Compositions, kits and related methods for the detection and/or monitoring of

• US 8,927,703 B2
• Filed: 04/02/2008
• Issued: 01/06/2015
• Est. Priority Date: 04/02/2007
• Status: Active Grant

• Current Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Kaplan, Shannon K., Livezey, Kristin, Bungo, Jennifer J., Hogan, James J.
• Primary Examiner(s): WOOLWINE, SAMUEL C
• Application Number:
  US12/061,438
• Publication Number:
  US 20080268452A1
• Time in Patent Office: 2,470 Days
• Field of Search: None
• US Class Current: 536/24.32
• CPC Class Codes: C12Q 1/689 for bacteria

Abstract

The present invention provides compositions, methods and kits for the species-specific detection of Pseudomonas aeruginosa.

Claims

1. A composition for use in a Pseudomonas aeruginosa nucleic acid amplification assay comprising a T7 provider oligo and a non-T7 primer oligo, wherein the T7 provider oligo is selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

1, SEQ ID NO;

11 and SEQ ID NO;

14, which targets a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases from 725-825 of E. coli 23s rRNA, and the non-T7 primer oligo is selected from the group consisting of SEQ ID NO;

22, SEQ ID NO;

24, SEQ ID NO;

19 and SEQ ID NO;

15, which targets the complement of a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases from 845-950 of E. coli 23s rRNA.

2. The composition of claim 1, where the T7 provider is selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

1, which targets a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases 725-775 of E. coli 23s rRNA, and the non-T7 primer is selected from the group consisting of SEQ ID NO;

22 and SEQ ID NO;

24, which targets the complement of a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases 900-950 of E. coli 23s rRNA.

3. The composition of claim 1, where the T7 provider is SEQ ID NO:

2 and the non-T7 primer is SEQ ID NO;

22.

4. The composition of claim 1, further comprising a detection oligo.

5. The composition of claim 4, where the detection oligo is a torch oligo or molecular beacon.

6. The composition of claim 5, where the torch oligo is selected from the group consisting of SEQ ID NO:

51, SEQ ID NO;

54, SEQ ID NO;

50 and SEQ ID NO;

56.

7. The composition of claim 1, further comprising an extend oligo.

8. The composition of claim 7, where the extend oligo is selected from the group consisting of SEQ ID NO:

43 and SEQ ID NO;

44.

9. The composition of claim 1, further comprising a blocker oligo.

10. The composition of claim 9, wherein the blocker oligo is selected from the group consisting of SEQ ID NO:

29, SEQ ID NO;

26, SEQ ID NO;

40 and SEQ ID NO;

42.

11. A composition for use in a transcription-based Pseudomonas aeruginosa nucleic acid amplification assay comprising the T7 provider oligo, SEQ ID NO:

2 and the non-T7 primer oligo, SEQ ID NO;

24.

12. The composition of claim 11, further comprising any one of the blocker oligo SEQ ID NO:

29, the torch oligo SEQ ID NO;

54, the extend oligo SEQ ID NO;

44, and the target capture oligo SEQ ID NO;

69.

13. The composition of claim 12, further comprising the target capture helper oligo SEQ ID NO:

73.

14. A kit for use in a Pseudomonas aeruginosa amplification assay comprising a T7 provider oligo and a non-T7 primer oligo, wherein the T7 provider oligo is selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

1, SEQ ID NO;

11 and SEQ ID NO;

14, which targets a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases from 725-825 of E. coli 23s rRNA, and the non-T7 primer oligo is selected from the group consisting of SEQ ID NO;

22, SEQ ID NO;

24, SEQ ID NO;

19 and SEQ ID NO;

15, which targets the complement of a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases from 845-950 of E. coli 23s rRNA.

15. The kit of claim 14, where the T7 provider is selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

1, which targets a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases 725-775 of E. coli 23s rRNA, and the non-T7 primer is selected from the group consisting of SEQ ID NO;

22 and SEQ ID NO;

24, which targets the complement of a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases 900-950 of E. coli 23s rRNA.

16. The kit of claim 14, where the T7 provider is SEQ ID NO:

2 and the non-T7 primer is SEQ ID NO;

22.

