Methods and compositions 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., Gardneralla vaginalis , and Eggerthella sp. in a subject sample. Also disclosed are methods and compositions for detecting Lactobacillus sp., Gardneralla vaginalis , and/or Eggerthella nucleic acid in a sample.
1. A multiplex method for determining the presence or absence of each of Lactobacillus sp., G. vaginalis , and Eggerthella sp. in a clinical sample, the method comprising:
(1) contacting a clinical sample, said sample suspected of containing at least one of Lactobacillus sp., G. vaginalis , and Eggerthella sp., with
(a) first, second, and third 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 consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:7, (ii) the second Lactobacillus -specific amplification oligomer comprises a second Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:6, and (iii) the third Lactobacillus -specific amplification oligomer comprises a third Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:8;
(b) 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 consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:13 and (ii) the second G. vaginalis -specific amplification oligomer comprises a second G. vaginalis -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:12; and
(c) first and second Eggerthella -specific amplification oligomers for amplifying a target region of an Eggerthella sp. target nucleic acid, wherein (i) the first Eggerthella -specific amplification oligomer comprises a first Eggerthella -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-51 of SEQ ID NO:17 and (ii) the second Eggerthella -specific amplification oligomer comprises a second Eggerthella -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:16;
wherein each of the first Lactobacillus -specific amplification oligomer, the third Lactobacillus -specific amplification oligomer, the first G. vaginalis -specific amplification oligomer, and the first Eggerthella specific amplification oligomer is a promoter primer or promoter provider further comprising a T7 promoter sequence located 5′ to the respective target-hybridizing sequence;
(2) performing an in vitro nucleic acid amplification reaction, wherein any Lactobacillus sp., G. vaginalis , and Eggerthella 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., G. vaginalis , and Eggerthella sp. 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., G. vaginalis , and Eggerthella sp. in the sample.
2. The method of claim 1 , wherein
the nucleotide sequence of the first Lactobacillus -specific amplification oligomer consists of the sequence of SEQ ID NO:7;
the nucleotide sequence of the third Lactobacillus -specific amplification oligomer consists of the sequence of SEQ ID NO:8;
the nucleotide sequence of the first G. vaginalis -specific amplification oligomer consists of the sequence of SEQ ID NO:13; and/or
the nucleotide sequence of the first Eggerthella -specific amplification oligomer consists of the sequence of SEQ ID NO:17.
3. The method of claim 1 , further comprising purifying the Lactobacillus, G. vaginalis , and Eggerthella target nucleic acids, if present, from other components in the sample before step (2).
4. The method of claim 1 , wherein the detecting step (3) comprises
contacting the one or more amplification products with a first Lactobacillus -specific detection probe that specifically hybridizes to the Lactobacillus sp. target region, a first G. vaginalis -specific detection probe that specifically hybridizes to the G. vaginalis target region, and a first Eggerthella -specific detection probe that specifically hybridizes to the Eggerthella sp. target region, and
detecting the presence or absence of any target-hybridized Lactobacillus -specific, G. vaginalis -specific, and/or Eggerthella -specific detection probe.
5. The method of claim 4 , wherein
the first Lactobacillus -specific detection probe comprises a target-hybridizing sequence consisting of SEQ ID NO:9,
the first G. vaginalis -specific detection probe comprises a target-hybridizing sequence consisting of residues 1-19 of SEQ ID NO:14, and/or
the first Eggerthella -specific detection probe comprises a target-hybridizing sequence consisting of SEQ ID NO:18.
6. The method of claim 4 , wherein each of the first Lactobacillus -specific detection probe, the first G. vaginalis -specific detection probe, and the first Eggerthella -specific detection probe comprises a label.
7. The method of claim 6 , wherein the label is a chemiluminescent label or a fluorescent label.
8. An oligomer combination for determining the presence or absence of each of Lactobacillus sp., G. vaginalis , and Eggerthella sp. in a clinical sample, the oligomer combination comprising:
(a) first, second, and third 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 consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:7, (ii) the second Lactobacillus -specific amplification oligomer comprises a second Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:6, and (iii) the third Lactobacillus -specific amplification oligomer comprises a third Lactobacillus -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:8;
(b) 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 consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:13 and (ii) the second G. vaginalis -specific amplification oligomer comprises a second G. vaginalis -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:12; and
(c) first and second Eggerthella -specific amplification oligomers for amplifying a target region of an Eggerthella sp. target nucleic acid, wherein (i) the first Eggerthella -specific amplification oligomer comprises a first Eggerthella -specific target-hybridizing sequence consisting of the nucleotide sequence of residues 28-51 of SEQ ID NO:17 and (ii) the second Eggerthella -specific amplification oligomer comprises a second Eggerthella -specific target-hybridizing sequence consisting of the nucleotide sequence of SEQ ID NO:16;
wherein each of the first Lactobacillus -specific amplification oligomer, the third Lactobacillus -specific amplification oligomer, the first G. vaginalis -specific amplification oligomer, and the first Eggerthella specific amplification oligomer is a promoter primer or promoter provider further comprising a T7 promoter sequence located 5′ to the respective target-hybridizing sequence.
9. The oligomer combination of claim 8 , wherein
the nucleotide sequence of the first Lactobacillus -specific amplification oligomer consists of the sequence of SEQ ID NO:7;
the nucleotide sequence of the third Lactobacillus -specific amplification oligomer consists of the sequence of SEQ ID NO:8;
the nucleotide sequence of the first G. vaginalis -specific amplification oligomer consists of the sequence of SEQ ID NO:13; and/or
the nucleotide sequence of the first Eggerthella -specific amplification oligomer consists of the sequence of SEQ ID NO:17.
10. The oligomer combination of claim 8 , further comprising a first Lactobacillus -specific detection probe that specifically hybridizes to the Lactobacillus sp. target region, a first G. vaginalis -specific detection probe that specifically hybridizes to the G. vaginalis target region, and a first Eggerthella -specific detection probe that specifically hybridizes to the Eggerthella sp. target region.
11. The method of claim 5 , wherein the detecting step (3) further comprises contacting the one or more amplification products with a second Lactobacillus -specific detection probe that specifically hybridizes to the Lactobacillus sp. target region, wherein the second Lactobacillus -specific detection probe comprises a target-hybridizing sequence consisting of residues 6-21 of SEQ ID NO:10.