COMPOSITIONS, METHODS AND KITS FOR DETERMINING THE PRESENCE OF CHLAMYDOPHILA PNEUMONIAE IN A TEST SAMPLE

• US 20070059735A1
• Filed: 06/06/2006
• Published: 03/15/2007
• Est. Priority Date: 06/06/2005
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Cunningham, Melissa
• Granted Patent: US 7,498,137 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

The present invention relates to oligonucleotides useful for determining the presence of Chlamydophila pneumoniae in a test sample. The oligonucleotides of the present invention may be incorporated into detection probes, capture probes and amplification oligonucleotides, and used in various combinations thereof.

Claims

1. A detection probe for use determining the presence of Chlamydophila pneumoniae in a test sample, said probe being up to 50 bases in length and comprising a target binding region that forms a probe:

target hybrid stable for detection with a target sequence contained within a target region selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3 and SEQ ID NO;

4 under stringent hybridization conditions, wherein said probe does not form a hybrid stable for detection with nucleic acid derived from Chlamydia trachomatis or Chlamydophila psittaci under said conditions.

2. The detection probe of claim 1, wherein the base sequence of said target binding region consists of or is contained within and includes at least 12 of 15 contiguous bases of a base sequence selected from the group consisting of SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23 and SEQ ID NO;

24.

3. The detection probe of claim 1, wherein said probe comprises a base sequence selected from the group consisting of SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23 and SEQ ID NO;

24.

4. The detection probe of claim 1, wherein the base sequence of said probe consists of or is contained within and includes at least 12 of 15 contiguous bases of a base sequence selected from the group consisting of SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23 and SEQ ID NO;

24.

5. The detection probe of claim 1, wherein the base sequence of said probe consists of a base sequence selected from the group consisting of SEQ ID NO:

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9, SEQ ID NO;

10, SEQ ID NO;

11, SEQ ID NO;

12, SEQ ID NO;

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

19, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23 and SEQ ID NO;

24.

6. The detection probe of claim 1, wherein said probe includes a detectable label.

7. The probe of claim 1, wherein said probe is a self-hybridizing probe under said conditions and in the absence of said target sequence.

8. The probe of claim 7, wherein said probe comprises a pair of interacting labels.

9. The probe of claim 1,wherein said target binding region includes at least one 2′

-O-methyl ribonucleotide.

10. The probe of claim 1, wherein said conditions include a temperature of about 60°

C. and a salt concentration of about 0.6 M to about 0.9 M.

11. A composition comprising said probe of claim 1 hybridized to nucleic acid derived from Chlamydophila pneumoniae under said conditions.

12. A method for determining the presence of Chlamydophila pneumoniae in a test sample, said method comprising the steps of:

a) contacting a test sample with said probe of claim 1 under said conditions; and

b) determining whether said probe;

target hybrid is present in said test sample as indication of the presence of Chlamydophila pneumoniae in said test sample.

13. A set of oligonucleotides for use in amplifying a target region of nucleic acid derived from Chlamydophila pneumoniae, said set of oligonucleotides comprising:

a first oligonucleotide having a target binding region that is up to 40 bases in length and contains an at least 12 contiguous base region that is perfectly complementary to an at least 12 contiguous base region present in a first target region selected from the group consisting of SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39 and SEQ ID NO;

40; and

a second oligonucleotide having a target binding region that is up to 40 bases in length and contains an at least 12 contiguous base region that is perfectly complementary to an at least 12 contiguous base region present in a second target region selected from the group consisting of SEQ ID NO;

73, SEQ ID NO;

74, SEQ ID NO;

75 and SEQ ID NO;

76,

wherein said first and second oligonucleotides stably hybridize to said first and second target sequences, respectively, under amplification conditions.