17. The kit of claim 14, further comprising a detection oligo.

18. The kit of claim 17, where the detection oligo is a torch oligo or molecular beacon.

19. The kit of claim 18, where the torch oligo is selected from the group consisting of SEQ ID NO:

51, SEQ ID NO;

54, SEQ ID NO;

50 and SEQ ID NO;

56.

20. The kit of claim 14, further comprising an extend oligo.

21. The kit of claim 20, where the extend oligo is selected from the group consisting of SEQ ID NO:

43 and SEQ ID NO;

44.

22. The kit of claim 14, further comprising a blocker oligo.

23. The kit of claim 22, wherein the blocker oligo is selected from the group consisting of SEQ ID NO:

29 SEQ ID NO;

26, SEQ ID NO;

40 and SEQ ID NO;

42.

24. A kit for use in a transcription-based Pseudomonas aeruginosa nucleic acid amplification assay comprising the T7 provider oligo, SEQ ID NO:

2 and the non-T7 primer oligo, SEQ ID NO;

24.

25. The kit of claim 24, further comprising anyone of the blocker oligo SEQ ID NO:

29, the torch oligo SEQ ID NO;

54, the extend oligo SEQ ID NO;

44, and the target capture oligo SEQ ID NO;

69.

26. The kit of claim 25, further comprising the target capture helper oligo SEQ ID NO:

73.

27. A method for detecting the presence of Pseudomonas aeruginosa in a sample, said method comprising performing a nucleic acid amplification assay using a T7 provider oligo and a non-T7 primer oligo, wherein the T7 provider oligo is selected from the group consisting of SEQ ID NO:

2, SEQ ID NO;

1, SEQ ID NO;

11 and SEQ ID NO;

14, which targets a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases from 725-825 of E. coli 23s rRNA, and the non-T7 primer oligo is selected from the group consisting of SEQ ID NO;

22, SEQ ID NO;

24, SEQ ID NO;

19 and SEQ ID NO;

15, which targets the complement of a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases from 845-950 of E. coli 23s rRNA.

28. The method of claim 27, where the T7 provider is selected from the group consisting of SEQ ID NO:

2 and SEQ ID NO;

1, which targets a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases 725-775 of E. coli 23s rRNA, and the non-T7 primer is selected from the group consisting of SEQ ID NO;

22 and SEQ ID NO;

24, which targets the complement of a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases 900-950 of E. coli 23s rRNA.

29. The method of claim 28, further comprising detecting amplified nucleic acid with a detection oligo.

30. The method of claim 29, where the detection oligo is a torch oligo or molecular beacon.

31. The method of claim 30, where the torch oligo is selected from the group consisting of SEQ ID NO:

51, SEQ ID NO;

54, SEQ ID NO;

50 and SEQ ID NO;

56.

32. The method of claim 30 wherein detecting said amplified nucleic acid occurs in real-time.

33. The method of claim 32 wherein P. aeruginosa nucleic acid is specifically detected in the presence of closely related Pseudomonads.

34. The method of claim 32 wherein the detection cut-off is set whereby P. aeruginosa nucleic acid and one or more closely related but not all Pseudomonads are detected.

35. The method of claim 28, further comprising a blocker oligo.

36. The method of claim 35, wherein the blocker oligo is selected from the group consisting of SEQ ID NO:

29, SEQ ID NO;

26, SEQ ID NO;

40 and SEQ ID NO;

42.

37. The method of claim 27, where the T7 provider is SEQ ID NO:

2, which targets a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases 739-766 of E. coli 23s rRNA, and the non-T7 primer is SEQ ID NO;

24, which targets the complement of a sequence in a region of Pseudomonas aeruginosa nucleic acid corresponding to bases 918-943 of E. coli 23s rRNA.

38. The method of claim 27, where the T7 provider is SEQ ID NO:

2 and the non-T7 primer is SEQ ID NO;

22.

39. The method of claim 27, further comprising an extend oligo.

40. The method of claim 39, where the extend oligo is selected from the group consisting of SEQ ID NO:

43 and SEQ ID NO;

44.

41. The method of claim 27, where the amplification assay is transcription-based.

42. The method of claim 41, where the method further includes the use of anyone of the blocker oligo SEQ ID NO:

29, the torch oligo SEQ ID NO;

54, the extend oligo SEQ ID NO;

44, and the target capture oligo SEQ ID NO;

69.

43. The method of claim 42, where the method further includes the use of the target capture helper oligo, SEQ ID NO:

73.