli 23S rRNA or the encoding DNA.

81. The method of claim 5, wherein said target region corresponds to bases 270-390 of E. coli 23S rRNA.

82. The method of claim 5, wherein said target region corresponds either to bases 535-560 of E. coli 23S rRNA or the encoding DNA.

83. The method of claim 5, wherein said target region corresponds to bases 535-560 of E. coli 23S rRNA.

84. The method of claim 5, wherein said target region corresponds to bases 1150-1200 of E. coli 23S rRNA or the encoding DNA.

85. The method of claim 5, wherein said target region corresponds to bases 1150-1200 of E. coli 23S rRNA.

86. The method of claim 5, wherein said target region corresponds to either bases 1440-1600 of E. coli 23S rRNA or the encoding DNA.

87. The method of claim 5, wherein said target region corresponds to bases 1440-1600 of E. coli 23S rRNA.

88. The method of claim 5, wherein said target region corresponds to bases 1710-1750 of E. coli 23S rRNA or the encoding DNA.

89. The method of claim 5, wherein said target region corresponds to bases 1710-1750 of E. coli 23S rRNA.

90. The method of claim 5, wherein said target region corresponds to bases 2190-2330 of E. coli 23S rRNA or the encoding DNA.

91. The method of claim 5, wherein said target region corresponds to bases 2190-2330 of E. coli 23S rRNA.

115. The method of claim 5, wherein said target region corresponds to bases 270-390 of E. coli 23S rRNA.

10. The method for making an oligonucleotide probe able to distinguish between a non-viral target species and a non-viral non-target species belonging to the same genus, which comprises producing said oligonucleotide to comprise a nucleotide sequence designed by:

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

bases 65-120 of E. coli 5S rRNA or the encoding DNA;

bases 60-105 of E. coli 16S rRNA or the encoding DNA;

bases 125-150 of E. coli 16S rRNA or the encoding DNA;

bases 175-210 of E. coli 16S rRNA or the encoding DNA;

bases 185-225 of E. coli 16S rRNA or the encoding DNA;

bases 190-230 of E. coli 16S rRNA or the encoding DNA;

bases 450-490 of E. coli 16S rRNA or the encoding DNA;

bases 455-485 of E. coli 16S rRNA or the encoding DNA;

bases 600-635 of E. coli 16S rRNA or the encoding DNA;

bases 820-860 of E. coli 16S rRNA or the encoding DNA;

bases 830-870 of E. coli 16S rRNA or the encoding DNA;

bases 980-1015 of E. coli 16S rRNA or the encoding DNA;

bases 995-1030 of E. coli 16S rRNA or the encoding DNA;

bases 1255-1290 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;

provided that if said target region corresponds to bases 65-120 of E. coli 5S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,

if said target region corresponds to bases 60-150 of E. coli 16S rRNA or the encoding DNA, then said target species is Chlamydia trachomatis,

if said target region corresponds to bases 125-150 of E. coli 16S rRNA or the encoding DNA, then said target species is Neisseria gonorrhoeae;

if said target region corresponds to bases 185-225 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycobacterium avium or Mycobaterium intracellulare,

if said target region corresponds to bases 190-230 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,

if said target region corresponds to bases 450-490 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,

if said target region region corresponds to bases 450-490 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,

if said target region corresponds to bases 455-485 of E. coli 16S rRNA or the encoding DNA, then said target species is Neisseria gonorrhoeae,

if said target region corresponds to bases 600-636 of E. coli 16S rRNA or the encoding DNA, then said target species is Chlamydia trachomatis,

if said target region corresponds to bases 820-860 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae,

if said target region corresponds to bases 830-870 of E. coli 16S rRNA or the encoding DNA, then said target species is Chlamydia trachomatis,

if said target region corresponds to bases 980-1015 of E. coli 16S rRNA or the encoding DNA, then said target species is Neisseria gonorrhoeae,

if said target region corresponds to bases 995-1030 of E. coli 16S rRNA or the encoding DNA, then said target species is Escherichia coli, and

if said target region corresponds to bases 1255-1290 of E. coli 16S rRNA or the encoding DNA, then said target species is Mycoplasma pneumoniae; and

b) substantially maximizing complementarity of said nucleotide sequence to said variable region present in said target species while substantially minimizing complementarity of said nucleotide sequence to said variable region present in said non-target species, such that a duplex formed between said oligonucleotide and said target species has a higher T_m than a duplex formed between said oligonucleotide and said non-target species.

