Compositions and methods for detecting bacterial nucleic acids and diagnosing bacterial vaginosis

Abstract

To provide compositions and methods for detecting bacterial nucleic acids and diagnosing bacterial vaginosis.SOLUTION: Disclosed is a method for diagnosing bacterial vaginosis in a subject comprising performing an assay to detect one or more of Lactobacillus spp., Atopobiumvaginae, and Gardnerallvaginalis in a sample of the subject. Also disclosed are compositions and methods for detecting Lactobacillus spp., Atopobiumvaginae, and / or Gardnerallvaginalis nucleic acids in a sample.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 722,627, filed August 24, 2018, which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] According to the National Health and Nutrition Examination Survey, bacterial vaginosis (BV) affects nearly one-third of women between the ages of 14 and 49. (See Allsworth and Peipert, Obstetrics and Gynecology 109:114-120, 2007.) BV is the most common cause of vaginal discharge and the reason many women seek medical attention. It is also associated with increased preterm birth, low birth weight, pelvic inflammatory disease, and STD infections, including HIV, as well as an increased risk of transmitting HIV to sexual partners. (See Srinivasan and Fredricks, Interdisciplinary Perspectives on Infectious Diseases, Vol. 2008, Article ID 750479, p. 22, 2008.) Women with bacterial vaginosis may have symptoms including foul-smelling vaginal discharge and inflammation, but half of women with diagnosable BV have no obvious symptoms (see Srivinvasan and Fredricks, above).

[0003] There is no known single pathogen that causes BV. Most researchers and the CDC believe that bacterial vaginosis is the result of a disruption of the normal flora of the vagina. Unlike common infections, this dysbiosis is not the result of an individual bacterial species. See CDC Fact Sheet, 2014 (BV-Fact-Sheet-March-2014.pdf, from the CDC website). Dysbiosis is a disruption of the normal microflora within the body's environment, such as the vagina. See Nibali et al., Journal of Oral Microbiology, 6:22962, 2014.

[0004] BV is diagnosed in the clinic using the Amsel Criteria and in the laboratory using the Nugent Scoring System. The latter relies on counting bacterial morphotypes with the aid of Gram staining. Thus, the Nugent score is a visual assessment of dysbiosis, scoring beneficial versus harmful bacteria. See Nugent et al., Journal of Clinical Microbiology 29:297-301, 1991. The Amsel Criteria evaluate samples for the presence of bead cells, pH, color, and odor, which are key symptoms associated with BV. See Amsel et al., Am. J. Med. 74:14-22, 1983. Wet mounts of samples are examined microscopically to detect bead cells, which are human epithelial cells coated with bacteria thought to consist primarily of Gardnerella vaginalis (G. vaginalis).

[0005] Molecular tests generally target multiple organisms that are strongly correlated with bacterial vaginosis. Which organisms are targeted varies from test to test. In almost all cases, abundant anaerobic bacteria are targeted, including Atopobium species, Gardnerella species, and Megasphaera species.

[0006] The FDA-approved BV test is available from BD Affirm TM VPIII Microbial Identification Test" (2010) and "BD MAX TM The only other test that has been shown to detect BV is the BD Affirm VPIII Vaginal Panel (2016). Both the Affirm and MAX products detect Gardnerella vaginalis (G. vaginalis) as the sole indicator of BV. A study by Cartwright et al. found that the BD Affirm VPIII test had a sensitivity of 67.6% and a specificity of 76.4% (Journal of Clinical Microbiology). of Clinical Microbiology 51:3694-3699, 2013). The BD Affirm VIII package insert indicates that the Affirm product has a sensitivity of 95.1% and a specificity of 83.3% when compared to the scoring Gram stain method, while the BD MAX package insert indicates that the MAX product has a sensitivity of 90.5% and a specificity of 85.8%.

[0007] Cartwright et al., cited above, used a multiplex assay to detect Atopobium vaginae BVAB-2 and Megasphaera-1 for the diagnosis of BV. They measured the performance of this assay against the combined Nugent and Amsel scores in 323 women (93% African American, 7% White non-Hispanic). They reported a sensitivity of 96.9% and a specificity of 92.6% when compared with the combined Nugent and Amsel scores. They did not report the results of this assay compared with the Nugent score alone. Summary of the Invention [Means for solving the problem]

