Nucleic acid amplification and detection of Mycobacterium species

• US 20050100915A1
• Filed: 09/18/2003
• Published: 05/12/2005
• Est. Priority Date: 12/17/1999
• Status: Active Grant

• Current Assignee: Francisco D. Delgado, Marc Rodrigue, Markus T. Jucker, Philippe Cleuziat, Steven T. Brentano
• Original Assignee: Francisco D. Delgado, Marc Rodrigue, Markus T. Jucker, Philippe Cleuziat, Steven T. Brentano
• Inventors: Brentano, Steven T., Rodrigue, Marc, Cleuziat, Philippe, Jucker, Markus T., Delgado, Francisco D.
• Granted Patent: US 7,294,489 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 Mycobacterium 16S rRNA;

amplifying the Mycobacterium 16S rRNA or Mycobacterium DNA in an in vitro nucleic acid amplification mixture comprising at least one polymerase activity, and a combination of at least two primers having sequences selected from the group consisting of a first primer of SEQ ID NO;

11 and a second primer that is an oligonucleotide consisting of 19 to 25 bases containing 18 contiguous bases of SEQ ID NO;

24 and three to seven bases 5′

to the 18 contiguous bases of SEQ ID NO;

24 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 1, wherein the amplifying step amplifies 16S rRNA or DNA encoding 16S rRNA from M. tuberculosis or a Mycobacterium other than tuberculosis (MOTT) species.

4. The method of claim 1, wherein the amplifying step amplifies 16S rRNA or DNA encoding 16S rRNA from M. abscessus, M. africanum, M. asiaticum, M. avium, M. bovis, M. celatum, M. chelonae, M. flavescens, M. fortuitum, M. gastri, M. gordonae, M. haemophilum, M. intracellulare, M. interjectum, M. intermedium, M. kansasii, M. malmoense, M. marinum, M. non-chromogenicum, M. paratuberculosis, M. phlei, M. scrofulaceum, M. shimodei, M. simiae, M. smegmatis, M. szulgai, M. terrae, M. triviale, M. tuberculosis, M. ulcerans or M. xenopi.

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

6. The method of claim 5, wherein the detecting step uses at least one labeled probe that hybridizes specifically to the amplified Mycobacterium nucleic acid.

7. The method of claim 5, wherein the detecting step uses a plurality of probes that hybridize specifically to the amplified Mycobacterium nucleic acid.

8. The method of claim 1, wherein the amplifying step uses a combination of at least a first primer and a second primer, wherein the first primer is SEQ ID NO:

11, and the second primer is selected from the group consisting of SEQ ID NO;

21, SEQ NO;

22, SEQ ID NO;

23 and SEQ ID NO;

24.

9. The method of claim 8, wherein the amplifying step uses a combination of a first primer and a second primer, wherein the second primer is SEQ ID NO:

21.

10. The method of claim 8, wherein the amplifying step uses a combination of at least a first primer and a second primer, wherein the second primer is SEQ ID NO:

22.

11. The method of claim 8, wherein the amplifying step uses a combination of the first primer and the second primer, wherein the second primer is SEQ ID NO:

23.

12. The method of claim 8, wherein the amplifying step uses a combination of the first primer and the second primer, wherein the second primer is SEQ ID NO:

24.

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 two oligonucleotides, wherein a first oligonucleotide contains a promoter sequence and a sequence that hybridizes to a Mycobacterium 16S rRNA or DNA sequence, and a second oligonucleotide is an oligonucleotide consisting of 19 to 25 bases, containing 18 contiguous bases of SEQ ID NO:

24 and three to seven bases 5′

to the 18 contiguous bases of SEQ ID NO;

24.

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

at least one first oligonucleotide having the sequence of SEQ ID NO;

11, and

at least one second oligonucleotide having the sequence of any one of SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23 and SEQ ID NO;

24.

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

at least one first oligonucleotide of SEQ ID NO;

11, and

at least one second oligonucleotide of SEQ ID NO;

21.

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

at least one first oligonucleotide of SEQ ID NO;

11, and

at least one second oligonucleotide of SEQ ID NO;

23.

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

at least one first oligonucleotide of SEQ ID NO;

11, and

at least one second oligonucleotide of SEQ ID NO;

24.

16. A kit containing one or more oligonucleotides having a sequence selected from the group consisting of SEQ ID NO:

21, SEQ ID NO;

22, SEQ ID NO;

23 and SEQ ID NO;

24.

17. The kit of claim 16, further containing an oligonucleotide of SEQ ID NO:

11.

18. The kit of claim 17, containing

at least one first oligonucleotide of SEQ ID NO:

11, and

at least one second oligonucleotide of any one of SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23 or SEQ ID NO;

25.