Nucleic acid amplification and detection of
View Patent ↗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.
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 of a target-specific sequence that consists of SEQ ID NO:5 that is joined to a 5′ promoter sequence, and the second oligonucleotide is an oligonucleotide consisting of 19 to 25 bases made up of contiguous bases 1 to 18 of SEQ ID NO:24 and optionally three to seven bases 5′ to the contiguous bases 1 to 18 of SEQ ID NO:24 and/or optionally one base 3′ to the contiguous bases 1 to 18 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 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. celatum or M. xenopi.
5. The method of claim 1 , wherein the detecting step uses at least one probe that hybridizes specifically to the amplified Mycobacterlum 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 consists of 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 second primer consists of SEQ ID NO:21.
10. The method of claim 8 , wherein the second primer consists of SEQ ID NO:22.
11. The method of claim 8 , wherein the second primer consists of SEQ ID NO:23.
12. The method of claim 8 , wherein the second primer consists of 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 one first oligonucleotide and at least one second oligonucleotide, wherein the first oligonucleotide consists of target-specific sequence that consists of SEQ ID NO:5 that is joined to a 5′ promoter sequence, and wherein the second oligonucleotide is an oligonucleotide consisting of 19 to 26 bases made up of contiguous bases 1 to 18 of SEQ ID NO:24 and optionally three to seven bases 5′ to the contiguous bases 1 to 18 of SEQ ID NO:24 and/or optionally one base 3′ to the contiguous bases 1 to 18 of SEQ ID NO:24.
14. The composition of claim 13 , wherein the composition comprises:
at least one first oligonucleotide consisting of SEQ ID NO:11, and
at least one second oligonucleotide consisting of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 or SEQ ID NO:24.
15. The composition of claim 14 , wherein the composition comprises:
the at least one first oligonucleotide consisting of SEQ ID NO:11, and
the at least one second oligonucleotide consisting of SEQ ID NO:21.
16. 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 that is mined to a 5′ promoter sequence, and wherein at least one second oligonucleotide consists of 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 , wherein the first oligonucleotide consists of SEQ ID NO:11.
18. The kit of claim 17 , wherein:
the first oligonucleotide consists of SEQ ID NO:11, and
the at least one second oligonucleotide consists of SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.
19. The composition of claim 14 , wherein the composition comprises:
the at least one first oligonucleotide consisting of SEQ ID NO:11, and
the at least one second oligonucleotide consisting of SEQ ID NO:23.
20. The composition of claim 14 , wherein the composition comprises:
the at least one first oligonucleotide consisting of SEQ ID NO:11, and
the at least one second oligonucleotide consisting of SEQ ID NO:24.