[0008] In one aspect, the present invention provides a method for determining the presence or absence of bacterial vaginosis (BV) in a subject. In some embodiments, the method generally comprises the following steps: (a) providing a sample from a subject suspected of having BV; (b) performing an assay to detect Lactobacillus spp., A. vaginae, and Gardnerella vaginalis in the sample; (c) assaying the Lactobacillus spp., A. vaginae, and Gardnerella vaginalis in the sample; and (d) assaying the Lactobacillus spp., A. vaginae, and Gardnerella vaginalis in the sample. (d) assigning a quantitative value for each of A. vaginae and G. vaginalis based on the detection assay; (d) subtracting the quantitative value for Lactobacillus species from the greater of the quantitative value for A. vaginae and the quantitative value for G. vaginalis; (e) assigning a single BV score based on step (d); and (f) determining the presence or absence of BV in the subject based on comparing the BV score to a cutoff value. In some embodiments, in step (c), a minimum quantitative value is imposed for Lactobacillus species, and / or A. vaginae and G. vaginalis. Typically, the quantitative values ​​for each of the Lactobacillus species, A. vaginae, and G. vaginalis are standardized and / or weighted. In some variations involving standardized quantitative values, the quantitative values ​​for each of the Lactobacillus species, A. vaginae, and G. vaginalis are in units of log copies, and standardization involves subtracting the median population value from the value determined from the detection assay. Step (d) may further include adding an adjustment constant.In certain embodiments, step (d) further comprises adding an internal standard (IC) adjustment factor to compensate for sample inhibition of the detection assay, the IC adjustment factor being based on the ratio of (i) the observed IC value generated from the detection assay to (ii) the expected IC value of the detection assay. In certain variations, the BV score is assigned using the following formula: BV score = C0 + W L Max(L S ,F LS )+W GA Max(A S ,G S ,F GAS )+W IC Log2 (IC ratio), where C0 is the tuning constant and W L is the weighting constant for Lactobacillus species, and Max(L S ,F LS ) is L S and F LS is the larger of S is the observed normalized quantitative value for Lactobacillus species, and F LS is the minimum standardized quantitative value imposed for Lactobacillus species, and W GA is the weighting constant for A. vaginae and G. vaginalis, and Max(A S ,G S ,F GAS ) is A S , G S , and F GAS is the larger of S is the observed normalized quantitative value for A. vaginae, and G S is the observed normalized quantitative value for Gardnerella vaginalis (G. vaginalis), and F GASis the minimum normalized quantitative value for A. vaginae and G. vaginalis (e.g., 0), and W IC is a weighting constant for the internal standard (IC), and the IC ratio is the ratio of (i) the observed internal standard (IC) value generated from the detection assay to (ii) the expected IC value of the detection assay.

[0009] In some embodiments of the methods for diagnosing BV described above, the assays for detecting Lactobacillus species, A. vaginae, and G. vaginalis are nucleic acid-based detection assays. Particularly suitable nucleic acid-based detection assays include amplification-based assays, such as assays involving isothermal amplification reactions (e.g., transcription-mediated amplification (TMA) reactions) that can be performed in real time. In certain embodiments, the nucleic acid-based detection assays target the 16S rRNA of Lactobacillus species, A. vaginae, and G. vaginalis. In certain variations, the nucleic acid-based detection assay is directed to (i) the 16S rRNA region of L. crispatus corresponding to the region of SEQ ID NO:1 from about nucleotide position 40 to about nucleotide position 265; (ii) the 16S rRNA region of L. jensenii corresponding to the region of SEQ ID NO:2 from about nucleotide position 43 to about nucleotide position 247; (iii) the 16S rRNA region of L. gasseri corresponding to the region of SEQ ID NO:3 from about nucleotide position 93 to about nucleotide position 298; (iv) the 16S rRNA region of A. vaginae corresponding to the region of SEQ ID NO:4 from about nucleotide position 540 to about nucleotide position 625; and / or (v) the 16S rRNA region of Gardnerella vaginalis corresponding to the region of SEQ ID NO:5 from about nucleotide position 172 to about nucleotide position 227. Targets the rRNA region.

[0010] In certain embodiments of the methods for diagnosing BV as described above that involve a nucleic acid-based detection assay, the assay is an amplification-based assay that includes the following steps: (1) contacting the sample with: and (ii) a second Lactobacillus-specific amplification oligomer that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7. (iii) a 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) a fourth Lactobacillus-specific amplification oligomer comprises a fourth Lactobacillus-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 9; and (ii) a second A. vaginae-specific amplification oligomer comprising a first A. vaginae-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:18; and a second A. vaginae-specific amplification oligomer comprising a second A. vaginae-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:17. first and second Gardnerella vaginalis (G. vaginalis)-specific amplification oligomers for amplifying a target region of a Gardnerella vaginalis (G. vaginalis) target nucleic acid, wherein (i) the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a first Gardnerella vaginalis (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 Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a second Gardnerella vaginalis (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 in which target nucleic acids of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis), if present in the sample, are used as templates to produce one or more amplification products corresponding to target regions of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis); (3) Detecting the presence or absence of one or more amplification products.

