METHODS FOR DETECTING BACTERIAL NUCLEIC ACID
Disclosed are methods for diagnosing Bacterial Vaginosis in a subject comprising performing an assay for the detection of any one or more of Lactobacillus sp., Atopobium vaginae , and Gardneralla vaginalis in a subject sample. Also disclosed are compositions and methods for detecting Lactobacillus sp., Atopobium vaginae , and/or Gardneralla vaginalis nucleic acid in a sample.
1 . A multiplex method for determining the presence or absence of each of Lactobacillus sp., A. vaginae , and G. vaginalis in a sample, the method comprising:
(1) contacting a sample, said sample suspected of containing at least one of Lactobacillus sp., A. vaginae , and G. vaginalis , with
(a) first, second, third, and fourth Lactobacillus -specific amplification oligomers for amplifying a target region of a Lactobacillus sp. target nucleic acid, wherein (i) the first Lactobacillus -specific amplification oligomer comprises a first Lactobacillus -specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:10; (ii) the second Lactobacillus -specific amplification oligomer comprises a second Lactobacillus -specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:7; (iii) the third Lactobacillus -specific amplification oligomer comprises a third Lactobacillus -specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:8; and (iv) the fourth Lactobacillus -specific amplification oligomer comprises a fourth Lactobacillus -specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:9;
(b) first and second A. vaginae -specific amplification oligomers for amplifying a target region of a A. vaginae target nucleic acid, wherein (i) the first A. vaginae -specific amplification oligomer comprises a first A. vaginae -specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:18 and (ii) the second A. vaginae -specific amplification oligomer comprises a second A. vaginae -specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:17; and
(c) first and second G. vaginalis -specific amplification oligomers for amplifying a target region of a G. vaginalis target nucleic acid, wherein (i) the first G. vaginalis -specific amplification oligomer comprises a first G. vaginalis -specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO:15 and (ii) the second G. vaginalis -specific amplification oligomer comprises a second G. vaginalis -specific target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:14;
(2) performing an in vitro nucleic acid amplification reaction, wherein any Lactobacillus sp., A. vaginae , and G. vaginalis target nucleic acid, if present in the sample, is used as a template for generating one or more amplification products corresponding to the Lactobacillus sp., A. vaginae , and G. vaginalis target regions; and
(3) detecting the presence or absence of the one or more amplification products, thereby determining the presence or absence of Lactobacillus sp., A. vaginae , and G. vaginalis in the sample.
2 . The multiplex method of claim 1 , wherein at least one of the first Lactobacillus -specific amplification oligomer, the first A. vaginae -specific amplification oligomer, and the first G. vaginalis -specific amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located 5′ to the respective target hybridizing sequence.
3 . The multiplex method of claim 2 , wherein the promoter sequence is a T7 promoter sequence,
optionally wherein the T7 promoter sequence has the nucleotide sequence of residues 1-27 of SEQ ID NO:10.
4 . The multiplex method of claim 1 , further comprising purifying the Lactobacillus sp., A. vaginae , and G. vaginalis target nucleic acids, if present, from other components in the sample before step (2).
5 . The multiplex method of claim 4 , wherein the purifying step comprises contacting the sample with a first capture probe oligomer comprising a target-hybridizing sequence that specifically hybridizes to a target sequence within the Lactobacillus sp. target nucleic acid and a second capture probe oligomer comprising a target-hybridizing sequence that specifically hybridizes to a target sequence within each of the A. vaginae and G. vaginalis target nucleic acids,
wherein each of the first and second capture probe target-hybridizing sequences is covalently attached to a sequence or moiety that binds to an immobilized probe.
6 . The multiplex method of claim 5 , wherein
the first capture probe target-hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO:6, and/or
the second capture probe target-hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO:13.
7 . The multiplex method of claim 1 , wherein the detecting step (3) comprises
(i) contacting the one or more amplification products with a first Lactobacillus -specific detection probe comprising a target-hybridizing sequence that specifically hybridizes to the Lactobacillus sp. target region, a first A. vaginae -specific detection probe comprising a target-hybridizing sequence that specifically hybridizes to the A. vaginae target region, and a first G. vaginalis -specific detection probe comprising a target-hybridizing sequence that specifically hybridizes to the G. vaginalis target region, and
(ii) detecting the presence or absence of any target-hybridized Lactobacillus -specific, A. vaginae -specific, and/or G. vaginalis -specific detection probe.
8 . The multiplex method of claim 7 , wherein the first Lactobacillus -specific detection probe target-hybridizing sequence specifically hybridizes to a target region of each of L. crispatus and L. jensenii target nucleic acid and the method further comprises contacting the one or more amplification products with a second Lactobacillus -specific detection probe comprising a target-hybridizing sequence that specifically hybridizes to a target region of L. gasseri target nucleic acid.
