Nucleic Acid Amplification and Detection of Mycobacterium Species

• US 20090029360A1
• Filed: 11/12/2007
• Published: 01/29/2009
• Est. Priority Date: 12/17/1999
• Status: Active Grant

• Current Assignee: Biomerieux SA (Compagnie Merieux Alliance SAS), Gen-Probe Incorporated (Hologic Incorporated)
• Original Assignee: Biomerieux SA (Compagnie Merieux Alliance SAS), Gen-Probe Incorporated (Hologic Incorporated)
• Inventors: Cleuziat, Philippe, Brentano, Steven T., Rodrigue, Marc, Jucker, Markus T., Delgado, Francisco D.
• Granted Patent: US 7,879,581 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

Oligonucleotides used to prime in vitro nucleic acid amplification of 16S rRNA sequences or DNA encoding 16S rRNA sequences for many species within the genus Mycobacterium are disclosed. Kits including such oligonucleotides are disclosed. Methods of detecting Mycobacterium species using the oligonucleotides in in vitro nucleic acid amplification are disclosed.

Claims

1. A method of detecting Mycobacterium species present in a biological sample, comprising the steps of:

providing a biological sample containing nucleic acid from at least one Mycobacterium species comprising a Mycobacterium 16S ribosomal RNA (rRNA) or DNA encoding a Mycobacterium 16S rRNA;

amplifying the Mycobacterium 16S rRNA or Mycobacterium DNA encoding the Mycobacterium 16S rRNA in an in vitro nucleic acid amplification mixture comprising at least one polymerase activity, and a combination of at least one first oligonucleotide and at least one second oligonucleotide, wherein the first oligonucleotide consists a target-specific sequence that consists of SEQ ID NO;

5 joined to a 5′

promoter sequence or SEQ ID NO;

11, and the second oligonucleotide consists of SEQ ID NO;

16 to produce amplified Mycobacterium nucleic acid; and

detecting the amplified Mycobacterium nucleic acid by detecting a label associated with the amplified Mycobacterium nucleic acid.

2. The method of claim 1, further comprising in the steps of:

adding to the biological sample at least one capture oligonucleotide that specifically hybridizes to the Mycobacterium 16S rRNA and an immobilized nucleic acid that hybridizes to the capture oligonucleotide under hybridizing conditions to produce a hybridization complex; and

separating the hybridization complex from other components of the biological sample before the amplifying step.

3. The method of claim 2, wherein the capture oligonucleotide consists of SEQ ID NO:

35.

4. The method of claim 1, wherein the amplifying step amplifies 16S rRNA or DNA encoding 16S rRNA from Mycobacterium tuberculosis.

5. The method of claim 1, wherein the amplifying step amplifies 16S rRNA or DNA encoding 16S rRNA from a Mycobacterium other than tuberculosis (MOTT) species.

6. The method of claim 5, wherein the amplifying step amplifies 16S rRNA or DNA encoding 16S rRNA from M. bovix, M. avium, M. intracellulare, M. kansasii, M. gastri, M. scrofulaceum, M. terrae, or M. xenopi.

7. The method of claim 1, wherein the detecting step uses at least one probe that hybridizes specifically to the amplified Mycobacterium nucleic acid.

8. The method of claim 7, wherein the detecting step uses at least one labeled probe that hybridizes specifically to the amplified Mycobacterium nucleic acid.

9. The method of claim 7, wherein the detecting step uses a plurality of probes that hybridize specifically to the amplified Mycobacterium nucleic acid.

10. The method of claim 1, wherein the second oligonucleotide consists of SEQ ID NO:

16 that contains a modified K-base at the twenty-fifth position.

11. The method of claim 1, wherein the amplifying step uses a combination of the first oligonucleotide that consists of SEQ ID NO:

11, and the second oligonucleotide that consists of SEQ ID NO;

16.

12. The method of claim 1, wherein the amplifying step uses a combination of the first oligonucleotide that consists of SEQ ID NO:

11, the second oligonucleotide that consists of SEQ ID NO;

16, and a third oligonucleotide that consists of SEQ ID NO;

37.

13. A composition for amplifying in an in vitro amplification reaction a Mycobacterium 16S rRNA sequence or a DNA encoding 16S rRNA, comprising a combination of at least one first oligonucleotide and at least one second oligonucleotide, wherein the first oligonucleotide consists of a target-specific sequence that consists of SEQ ID NO:

5 joined to a 5′

promoter sequence or SEQ ID NO;

11, and wherein the second oligonucleotide consists of SEQ ID NO;

16.

14. The composition of claim 13, wherein the composition comprises:

the first oligonucleotide consisting of SEQ ID NO;

11, and

the second oligonucleotide consisting of SEQ ID NO;

16.

15. The composition of claim 13, wherein the second oligonucleotide consisting of SEQ ID NO:

16 contains a modified K-base at the twenty-fifth position.

16. The composition of claim 13, wherein the composition further comprises a third oligonucleotide consisting of SEQ ID NO:

37.

17. A kit containing at least a pair of oligonucleotides, wherein at least one first oligonucleotide consists of a target-specific sequence that consists of SEQ ID NO:

5 joined to a 5′

promoter sequence or SEQ ID NO;

11, and wherein at least one second oligonucleotide consists of SEQ ID NO;

16.

18. The kit of claim 17, wherein the oligonucleotide that consists of SEQ ID NO:

16 contains a modified K-base at the twenty-fifth position.

19. The kit of claim 17, further containing a third oligonucleotide consisting of SEQ ID NO:

37.

20. The kit of claim 17, further containing a third oligonucleotide consisting of SEQ ID NO:

35.