[0011] In some variations of the method for diagnosing BV comprising an amplification-based detection assay as described above, the first Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO:10, the second Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:7, the third Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:8, and the fourth Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:9. The first A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO:18, the second A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:17, the first G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO:15, and / or the second G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.In some such embodiments, a first Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, a second Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 7, a third Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 8, and a fourth Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 9, and the first The A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO:18, the second A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:17, the first G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO:15, and / or the second G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.

[0012] In some embodiments of the method for diagnosing BV including the amplification-based detection assay described above, at least one of the first Lactobacillus-specific amplification oligomer, the first A. vaginae-specific amplification oligomer, and the first Gardnerella vaginalis-specific amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located 5' to its respective target hybridizing sequence. A particularly suitable promoter sequence is a T7 promoter sequence, such as the promoter sequence having the nucleotide sequence of residues 1-27 of SEQ ID NO:10 (or, for example, residues 1-27 of SEQ ID NO:18 or residues 9-35 of SEQ ID NO:15). In certain variations, the first Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10, the first A. vaginae-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 18, and / or the first G. vaginalis-specific amplification oligomer comprises the nucleotide sequence of residues 9-52 of SEQ ID NO: 15. In some embodiments, the first G. vaginalis-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

[0013] In certain embodiments of methods for diagnosing BV including amplification-based detection assays as described above, the methods further include purifying the target nucleic acids of Lactobacillus spp., A. vaginae, and G. vaginalis, if present, from other components in the sample prior to step (2). In some such embodiments, the purification step includes contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe. For example, a sample can be contacted with a first capture probe oligomer comprising a target hybridizing sequence that specifically hybridizes to a target sequence within a target nucleic acid of a Lactobacillus species and a second capture probe target hybridizing sequence that comprises a target hybridizing sequence that specifically hybridizes to a target sequence within each of the target nucleic acids of A. vaginae and G. vaginalis, each of the first and second capture probe target hybridizing sequences being covalently linked to a sequence or moiety that binds to the immobilized probe; in some such embodiments, the first capture probe target hybridizing sequence specifically hybridizes to a target sequence within each of the target nucleic acids of L. crispatus, L. jensenii, and L. gasseri. In certain variations comprising first and second target capture probes as described above, 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. In some embodiments, the first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:6, and / or the second capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0014] In some embodiments of the method for diagnosing BV comprising an amplification-based detection assay as described above, the detecting step (3) comprises: (i) a first Lactobacillus-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of a Lactobacillus species; a first A. vaginae-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of A. vaginae; and a Gardnerella (ii) contacting the one or more amplification products with a first Gardnerella vaginalis (G. vaginalis)-specific detection probe comprising a target-hybridizing sequence that specifically hybridizes to a target region of G. vaginalis; and (iii) detecting the presence or absence of the target-hybridized Lactobacillus-specific detection probe, A. vaginae-specific detection probe, and / or Gardnerella vaginalis (G. vaginalis)-specific detection probe. In some embodiments, 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 substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO:16; in more specific variations, the first A. vaginae-specific detection probe target hybridizing sequence comprises nucleotides from 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.In some embodiments, the first Lactobacillus-specific detection probe target hybridizing sequence specifically hybridizes to a target region of each of the target nucleic acids of L. crispatus and L. jensenii, 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 the target nucleic acid of L. gasseri; in some such embodiments, the first Lactobacillus-specific detection probe target hybridizing sequence specifically hybridizes to a target region of the target nucleic acid of L. gasseri. The target hybridizing sequence of a first Lactobacillus-specific detection probe substantially corresponds to the nucleotide sequence of residues 1-17 of SEQ ID NO:11, and / or the target hybridizing sequence of a second Lactobacillus-specific detection probe substantially corresponds to the nucleotide sequence of residues 7-23 of SEQ ID NO:12, and in more particular variations, the target hybridizing sequence of a first Lactobacillus-specific detection probe comprises the nucleotide sequence of residues 1-17 of SEQ ID NO:11, and / or the target hybridizing sequence of a second Lactobacillus-specific detection probe comprises the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0015] In certain embodiments of the method for diagnosing BV, which includes the use of a first Lactobacillus-specific detection probe, a first A. vaginae-specific detection probe, and a first Gardnerella vaginalis-specific detection probe, each of the probes comprises a label. In some embodiments, which further include the use of a second Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe comprises a label. Particularly suitable labels include chemiluminescent and fluorescent labels.