11. The method of claim 10, wherein said nontarget species is selected based on it being the species most closely related phylogenetically to said target species.

12. The method of claim 10, wherein said target region corresponds to either bases 125-150 of E. coli 16S rRNA or the encoding DNA.

13. The method of claim 12, wherein said target region corresponds to bases 125-150 of E. coli 16S rRNA.

14. The method of claim 10, wherein said target region corresponds to either bases 1255-1290 of E. coli 16S rRNA or the encoding DNA.

15. The method of claim 14, wherein said target region corresponds to bases 1255-1290 of E. coli 16S rRNA.

16. The method of claim 10, wherein said target region corresponds to either bases 270-390 of E. coli 23S rRNA or the encoding DNA.

17. The method of claim 16, wherein said target region corresponds to bases 270-390 of E. coli 23S rRNA.

18. The method of claim 10, wherein said target region corresponds to either bases 535-560 of E. coli 23S rRNA or the encoding DNA.

19. The method of claim 18, wherein said target region corresponds to bases 535-560 of E. coli 23S rRNA.

20. The method of claim 10, wherein said target region corresponds to either bases 1150-1200 of E. coli 23S rRNA or the encoding DNA.

21. The method of claim 20, wherein said target region corresponds to bases 1150-1200 of E. coli 23S rRNA.

22. The method of claim 10, wherein said target region corresponds to either bases 1440-1600 of E. coli 23S rRNA or the encoding DNA.

23. The method of claim 22, wherein said target region corresponds to bases 1440-1600 of E. coli 23S rRNA.

24. The method of claim 10, wherein said target region corresponds to either bases 1710-1750 of E. coli 23S rRNA or the encoding DNA.

25. The method of claim 24, wherein said target region corresponds to bases 1710-1750 of E. coli 23S rRNA.

26. The method of claim 10, wherein said target region corresponds to either bases 2190-2330 of E. coli 23S rRNA or the encoding DNA.

27. The method of claim 26, wherein said target region corresponds to bases 2190-2330 of E. coli 23S rRNA.

28. The method of any of claims 12, 13 or 14-27, further comprising the step of labeling said oligonucleotide with either an isotopic label or a non-isotopic label.

29. The method of claim 10, further comprising the step of labeling said oligonucleotide with a non-isotopic label selected from the group consisting of fluorescent molecules, chemiluminescent molecules, enzymes, cofactors, enzyme substrates, and haptens.

30. The method of claim 29, wherein said non-isotopic label is an acridinium ester.

92. The method of claim 10, wherein said target species is Chlamydia trachomatis and said target region corresponds to bases 60-105 o fE. coli 16S rRNA.

93. The method of claim 10, wherein said target species is Neisseria gonorrhoeae and said target region corresponds to bases 125-150 of E. coli 16S rRNA.

94. The method of claim 10, wherein said target species wherein said target species is either Mycobacterium avium, Mycobacterium intracellulare, Mycoplasma pneumoniae, or Chlamydia trachomatis,

provided that if said target species is Mycobacterium avium or Mycobacterium intracellulare, then said target region corresponds to bases 185-225 of E. coli 16S rRNA.

if said target species is Mycoplasma pneumoniae, then said target region corresponds to bases 190-230 of E. coli 16 rRNA, and

if said target species is Chlamydia trachomatis, then said target region corresponds to bases 175-210 of E. coli 16S rRNA.

95. The method of claim 10, wherein said target species is either Mycoplasma pneumoniae or Neisseria gonorrhoeae,

provided that if said target species is Mycoplasma pneumoniae, then said target region corresponds to bases 450-490 of E. coli 16S rRNA, and

if said target species is Neisseria gonorrhoeae, then said target region corresponds to bases 455-485 of E. coli 16S rRNA.

