Compositions and methods for detecting BV-associated bacterial nucleic acid
View Patent ↗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 two oligomers for detecting in a sample a Gardnerella vaginalis (GV) 16S rRNA or a gene encoding the 16S rRNA, the oligomer combination comprising:
first and second amplification oligomers for amplifying a GV nucleic acid target region corresponding to the GV 16S rRNA or the gene encoding the 16S-rRNA, wherein
(a) the first amplification oligomer comprises a nucleotide sequence, wherein the nucleotide sequence consists of a first target-hybridizing sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, and wherein the first amplification oligomer does not comprise an additional target hybridizing sequence; and
(b) the second amplification oligomer is a promoter primer or promoter provider and comprises a first region nucleotide sequence and a second region nucleotide sequence, wherein (i) the first region nucleotide sequence consists of a second target-hybridizing sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:39, and (ii) the second region nucleotide sequence consists of a promoter sequence located 5′ to the second target-hybridizing sequence, and wherein the second amplification oligomer does not comprise an additional target hybridizing sequence.
2. The combination of at least two oligomers as in claim 1 , wherein the at least two oligomers are in a kit for amplification of a Gardnerella vaginalis target nucleic acid.
3. A method for detecting in a sample a Gardnerella vaginalis (GV) target nucleic acid, wherein the target nucleic acid is a GV 16S rRNA or a gene encoding the 16S rRNA, the method comprising:
(a) contacting a sample, the sample suspected of containing a GV bacterium, with at least two oligomers for amplifying a GV nucleic acid target region corresponding to the target nucleic acid, the oligomer combination comprising
(i) a first amplification oligomer comprising a nucleotide sequence, wherein the nucleotide sequence consists of a first target-hybridizing sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:8, and wherein the first amplification oligomer does not comprise an additional target hybridizing sequence; and
(ii) a second amplification oligomer that is a promoter primer or promoter provider and that comprises a first region nucleotide sequence and a second region nucleotide sequence, wherein (i) the first region nucleotide sequence consists of a second target-hybridizing sequence selected from the group consisting of SEQ ID NO:36, SEQ ID NO:38, and SEQ ID NO:39, and (ii) the second region nucleotide sequence consists of a promoter sequence located 5′ to the second target-hybridizing sequence, and wherein the second amplification oligomer does not comprise a target hybridizing region in addition to the second target hybridizing region;
(b) performing an in vitro nucleic acid amplification reaction, wherein any GV target nucleic acid present in the sample is used as a template for generating an amplification product; and
(c) detecting the presence or absence of the amplification product, thereby indicating the presence or absence of GV in the sample.
4. The method of claim 3 , wherein the promoter sequence is a T7 promoter sequence has the sequence shown in SEQ ID NO:87.
5. The method of claim 4 , wherein the second amplification oligomer comprises a nucleotide sequence that consists of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:5.
6. The method of claim 3 , further comprising a step of purifying the GV target nucleic acid from other components in the sample before step (a), 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 the target-hybridizing sequence is selected from the group consisting of SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO:46.
7. The method of claim 3 , wherein the detecting step (c) comprises contacting the 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 GV in the sample.
8. The method of claim 7 , wherein the detection probe oligomer is selected from the group consisting of:
a detection probe oligomer comprising a target-hybridizing sequence that is from about 14 to about 35 nucleotides in length and is configured to specifically hybridize to a target sequence contained within SEQ ID NO:88 from about nucleotide position 164 to about nucleotide position 205;
a detection probe oligomer comprising a target hybridizing sequence contained in the sequence of SEQ ID NO:84 and includes at least the sequence of SEQ ID NO:83; and
a detection probe oligomer comprising a target hybridizing sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24.
9. The method of claim 8 , wherein the detection probe 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 of (a), (b), and (c).
10. The method of claim 8 , wherein the detecting step (c) occurs during the amplifying step (b).
11. The method of claim 8 , wherein the detection probe further comprises a non-target-hybridizing sequence.
12. The method of claim 11 , wherein the detection probe is a hairpin detection probe a TaqMan detection probe, a molecular beacon, or a molecular torch.
13. The method of claim 3 , wherein the amplification reaction at step (b) is an isothermal amplification reaction, or is a PCR amplification reaction.
14. The method of claim 13 , wherein the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction or wherein the isothermal amplification reaction is a reverse TMA reaction.
15. The combination of claim 1 , wherein the promoter sequence consists of SEQ ID NO:87.
16. The combination of claim 1 , wherein the second amplification oligomer comprises a nucleotide sequence consisting of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 5.