[0016] In some embodiments of methods for diagnosing BV that involve the use of labeled detection probes as described above, the detection step (3) occurs during the amplification step (2). In some such variations, each detection probe comprises a fluorescent label and a quencher. Particularly suitable detection probes that comprise a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0017] In certain embodiments of the methods for diagnosing BV that include the use of detection probes as described above, at least one of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridizing sequence. In some embodiments that further include the use of a second Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe further comprises a non-target hybridizing sequence. In some such variations, each of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first G. vaginalis-specific detection probe (or each of the first Lactobacillus-specific detection probe, the second 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.

[0018] In some embodiments of the methods for diagnosing BV as described above, the method includes detecting 10 or fewer bacterial genera associated with BV. For example, in certain variations, the method includes detecting 5 or fewer bacterial genera associated with BV. In certain variations, the method does not include detecting bacterial genera associated with BV other than Lactobacillus, Atopobium, and Gardnerella.

[0019] In some embodiments of the methods for diagnosing BV as described above, if the presence of BV is indicated in the subject, the method further comprises administering to the subject a treatment regimen for BV.

[0020] In some embodiments of the methods for diagnosing BV as described above, the method is a method for monitoring BV in a subject, and the subject is receiving a treatment regimen for BV prior to step (a). In some such variations, if the presence of BV is indicated in the subject, the method further includes either (i) administering a treatment regimen for BV to the subject, or (ii) administering a different treatment regimen for BV to the subject.

[0021] In one aspect, the present invention provides a multiplex method for determining the presence or absence of each of Lactobacillus species, A. vaginae, and Gardnerella vaginalis in a sample, the method generally comprising the steps of: (1) contacting a sample suspected of containing at least one of Lactobacillus species, A. vaginae, and Gardnerella vaginalis with: (a) first, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying a target region of a target nucleic acid of a Lactobacillus species, wherein (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 7. (iii) a 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) a 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 an 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; (c) first and second Gardnerella vaginalis (G. vaginalis)-specific amplification oligomers for amplifying a target region of a Gardnerella vaginalis (G. vaginalis) target nucleic acid, wherein (i) the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a first Gardnerella vaginalis (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 Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a second Gardnerella vaginalis (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 in which target nucleic acids of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis), if present in the sample, are used as templates to produce one or more amplification products corresponding to target regions of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis); (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Lactobacillus species, A. vaginae, and Gardnerella vaginalis in the sample.

[0022] In some variations of such multiplex methods, the first Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 8, and the fourth Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 9; The first A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO:18, the second A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:17, the first G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO:15, and / or the second G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.In some such embodiments, a first Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, a second Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 7, a third Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 8, and a fourth Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 9, and the first The A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO:18, the second A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:17, the first G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO:15, and / or the second G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.

[0023] In some embodiments of the multiplex method, at least one of the first Lactobacillus-specific amplification oligomer, the first A. vaginae-specific amplification oligomer, and the first Gardnerella vaginalis-specific amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' to its respective target hybridizing sequence. A particularly suitable promoter sequence is a T7 promoter sequence, such as the promoter sequence having the nucleotide sequence of residues 1-27 of SEQ ID NO:10 (or, for example, residues 1-27 of SEQ ID NO:18 or residues 9-35 of SEQ ID NO:15). In certain variations, the first Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10, the first A. vaginae-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 18, and / or the first G. vaginalis-specific amplification oligomer comprises the nucleotide sequence of residues 9-52 of SEQ ID NO: 15. In some embodiments, the first G. vaginalis-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