96. The method of claim 10, wherein said target species is Chlamydia trachomatis and said target region corresponds to bases 600-635 of E. coli 16S rRNA.

97. The method of claim 10, wherein said target species is either Mycoplasma pneumoniae or Chlamydia trachomatis,

provided that if said target species is Mycoplasma pneumoniae, then said target region corresponds to bases 820-860 of E. coli 16S rRNA, and

if said target species is Chlamydia trachomatis, then said target region corresponds to bases 830-870 of E. coli 16S rRNA.

98. The method of claim 10, wherein said target species is either Escherichia coli or Neisseria gonorrhoeae,

provided that if said target species is Escherichia coli, then said target region corresponds to bases 995-1030 of E. coli 16S rRNA, and

if said target species is Neisseria gonorrhoeae, then said target region corresponds to bases 980-1015 of E. coli 16S rRNA.

99. The method of claim 10, wherein said target species is either Proteus mirabilis, Chlamydia trachomatis or Enterobacter cloacae,

provided that if said target species is Proteus mirabilis, then said target region corresponds to bases 270-305 of E. coli 23S rRNA,

if said target species is Chlamydia trachomatis, then said target region corresponds to either bases 275-320, or 330-365, of E. coli 23S rRNA, and

if said target species is Enterobacter cloacae, then said target region corresponds to bases 305-340 of E. coli 23S rRNA.

100. The method of claim 10, wherein said target species is Chlamydia trachomatis and said target region corresponds to bases 1160-1190 of E. coli 23S rRNA.

101. The method of claim 10, wherein said target species is Chlamydia trachomatis and said target region corresponds to either bases 1450-1490, or 1510-1545, of E. coli 23S rRNA.

113. The method of claim 10, wherein said target species is Chlamydia trachomatis and said target region corresponds to bases 60-105 of E. coli 16S rRNA or the encoding DNA.

114. The method of claim 10, wherein said target species is Neisseria gonorrhoeae and said target regions corresponds to bases 125-150 of E. coli 16S rRNA or the encoding DNA.

116. The method of claim 10, wherein said target species is either Mycoplasma pneumoniae or Neisseria gonorrhoeae,

provided that if said target species is Mycoplasma pneumoniae, then said target region corresponds to bases 450-490 of E. coli 16S rRNA or the encoding DNA, and

if said target species is Neisseria gonorrhoeae, then said target region corresponds to bases 455-485 of E. coli 16S rRNA or the encoding DNA.

117. The method of claim 10, wherein said target species is Chlamydia trachomatis and said target region corresponds to bases 600-635 of E. coli 16S rRNA or the encoding DNA.

118. The method of claim 10, wherein said target species is either Mycoplasma pneumoniae or Chlamydia trachomatis,

provided that if said target species is Mycoplasma pneumoniae, then said target region corresponds to bases 820-860 of E. coli 16S rRNA or the encoding DNA, and

if said target species is Chlamydia trachomatis, then said target region corresponds to bases 830-870 of E. coli 16S rRNA or the encoding DNA.

119. The method of claim 10, wherein said target species is either Escherichia coli or Neisseria gonorrhoeae,

provided that if said target species is Escherichia coli, then said target region corresponds to bases 995-1030 of E. coli 16S rRNA or the encoding DNA, and

if said target species is Neisseria gonorrhoeae, then said target region corresponds to bases 980-1015 of E. coli 16S rRNA or the encoding DNA.

120. The method of claim 10, wherein said target species is either Proteus mirabilis, Chlamydia trachomatis or Enterobacter cloacae,

provided that if said target species is Proteus mirabilis, then said target region corresponds to bases 270-305 of E. coli 23S rRNA or the encoding DNA,

if said target species is Chlamydia trachomatis, then said target region corresponds to either bases 275-320, or 330-365, of E. coli 23S rRNA or the encoding DNA, and

if said target species is Enterobacter cloacae, then said target region corresponds to bases 305-340 of E. coli 23S rRNA or the encoding DNA.