14. The set of oligonucleotides of claim 13, wherein:

said target binding region of said first oligonucleotide comprises a base sequence having at least 12 contiguous bases of a base sequence selected from the group consisting of SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, SEQ ID NO;

62, SEQ ID NO;

63, SEQ ID NO;

64, SEQ ID NO;

65, SEQ ID NO;

66, SEQ ID NO;

67, SEQ ID NO;

68, SEQ ID NO;

69, SEQ ID NO;

70, SEQ ID NO;

71 and SEQ ID NO;

72; and

said target binding region of said second oligonucleotide comprises a base sequence having at least 12 contiguous bases of a base sequence selected from the group consisting of SEQ ID NO;

77, SEQ ID NO;

78, SEQ ID NO;

79, SEQ ID NO;

80, SEQ ID NO;

81, SEQ ID NO;

82, SEQ ID NO;

83, SEQ ID NO;

84, SEQ ID NO;

85, SEQ ID NO;

86, SEQ ID NO;

87, SEQ ID NO;

88, SEQ ID NO;

89, SEQ ID NO;

90, SEQ ID NO;

91, SEQ ID NO;

92, SEQ ID NO;

93, SEQ ID NO;

94, SEQ ID NO;

95 and SEQ ID NO;

96.

15. The set of oligonucleotides of claim 13, wherein:

the base sequence of said first oligonucleotide consists of or is contained within a base sequence selected from the group consisting of SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, SEQ ID NO;

62, SEQ ID NO;

63, SEQ ID NO;

64, SEQ ID NO;

65, SEQ ID NO;

66, SEQ ID NO;

67, SEQ ID NO;

68, SEQ ID NO;

69, SEQ ID NO;

70, SEQ ID NO;

71 and SEQ ID NO;

72, and, optionally, a 5′

sequence that is recognized by an RNA polymerase or that enhances initiation or elongation by an RNA polymerase; and

the base sequence of said second oligonucleotide consists of or is contained within a base sequence selected from the group consisting of SEQ ID NO;

77, SEQ ID NO;

78, SEQ ID NO;

79, SEQ ID NO;

80, SEQ ID NO;

81, SEQ ID NO;

82, SEQ ID NO;

83, SEQ ID NO;

84, SEQ ID NO;

85, SEQ ID NO;

86, SEQ ID NO;

87, SEQ ID NO;

88, SEQ ID NO;

89, SEQ ID NO;

90, SEQ ID NO;

91, SEQ ID NO;

92, SEQ ID NO;

93, SEQ ID NO;

94, SEQ ID NO;

95 and SEQ ID NO;

96, and, optionally, a 5′

sequence that is recognized by an RNA polymerase or that enhances initiation or elongation by an RNA polymerase.

16. The set of oligonucleotides of claim 13, wherein:

the base sequence of said first oligonucleotide consists of a base sequence selected from the group consisting of SEQ ID NO;

45, SEQ ID NO;

46, SEQ ID NO;

47, SEQ ID NO;

48, SEQ ID NO;

49, SEQ ID NO;

50, SEQ ID NO;

51, SEQ ID NO;

52, SEQ ID NO;

53, SEQ ID NO;

54, SEQ ID NO;

55, SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

59, SEQ ID NO;

60, SEQ ID NO;

61, SEQ ID NO;

62, SEQ ID NO;

63, SEQ ID NO;

64, SEQ ID NO;

65, SEQ ID NO;

66, SEQ ID NO;

67, SEQ ID NO;

68, SEQ ID NO;

69, SEQ ID NO;

70, SEQ ID NO;

71 and SEQ ID NO;

72, and, optionally, a 5′

sequence that is recognized by an RNA polymerase or that enhances initiation or elongation by an RNA polymerase; and

the base sequence of said second oligonucleotide consists of a base sequence selected from the group consisting of SEQ ID NO;

77, SEQ ID NO;

78, SEQ ID NO;

79, SEQ ID NO;

80, SEQ ID NO;

81, SEQ ID NO;

82, SEQ ID NO;

83, SEQ ID NO;

84, SEQ ID NO;

85, SEQ ID NO;

86, SEQ ID NO;

87, SEQ ID NO;

88, SEQ ID NO;

89, SEQ ID NO;

90, SEQ ID NO;

91, SEQ ID NO;

92, SEQ ID NO;

93, SEQ ID NO;

94, SEQ ID NO;

95 and SEQ ID NO;

96, and, optionally, a 5′

sequence that is recognized by an RNA polymerase or that enhances initiation or elongation by an RNA polymerase.

17. A method for amplifying a target region of nucleic acid derived from Chlamydophila pneumoniae present in a test sample, said method comprising the steps of:

a) contacting said test sample with said oligonucleotides of claim 13;

and

b) exposing said test sample to said conditions for a period of time sufficient to amplify said target region.