[0024] In certain embodiments of the multiplex method described above, the method further comprises purifying the target nucleic acids of Lactobacillus spp., A. vaginae, and G. vaginalis, if present, from other components in the sample prior to step (2). In some such embodiments, the purification step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe. For example, a sample can be contacted with a first capture probe oligomer comprising a target hybridizing sequence that specifically hybridizes to a target sequence within a target nucleic acid of a Lactobacillus species and a second capture probe target hybridizing sequence that comprises a target hybridizing sequence that specifically hybridizes to a target sequence within each of the target nucleic acids of A. vaginae and G. vaginalis, each of the first and second capture probe target hybridizing sequences being covalently linked to a sequence or moiety that binds to the immobilized probe; in some such embodiments, the first capture probe target hybridizing sequence specifically hybridizes to a target sequence within each of the target nucleic acids of L. crispatus, L. jensenii, and L. gasseri. In certain variations comprising first and second target capture probes as described above, 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. In some embodiments, the first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:6, and / or the second capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0025] In some embodiments of the multiplex method described above, the detecting step (3) comprises: (i) a first Lactobacillus-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of a Lactobacillus species, a first A. vaginae-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of A. vaginae, and a second Gardnerella vaginalis (G. vaginalis)-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of A. vaginalis; and (ii) detecting the presence or absence of the target-hybridized Lactobacillus-specific detection probe, Atopobium venegae (A. vaginae)-specific detection probe, and / or Gardnerella vaginalis (G. vaginalis)-specific detection probe. In some embodiments, 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 substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO:16; in more specific variations, the first A. vaginae-specific detection probe target hybridizing sequence comprises nucleotides from 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.In some embodiments, the first Lactobacillus-specific detection probe target hybridizing sequence specifically hybridizes to a target region of each of the target nucleic acids of L. crispatus and L. jensenii, 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 the target nucleic acid of L. gasseri; in some such embodiments, the first Lactobacillus-specific detection probe target hybridizing sequence specifically hybridizes to a target region of the target nucleic acid of L. gasseri. The target hybridizing sequence of a first Lactobacillus-specific detection probe substantially corresponds to the nucleotide sequence of residues 1-17 of SEQ ID NO:11, and / or the target hybridizing sequence of a second Lactobacillus-specific detection probe substantially corresponds to the nucleotide sequence of residues 7-23 of SEQ ID NO:12, and in more particular variations, the target hybridizing sequence of a first Lactobacillus-specific detection probe comprises the nucleotide sequence of residues 1-17 of SEQ ID NO:11, and / or the target hybridizing sequence of a second Lactobacillus-specific detection probe comprises the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0026] In certain embodiments of multiplex methods that include the use of a first Lactobacillus-specific detection probe, a first A. vaginae-specific detection probe, and a first Gardnerella vaginalis-specific detection probe, each of the probes comprises a label. In some embodiments that further include the use of a second Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe comprises a label. Particularly suitable labels include chemiluminescent and fluorescent labels.

[0027] In some embodiments of multiplex methods involving the use of labeled detection probes as described above, the detection step (3) occurs during the amplification step (2). In some such variations, each detection probe comprises a fluorescent label and a quencher. Particularly suitable detection probes comprising a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0028] In certain embodiments of multiplex methods involving the use of detector probes as described above, at least one of the first Lactobacillus-specific detector probe, the first A. vaginae-specific detector probe, and the first Gardnerella vaginalis-specific detector probe further comprises a non-target hybridizing sequence. In some embodiments further involving the use of a second Lactobacillus-specific detector probe, the second Lactobacillus-specific detector probe further comprises a non-target hybridizing sequence. In some such variations, each of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first G. vaginalis-specific detection probe (or each of the first Lactobacillus-specific detection probe, the second 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.

[0029] In some embodiments of the multiplex method described above, the amplification reaction in step (2) is an isothermal amplification reaction. In certain variations, the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction. In certain embodiments, the isothermal amplification reaction is a real-time amplification reaction.

[0030] In another aspect, the present invention provides a composition or kit for determining the presence or absence of each of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis in a sample. The composition or kit generally comprises the following oligomers: (a) first, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying a target region of a target nucleic acid of a Lactobacillus species, wherein (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 7. (iii) a 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) a 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 an 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; (c) first and second Gardnerella vaginalis (G. vaginalis)-specific amplification oligomers for amplifying a target region of a Gardnerella vaginalis (G. vaginalis) target nucleic acid, wherein (i) the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a first Gardnerella vaginalis (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 Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a second Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:14.

[0031] In some variations of such compositions or kits, the first Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 8, and the fourth Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 9; The first A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO:18, the second A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:17, the first G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO:15, and / or the second G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.In some such embodiments, a first Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, a second Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 7, a third Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 8, and a fourth Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 9, and the first The A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO:18, the second A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:17, the first G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO:15, and / or the second G. vaginalis-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.