121. The method of claim 10, wherein said target species is Chlamydia trachomatis and said target region corresponds to bases 1160-1190 of E. coli 23S rRNA or the encoding DNA.

122. The method of claim 10, wherein said target species is Chlamydia trachomatis and said target region corresponds to either bases 1450-1490, or 1510-1545, of E. coli 23S rRNA or the encoding DNA.

133. The method of claim 10, wherein said target region corresponds bases 65-120 of E. coli 5S rRNA or the encoding DNA.

134. The method of claim 133, wherein said target region corresponds bases 65-120 of E. coli 5S rRNA.

139. The method of claim 10, wherein said target region corresponds to either bases 125-150 of E. coli 16S rRNA or the encoding DNA.

140. The method of claim 139, wherein said target region corresponds to bases 125-150 of E. coli 16S rRNA.

31. A method for making an oligonucleotide provbe able to distinguish two or more non-viral target species belonging to a first genus from one or more non-viral non-target species belonging to a second genus, which comprises producing said oligonucleotide probe to comprise a nucleotide sequence designed by:

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

bases 185-225 of E. coli 16S rRNA or the encoding DNA;

bases 405-428 of E. coli 16S rRNA or the encoding DNA;

bases 440-475 of E. coli 16S rRNA or the encoding DNA;

bases 630-675 of E. coli 16S rRNA or the encoding DNA;

bases 705-735 of E. coli 16S rRNA or the encoding DNA;

bases 825-860 of E. coli 16S rRNA or the encoding DNA;

bases 975-1020 of E. coli 16S rRNA or the encoding DNA;

bases 980-1010 of E. coli 16S rRNA or the encoding DNA;

bases 1025-1060 of E. coli 16S rRNA or the encoding DNA;

bases 1125-1155 of E. coli 16S rRNA or the encoding DNA;

bases 270-390 of E. coli 23S rRNA or the encoding DNA;

bases 350-395 of E. coli 23S rRNA or the encoding DNA;

bases 365-405 of E. coli 23S rRNA or the encoding DNA;

bases 535-560 of E. coli 23S rRNA or the encoding DNA;

bases 540-575 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 1570-1610 of E. coli 23S rRNA or the encoding DNA;

bases 1585-1620 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;

provided that if said target region corresponds to bases 185-225 of E. coli 16S rRNA or the encoding DNA, then said first genus is Mycobacterium;

if said target region corresponds to bases 405-428 of E. coli 16S rRNA or the encoding DNA, then said first genus is Campylobacter,

if said target region corresponds to bases 440-475 of E. coli 16rRNA or the encoding DNA, then said first genus is Campylobacter,

if said target region corersponds to bases 630-675 of E. coli 16S rRNA or the encoding DNA, then said first genus is Legionella,

if said target region corresponds to bases 705-735 of E. coli 16S rRNA or the encoding DNA, then said first genus is Campylobacter,

if said target region corresponds to bases 825-860 of i E. coli 16S rRNA or the encoding DNA, then said first genus is Streptococcus,

if said target region corresponds to bases 975-1020 of E. coli 16S rRNA or the encoding DNA, then said first genus is Legionella,

if said target region corresponds to bases 980-1010 of E. coli 16S rRNA or the encoding DNA, then said first genus is Campylobacter,

if said target region corresponds to bases 1025-1060 of E. coli 16S rRNA or the encoding DNA, then said first genus Mycobacterium,

if said target region corresponds to bases 1125-1155 of E. coli 16S rRNA or the encoding DNA, then said first genus is Salmonella,

if said target region corresponds to bases 350-395 of E. coli 23S rRNA or the encoding DNA, then said first genus is Legionella,

if said target region corresponds to bases 365-405 of E. coli 23S rRNA or the encoding DNA, then said first genus is Pseudomonas,

if said target region corresponds to bases 540-575 of E. coli 23S rRNA or the encodng DNA, then said first genus is Mycobacterium,

if said targer region corresponds to bases 1570-1610 of E. coli 23S rRNA or the encoding DNA, then said first genus is Mycobacterium, and

if said target region corresponds to bases 1585-1620 of E. coli 23S rRNA or the encoding DNA, then said first genus is Legionella; and

b) substantially maximizing complementarity of said nucleotide sequence to said variable region present in each of said two or more target species while substantially minimizing the complementarity of said nucleotide sequence to said variable region present in said one or more non-target species, such that a probe;

target duplex formed between said oligonucleotide probe and each of said two or more target species has a higher T_m than a duplex formed between said oligonucletodide probe and said one or more non-target species;

wherein the percent similarity between the rRNA of said first genus and said second genus is between 90% and 99%.