9 . The multiplex method of claim 8 , wherein the first Lactobacillus -specific detection probe target-hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-17 of SEQ ID NO:11 and/or the second Lactobacillus -specific detection probe target-hybridizing sequence substantially corresponds to the nucleotide sequence of residues 7-23 of SEQ ID NO:12.
10 . The multiplex method of claim 7 , wherein
the first A. vaginae -specific detection probe target-hybridizing sequence substantially corresponds to the nucleotide sequence of residues 6-21 of SEQ ID NO:19, and/or
the first G. vaginalis -specific detection probe target-hybridizing sequence comprises the nucleotide sequence of residues 1-18 of SEQ ID NO:16.
11 . The multiplex method of claim 7 , wherein the detecting step (3) occurs during the amplifying step (2).
12 . The multiplex method of claim 11 , wherein each detection probe comprises a fluorescent label and a quencher.
13 . The multiplex method of claim 7 , wherein at least one of the first Lactobacillus -specific detection probe, the first A. vaginae -specific detection probe, and the first G. vaginalis -specific detection probe further comprises a non-target-hybridizing sequence.
14 . The multiplex method of claim 13 , wherein each of the first Lactobacillus -specific detection probe, the first A. vaginae -specific detection probe, and the first G. vaginalis -specific detection probe is a molecular torch or a molecular beacon.
15 . The multiplex method of claim 13 , wherein the first Lactobacillus -specific detection probe target-hybridizing sequence specifically hybridizes to a target region of each of L. crispatus and L. jensenii target nucleic acid and the method further comprises contacting the one or more amplification products with a second Lactobacillus -specific detection probe comprising a target-hybridizing sequence that specifically hybridizes to a target region of L. gasseri target nucleic acid, wherein the second Lactobacillus -specific detection probe further comprises a non-target-hybridizing sequence,
optionally wherein the second Lactobacillus -specific detection probe is a molecular torch or a molecular beacon.
16 . The multiplex method of claim 1 , wherein the amplification reaction at step (2) is an isothermal amplification reaction,
optionally wherein the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction.
17 . The multiplex method of claim 16 , wherein the isothermal amplification reaction is a real-time amplification reaction.
18 . A method for determining the presence or absence of Lactobacillus sp. in a sample, the method comprising:
(1) contacting a sample, said sample suspected of containing Lactobacillus sp., with first, second, third, and fourth amplification oligomers for amplifying a target region of a Lactobacillus sp. target nucleic acid, wherein (i) the first amplification oligomer comprises a first target-hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:10; (ii) the amplification oligomer comprises a second target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:7; (iii) the third amplification oligomer comprises a third target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:8; and (iv) the fourth amplification oligomer comprises a fourth target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:9;
(2) performing an in vitro nucleic acid amplification reaction, wherein any Lactobacillus sp. target nucleic acid, if present in the sample, is used as a template for generating one or more amplification products corresponding to the Lactobacillus sp. target region; and
(3) detecting the presence or absence of the one or more amplification products, thereby determining the presence or absence of Lactobacillus sp. in the sample.
19 . A method for determining the presence or absence of A. vaginae in a sample, the method comprising:
(1) contacting a sample, said sample suspected of containing A. vaginae , with first and second amplification oligomers for amplifying a target region of a A. vaginae target nucleic acid, wherein (i) the first amplification oligomer comprises a first target-hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:18 and (ii) the second amplification oligomer comprises a second target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:17;
(2) performing an in vitro nucleic acid amplification reaction, wherein any A. vaginae target nucleic acid, if present in the sample, is used as a template for generating one or more amplification products corresponding to the A. vaginae target region; and
(3) detecting the presence or absence of the one or more amplification products, thereby determining the presence or absence of A. vaginae in the sample.
20 . A method for determining the presence or absence of G. vaginalis in a sample, the method comprising:
(1) contacting a sample, said sample suspected of containing G. vaginalis , with first and second amplification oligomers for amplifying a target region of a G. vaginalis target nucleic acid, wherein (i) the first amplification oligomer comprises a first target-hybridizing sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO:15 and (ii) the second amplification oligomer comprises a second target-hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:14;
(2) performing an in vitro nucleic acid amplification reaction, wherein any G. vaginalis target nucleic acid, if present in the sample, is used as a template for generating one or more amplification products corresponding to the G. vaginalis target region; and
(3) detecting the presence or absence of the one or more amplification products, thereby determining the presence or absence of G. vaginalis in the sample.