[0032] In some embodiments of the compositions or kits described above, at least one of the first Lactobacillus-specific amplification oligomer, the first A. vaginae-specific amplification oligomer, and the first Gardnerella vaginalis-specific amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' to its respective target hybridizing sequence. A particularly suitable promoter sequence is a T7 promoter sequence, such as the promoter sequence having the nucleotide sequence of residues 1-27 of SEQ ID NO:10 (or, for example, residues 1-27 of SEQ ID NO:18 or residues 9-35 of SEQ ID NO:15). In certain variations, the first Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10, the first A. vaginae-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 18, and / or the first G. vaginalis-specific amplification oligomer comprises the nucleotide sequence of residues 9-52 of SEQ ID NO: 15. In some embodiments, the first G. vaginalis-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

[0033] In certain embodiments of the compositions or kits described above, the compositions or kits further comprise at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe. For example, a composition or kit can include a first capture probe oligomer comprising a target hybridizing sequence that specifically hybridizes to a target sequence within a target nucleic acid of a Lactobacillus species and a second capture probe target hybridizing sequence that comprises a target hybridizing sequence that specifically hybridizes to a target sequence within each of the target nucleic acids of A. vaginae and G. vaginalis, wherein each of the first and second capture probe target hybridizing sequences is covalently linked to a sequence or moiety that binds to the immobilized probe; in some such embodiments, the first capture probe target hybridizing sequence specifically hybridizes to a target sequence within each of the target nucleic acids of L. crispatus, L. jensenii, and L. gasseri. In certain variations comprising first and second target capture probes as described above, 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. In some embodiments, the first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:6, and / or the second capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0034] In some embodiments of the compositions or kits described above, the compositions or kits further comprise a first Lactobacillus-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of a Lactobacillus species, a first A. vaginae-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of A. vaginae, and a first Gardnerella vaginalis-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of G. vaginalis. In some embodiments, 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 substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO:16; in more specific variations, the first A. vaginae-specific detection probe target hybridizing sequence comprises nucleotides from 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.In some embodiments, the first Lactobacillus-specific detection probe target hybridizing sequence specifically hybridizes to a target region of each of the target nucleic acids of L. crispatus and L. jensenii, and the composition or kit further comprises a second Lactobacillus-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of the target nucleic acid of L. gasseri; in some such embodiments, the first Lactobacillus-specific detection probe target hybridizing sequence specifically hybridizes to a target region of the target nucleic acid of L. gasseri. The 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; in more particular variations, the first Lactobacillus-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1-17 of SEQ ID NO:11, and / or the second Lactobacillus-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0035] In certain embodiments of compositions or kits comprising a first Lactobacillus-specific detection probe, a first A. vaginae-specific detection probe, and a first Gardnerella vaginalis-specific detection probe, each of the probes comprises a label. In some embodiments further comprising a second Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe comprises a label. Particularly suitable labels include chemiluminescent and fluorescent labels. In some variations, each detection probe comprises a fluorescent label and a quencher, and particularly suitable detection probes comprising a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0036] In certain embodiments of compositions or kits comprising such detection probes, at least one of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridizing sequence. In some embodiments further comprising a second Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe further comprises a non-target hybridizing sequence. In some such variations, each of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first G. vaginalis-specific detection probe (or each of the first Lactobacillus-specific detection probe, the second 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.

[0037] In yet another aspect, the present invention provides a method for determining the presence or absence of Lactobacillus spp. in a sample, which method generally comprises the steps of: (1) contacting a sample suspected of containing Lactobacillus spp. with first, second, third, and fourth amplification oligomers for amplifying a target region of a target nucleic acid of Lactobacillus spp., 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 in which a target nucleic acid of Lactobacillus spp., if present in the sample, is used as a template to produce one or more amplification products corresponding to a target region of Lactobacillus spp.; (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Lactobacillus species in the sample.

[0038] In some variations of the methods for detecting Lactobacillus species as described above, the first Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 8, and / or the fourth target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 9. In some such embodiments, the first target hybridizing sequence consists of the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 7, the third target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 8, and / or the fourth target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 9.

[0039] In some embodiments of the methods for detecting Lactobacillus species as described above, the first amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' to the respective target hybridizing sequence. A particularly suitable promoter sequence is a T7 promoter sequence, such as the promoter sequence having the nucleotide sequence of residues 1-27 of SEQ ID NO: 10. In certain variations, the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10.