18. The method of claim 17 further comprising the steps of:

c) contacting said test sample with a detection probe, said probe being up to 50 bases in length and comprising a target binding region that forms a probe;

target hybrid stable for detection with a target sequence contained within a target region selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3 and SEQ ID NO;

4 under stringent hybridization conditions, wherein said probe does not form a hybrid stable for detection with nucleic acid derived from Chlamydia trachomatis or Chlamydophila psittaci under said stringent hybridization conditions, and wherein said probe is provided to said test sample prior to, at the same time as, or after step (a); and

d) determining whether said probe;

target hybrid is present in said test sample as an indication of the presence of Chlamydophila pneumoniae in said test sample.

19. The method of claim 17 further comprising the step of contacting said test sample with a capture probe under assay conditions, said capture probe being up to 100 bases in length and comprising a target binding region that forms a stable hybrid with a target sequence contained within a target region selected from the group consisting of SEQ ID NO:

25, SEQ ID NO;

26, SEQ ID NO;

27 and SEQ ID NO;

28 under said assay conditions.

20. The method of claim 19 further comprising the steps of:

c) contacting said test sample with a detection probe, said probe being up to 50 bases in length and comprising a target binding region that forms a probe;

target hybrid stable for detection with a target sequence contained within a target region selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3 and SEQ ID NO;

4 under stringent hybridization conditions, wherein said probe does not form a hybrid stable for detection with nucleic acid derived from Chlamydia trachomatis or Chlamydophila psittaci under said stringent hybridization conditions, and wherein said probe is provided to said test sample prior to, at the same time as, or after step (a); and

d) determining whether said probe;

target hybrid is present in said test sample as an indication of the presence of Chlamydophila pneumoniae in said test sample.

21. A kit for use in determining the presence of Chlamydophila pneumoniae in a test sample, said kit comprising:

a detection probe up to 50 bases in length and comprising a target binding region that forms a hybrid stable for detection with a target sequence contained within a first target region selected from the group consisting of SEQ ID NO;

1, SEQ ID NO;

2, SEQ ID NO;

3 and SEQ ID NO;

4 under stringent hybridization conditions, wherein said probe does not form a hybrid stable for detection with nucleic acid derived from Chlamydia trachomatis or Chlamydophila psittaci under said conditions;

a first oligonucleotide having a target binding region that is up to 40 bases in length and contains an at least 12 contiguous base region that is perfectly complementary to an at least 12 contiguous base region present in a second target region selected from the group consisting of SEQ ID NO;

37, SEQ ID NO;

38, SEQ ID NO;

39 and SEQ ID NO;

40; and

a second oligonucleotide having a target binding region that is up to 40 bases in length and contains an at least 12 contiguous base region that is perfectly complementary to an at least 12 contiguous base region present in a third target region selected from the group consisting of SEQ ID NO;

73, SEQ ID NO;

74, SEQ ID NO;

75 and SEQ ID NO;

76, wherein said first and second oligonucleotides stably hybridize to said second and third target regions, respectively, under amplification conditions.

22. The kit of claim 21 further comprising a capture probe up to 100 bases in length and comprising a target binding region that forms a stable hybrid with a target sequence contained within a fourth target region selected from the group consisting of SEQ ID NO:

25, SEQ ID NO;

26, SEQ ID NO;

27 and SEQ ID NO;

28 under said assay conditions.

23. The kit of claim 22, wherein the base sequence of said target binding region of said capture probe consists of or is contained within and includes at least 12 of 15 contiguous bases of a base sequence selected from the group consisting of SEQ ID NO:

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35 and SEQ ID NO;

36.

24. The kit of claim 22, wherein the base sequence of said target binding region of said capture probe comprises a base sequence selected from the group consisting of SEQ ID NO:

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35 and SEQ ID NO;

36.

25. The kit of claim 21, wherein the base sequence of said capture probe consists of a base sequence selected from the group consisting of SEQ ID NO:

29, SEQ ID NO;

30, SEQ ID NO;

31, SEQ ID NO;

32, SEQ ID NO;

33, SEQ ID NO;

34, SEQ ID NO;

35 and SEQ ID NO;

36 and an immobilized probe binding region having a base sequence that does not form a stable hybrid with nucleic acid derived from Chlamydophila pneumoniae, Chlamydia trachomatis or Chlamydophila psittaci under said assay conditions.