32. The method of claim 31, wherein said second genus is selected based on it being the phylogenetically most closely related genus to said first genus.

33. The method of claim 31, wherein said target region corresponds to either bases 270-390 of E. coli 23S rRNA or the encoding DNA.

34. The method of claim 33, wherein said target region corresponds to bases 270-390 of E. coli 23S rRNA.

35. The method of claim 31, wherein said target region corresponds to either bases 535-560 of E. coli 23S rRNA or the encoded DNA.

36. The method of claim 35, wherein said target region corresponds to bases 535-560 of E. coli 23S rRNA.

37. The method of claim 31, wherein said target region corresponds to either bases 1150-1200 of E. coli 23S rRNA or the encoding DNA.

38. The method of claim 37, wherein said target region corresponds to bases 1150-1200 of E. coli 23S rRNA.

39. The method of claim 31, wherein said target region corresponds to either bases 1440-1600 of E. coli 23S rRNA or the encoding DNA.

40. The method of any of claim 39, wherein said target region corresponds to bases 1440-1600 of E. coli 23S rRNA.

41. The method of claim 31, wherein said target region corresponds to either bases 1710-1750 of E. coli 23S rRNA or the encoding DNA.

42. The method of claim 41, wherein said target region corresponds to bases 1710-1750 of E. coli 23S rRNA.

43. The method of claim 31, wherein said target region corresponds to either bases 2190-2330 of E. coli 23S rRNA or the encoding DNA.

44. The method of claim 43, wherein said target region corresponds to bases 2190-2330 of E. coli 23S rRNA.

45. The method of claims 33-44, further comprising the step of labeling said oligonucleotide with either an isotopic label or a non-isotopic label.

46. The method of claim 31, further comprising the step of labeling said oligonucleotide with a non-isotopic label selected from the group consisting the fluorescent molecules, chemiluminescent molecules, enzymes, confactors, enzyme substrates, and haptens.

47. The method of claim 46, wherein said non-isotopic label is an acridinium ester.

103. The method of claim 31, wherein said first genus is Mycobacterium and said target region corresponds to bases 185-225 of E. coli 16S rRNA.

104. The method of claim 31, wherein said first genus is Campylobacter and said target region corresponds to either bases 405-428, or 440-475, of E. coli 16S rRNA.

105. The method of claim 31, wherein said first genus is Legionella and said target region corresponds to bases 630-675 of E. coli 16S rRNA.

106. The method of claim 31, wherein said first genus is Streptococcus and said target region corresponds to bases 825-860 of E. coli 16S rRNA.

107. The method of claim 31, wherein said first genus is either Legionella, Campylobacter or Mycobacterium,

provided that if said target species is Legionella, then said target region corresponds to bases 975-1020 of E. coli 16S rRNA,

if said target species is Campylobacter, then said target region corresponds to bases 980-1010, of E. coli 16S rRNA, and

if said target species is Mycobacterium, then said target region corresponds to bases 1025-1060 of E. coli 16S rRNA.

108. The method of claim 31, wherein said first genus is either Legionella, Salmonella, or Pseudomonas,

provided that if said first genus is Legionella, then said target region corresponds to bases 350-395 of E. coli 23S rRNA,

if said first genus is Salmonella, then said target region corresponds to bases 335-375 of E. coli 23S rRNA, and

if said first genus is Pseudomonas, then said target region corresponds to bases 365-405 of E. coli 23S rRNA.

109. The method of claim 31, wherein said first genus is Mycobacterium and said target region corresponds to bases 540-575 of E. coli 23S rRNA.