[0040] In certain embodiments of the methods for detecting Lactobacillus spp., as described above, the method further comprises purifying the target nucleic acid of the Lactobacillus spp., if present, from other components in the sample prior to step (2). In some such embodiments, the purification step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe, wherein the capture probe target-hybridizing sequence specifically hybridizes to a target sequence within the target nucleic acid of the Lactobacillus spp. In some embodiments, the capture probe target hybridizing sequence specifically hybridizes to a target sequence within each of the target nucleic acids of L. crispatus, L. jensenii, and L. gasseri; in some such variations, the first capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO:6; and in more specific embodiments, the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:6.

[0041] In some embodiments for detecting Lactobacillus spp. as described above, the detecting step (3) includes (i) contacting one or more amplification products with a first detection probe comprising a target-hybridizing sequence that specifically hybridizes to a target region of Lactobacillus spp., and detecting the presence or absence of the target-hybridizing detection probe. In some embodiments, the first detection probe target hybridizing sequence specifically hybridizes to a target region of each of the target nucleic acids of L. crispatus and L. jensenii, and the method further comprises contacting one or more amplification products with a second detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of the target nucleic acid of L. gasseri. In some such embodiments, the first detection probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-17 of SEQ ID NO:11 and / or the second detection probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 7-23 of SEQ ID NO:12. In more particular variations, the first detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1-17 of SEQ ID NO:11 and / or the second detection probe target hybridizing sequence comprises the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0042] In certain embodiments of the method for detecting Lactobacillus species as described above, the method comprises using a first detection probe, and the first detection probe comprises a label.In some embodiments, the method further comprises contacting a second probe with one or more amplification oligomers, the second probe comprises a label.Particularly suitable labels include chemiluminescent and fluorescent labels.

[0043] In some embodiments of the method for detecting Lactobacillus species, including the use of labeled detection probes, the detection step (3) occurs during the amplification step (2). In some such variations, each detection probe comprises a fluorescent label and a quencher. Particularly suitable detection probes comprising a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0044] In certain embodiments of the method for detecting Lactobacillus species as described above, the method comprises using labeled detection probe, wherein the first detection probe further comprises a non-target hybridization sequence.In some embodiments further comprising using a second detection probe, the second detection probe further comprises a non-target hybridization sequence.In some such variations, the first detection probe (or each of the first and second detection probes) is a molecular torch or a molecular beacon.

[0045] In yet another aspect, the present invention provides a method for determining the presence or absence of A. vaginae in a sample, the method generally comprising the steps of: (1) contacting a sample suspected of containing A. vaginae with first and second amplification oligomers for amplifying a target region of an 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) conducting an in vitro nucleic acid amplification reaction in which A. vaginae target nucleic acid, if present in the sample, is used as a template to produce one or more amplification products corresponding to target regions of A. vaginae; (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of A. vaginae in the sample.

[0046] In some variations of the methods for detecting A. vaginae as described above, the first target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and / or the second target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 17. In some such embodiments, the first target hybridizing sequence consists of the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and / or the second target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 17.

[0047] In some embodiments of the methods for detecting A. vaginae, such as those described above, the first amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' to its respective target hybridizing sequence. A particularly suitable promoter sequence is a T7 promoter sequence, such as the promoter sequence having the nucleotide sequence of residues 1-27 of SEQ ID NO:18. In certain variations, the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO:18.

[0048] In certain embodiments of the methods for detecting A. vaginae, as described above, the method further comprises, prior to step (2), purifying the A. vaginae target nucleic acid, if present, from other components in the sample. In some such embodiments, the purification 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 the immobilized probe, wherein the capture probe target hybridizing sequence specifically hybridizes to a target sequence within the A. vaginae target nucleic acid. In some embodiments, the capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO:13. In more specific variations, the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0049] In some embodiments for detecting A. vaginae as described above, the detecting step (3) comprises contacting one or more amplification products with a detection probe comprising a target-hybridizing sequence that specifically hybridizes to a target region of A. vaginae and detecting the presence or absence of the target-hybridizing detection probe. In some embodiments, the detection probe target-hybridizing sequence substantially corresponds to the nucleotide sequence of residues 6-21 of SEQ ID NO:19. In more specific variations, the detection probe target-hybridizing sequence comprises the nucleotide sequence of residues 6-21 of SEQ ID NO:19.

[0050] In certain embodiments of the methods for detecting A. vaginae, as described above, the methods include the use of a detection probe, wherein the detection probe comprises a label. Particularly suitable labels include chemiluminescent and fluorescent labels and chemiluminescent labels.

