Compositions and methods for detecting BV-associated bacterial nucleic acid
Disclosed are nucleic acid oligomers, including amplification oligomers, capture probes, and detection probes, for detection of a 16S rRNA or its encoding gene from bacterial species associated with bacterial vaginosis. Also disclosed are methods of specific nucleic acid amplification and detection using the disclosed oligomers, as well as corresponding reaction mixtures and kits.
1. A combination of at least three oligomers for detecting in a sample a Megasphaera sp. 16S rRNA or a gene encoding a Megasphaera sp.16S rRNA, said oligomer combination comprising:
a first amplification oligomer comprising a first target-hybridizing sequence selected from the group consisting of SEQ ID NO:13 and SEQ ID NO:14; a second amplification oligomer comprising a second target-hybridizing sequence that is SEQ ID NO:19, and wherein the second amplification oligomer is a promoter primer further comprising a T7 promoter sequence located 5′ to the target-hybridizing sequence; and a detection probe oligomer comprising a target-hybridizing sequence that is contained within the sequence of SEQ ID NO:31 and includes at least the sequence of SEQ ID NO:30.
2. The combination of at least three oligomers of claim 1 , wherein the nucleotide sequence of the second amplification oligomer consists of the sequence shown in SEQ ID NO:16.
3. The combination of at least three oligomers of claim 1 , wherein the detection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:25.
4. A kit comprising the combination of at least three oligomers as in claim 1 .
5. A method for detecting in a sample a Megasphaera sp. target nucleic acid, wherein the target nucleic acid is a Megasphaera sp. 16S rRNA or a gene encoding said 16S rRNA, said method comprising:
(a) providing a sample, wherein said sample is suspected of containing a Megasphaera sp. bacterium;
(b) contacting said sample with at least two oligomers for amplifying a Megasphaera sp. nucleic acid target region corresponding to the target nucleic acid, said oligomer combination comprising
a first amplification oligomer comprising a first target-hybridizing sequence selected from the group consisting of SEQ ID NO:13 and SEQ ID NO:14; and a second amplification oligomer comprising a second target-hybridizing sequence that is SEQ ID NO:19, and wherein the second amplification oligomer is a promoter primer further comprising a promoter sequence located 5′ to the target-hybridizing sequence;
(c) performing an in vitro nucleic acid amplification reaction, wherein any Megasphaera target nucleic acid present in said sample is used as a template for generating an amplification product; and
(d) detecting the presence or absence of the amplification product during the amplifying step (c), thereby indicating the presence or absence of Megasphaera sp. in said sample, wherein the detecting step comprises contacting said in vitro nucleic acid amplification reaction with a detection probe oligomer configured to specifically hybridize to the amplification product under conditions whereby the presence or absence of the amplification product is determined, thereby indicating the presence or absence of Megasphaera sp. in said sample, and wherein the detection probe oligomer comprises a target-hybridizing sequence that is contained within the sequence of SEQ ID NO:31 and includes at least the sequence of SEQ ID NO:30.
6. The method of claim 5 , wherein the promoter sequence is a T7 promoter sequence.
7. The method of claim 5 , wherein the nucleotide sequence of the second amplification oligomer consists of the sequence shown in SEQ ID NO:16.
8. The method of claim 5 , further comprising purifying the Megasphaera target nucleic acid from other components in the sample before step (b).
9. The method of claim 8 , wherein the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently attached to a sequence or moiety that binds to an immobilized probe, wherein said target-hybridizing sequence is selected from the group consisting of SEQ ID NOs:7-12.
10. The method of claim 5 , wherein the detection probe oligomer target-hybridizing sequence is selected from the group consisting of SEQ ID NO:23 and SEQ ID NO:25.
11. The method of claim 5 , wherein the detection probe oligomer comprises a label selected from the group consisting of
(a) a chemiluminescent label;
(b) a fluorescent label;
(c) a quencher; and
(d) a combination of two or more labels, wherein each of the two or more labels is independently selected from the group consisting of (a), (b), and (c).
12. The method of claim 5 , wherein the amplification reaction at step (c) is an isothermal amplification reaction.
13. The combination of at least three oligomers of claim 1 , wherein the detection probe oligomer target-hybridizing sequence is SEQ ID NO:25.
14. The method of claim 5 , wherein the detection probe oligomer target-hybridizing sequence is SEQ ID NO:25.