110. The method of claim 31, wherein said first genus is Mycobacterium and said target region corresponds to bases 1155-1190 of E. coli 23S rRNA.

111. The method of claim 31, wherein said first genus is either Legionella or Mycobacterium,

provided that if said first genus is Mycobacterium, then said target region corresponds to either bases 1440-1475, 1515-1555, or 1570-1610, of E. coli 23S rRNA, and

if said first genus is Legionella, then said target region corresponds to bases 1585-1620 of E. coli 23S rRNA.

112. The method of claim 31, wherein said first genus is either Legionella or Mycobacterium,

provided that if said first genus is Mycobacterium, then said target region corresponds to bases 2195-2235 of E. coli 23S rRNA, and

if said first genus is Legionella, then said target region corresponds to bases 2280-2330 of E. coli 23S rRNA.

123. The method of claim 31, wherein said first genus is Mycobacterium and said target region corresponds to bases 185-225 of E. coli 16S rRNA or the encoding DNA.

124. The method of claim 31, wherein said first genus is Campylobacter and said target region corresponds to either bases 405-428, or 440-475, of E. coli 16S rRNA or the encoding DNA.

125. The method of claim 31, wherein said first genus is Legionella and said target region corresponds to bases 630-675 of E. coli 16S rRNA or the encoding DNA.

126. The method of claim 31, wherein said first genus is Streptococcus and said target region corresponds to bases 825-860 of E. coli 16S rRNA or the encoding DNA.

127. The method of claim 31, wherein said first genus is either Legionella, Campylobacter or Mycobacterium,

provided that if said target species is Legionella, then said target region corresponds to bases 975-1020 of E. coli 16S rRNA or the encoding DNA,

if said target species is Campylobacter, then said target region corresponds to bases 980-1010 of E. coli 16S rRNA or the encoding DNA, and

if said target species is Mycobacterium, then said target region corresponds to bases 1025-1060 of E. coli 16S rRNA or the encoding DNA.

128. The method of claim 31, wherein said first genus is either Legionella, Salmonella, or Pseudomonas,

provided that if said first genus is Legionella, then said target region corresponds to bases 350-395 of E. coli 23S rRNA or the encoding DNA,

if said first genus is Salmonella, then said target region corresponds to bases 335-375, of E. coli 23S rRNA or the encoding DNA, and

if said first genus is Pseudomonas, then said target region corresponds to bases 365-405 of E. coli 23S rRNA or the encoding DNA.

129. The method of claim 31, wherein said first genus is Mycobacterium and said target region corresponds to bases 540-575 of E. coli 23S rRNA or the encoding DNA.

130. The method of claim 31, wherein said first genus is Mycobacterium and said target region corresponds to bases 1155-1190 of E. coli 23S rRNA or the encoding DNA.

131. The method of claim 31, wherein said first genus is either Legionella or Mycobacterium,

provided that if said first genus is Mycobacterium, then said target region corresponds to either bases 1440-1475, 1515-1555, or 1570-1610, of E. coli 23S rRNA or the encoding DNA, and

if said first genus is Legionella, then said target region corresponds to bases 1585-1620 of E. coli 23S rRNA or the encoding DNA.

132. The method of claim 31, wherein said first genus is either Legionella or Mycobacterium,

provided that if said first genus is Mycobacterium, then said target region corresponds to bases 2195-2235 of E. coli 23S rRNA or the encoding DNA, and

if said first genus is Legionella, then said target region corresponds to bases 2280-2330 of E. coli 23S rRNA or the encoding DNA.

135. The method of claim 31, wherein said first genus is Campylobacter and said target region corresponds to bases 705-735 of E. coli 16S rRNA or the encoding DNA.

136. The method of claim 135, wherein said target region corresponds to bases 705-735 of E. coli 16S rRNA.

137. The method of claim 31, wherein said first genus is Salmonella and said target region corresponds to bases 1125-1155 of E. coli 16S rRNA or the encoding DNA.

138. The method of claim 137, wherein said target region corresponds to bases 1125-1155 of E. coli 16S rRNA.

102. The method of claimss 24 o 25, wherein said target species is Chlamydia trachomatis.