[0051] In some embodiments of the methods for detecting A. vaginae, such as those described above, that involve the use of labeled detection probes, the detection step (3) occurs during the amplification step (2). In some such variations, the detection probe comprises a fluorescent label and a quencher. Particularly suitable detection probes that comprise a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0052] In certain embodiments of the methods for detecting A. vaginae, including the use of a detection probe, as described above, the detection probe further comprises a non-target hybridizing sequence. In some such variations, the detection probe is a molecular torch or molecular beacon.

[0053] In yet another aspect, the present invention provides a method for determining the presence or absence of Gardnerella vaginalis (G. vaginalis) in a sample, the method generally comprising the steps of: (1) contacting a sample suspected of containing Gardnerella vaginalis (G. vaginalis) with first and second amplification oligomers for amplifying a target region of a Gardnerella vaginalis (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 in which a Gardnerella vaginalis (G. vaginalis) target nucleic acid, if present in the sample, is used as a template to produce one or more amplification products corresponding to a target region of Gardnerella vaginalis (G. vaginalis); (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Gardnerella vaginalis (G. vaginalis) in the sample.

[0054] In some variations of the methods for detecting Gardnerella vaginalis (G. vaginalis) as described above, the first target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or the second target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 14. In some such embodiments, the first target hybridizing sequence consists of the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or the second target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 14.

[0055] In some embodiments of the methods for detecting Gardnerella vaginalis (G. vaginalis) as described above, the first amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' to its respective target hybridizing sequence. A particularly suitable promoter sequence is a T7 promoter sequence, such as a promoter sequence having the nucleotide sequence of residues 9-36 of SEQ ID NO:15. In certain variations, the first amplification oligomer comprises the nucleotide sequence of residues 9-52 of SEQ ID NO:15, and in some such embodiments, the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO:15.

[0056] In certain embodiments of the methods for detecting Gardnerella vaginalis (G. vaginalis) as described above, the method further comprises, prior to step (2), purifying the Gardnerella vaginalis (G. vaginalis) target nucleic acid, if present, from other components in the sample. In some such embodiments, the purification 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 the immobilized probe, wherein the capture probe target hybridizing sequence specifically hybridizes to a target sequence within the Gardnerella vaginalis (G. vaginalis) target nucleic acid. In some embodiments, the capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO:13. In more specific variations, the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0057] In some embodiments for detecting Gardnerella vaginalis (G. vaginalis) as described above, the detecting step (3) comprises contacting one or more amplification products with a detection probe comprising a target-hybridizing sequence that specifically hybridizes to a target region of Gardnerella vaginalis (G. vaginalis) and detecting the presence or absence of the target-hybridizing detection probe. In some embodiments, the detection probe target-hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO:16. In more specific variations, the detection probe target-hybridizing sequence comprises the nucleotide sequence of residues 1-18 of SEQ ID NO:16.

[0058] In certain embodiments of the methods for detecting Gardnerella vaginalis (G. vaginalis) as described above, the methods include the use of a detection probe, wherein the detection probe comprises a label. Particularly suitable labels include chemiluminescent and fluorescent labels and chemiluminescent labels.

[0059] In some embodiments of the methods for detecting Gardnerella vaginalis (G. vaginalis) described above, including the use of a labeled detection probe, the detection step (3) occurs during the amplification step (2). In some such variations, the detection probe comprises a fluorescent label and a quencher. Particularly suitable detection probes comprising a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0060] In certain embodiments of the methods for detecting Gardnerella vaginalis (G. vaginalis) that involve the use of a detection probe, as described above, the detection probe further comprises a non-target hybridizing sequence. In some such variations, the detection probe is a molecular torch or molecular beacon.

[0061] In some embodiments of the methods for detecting Lactobacillus spp., A. vaginae, or G. vaginalis, as described above, the amplification reaction in step (2) is an isothermal amplification reaction. In certain variations, the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction. In certain embodiments, the isothermal amplification reaction is a real-time amplification reaction.

[0062] These and other aspects of the present invention will become apparent upon reference to the following detailed description of the invention. Additional disclosed embodiments are as follows:

[0063] Embodiment 1 is a multiplex method for determining the presence or absence of each of Lactobacillus spp., Atopobium vaginae (A. vaginae), and Gardnerella vaginalis (G. vaginalis) in a sample, the method comprising: (1) contacting a sample suspected of containing at least one of Lactobacillus species, A. vaginae, and Gardnerella vaginalis with: (a) first, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying a target region of a target nucleic acid of a Lactobacillus species, wherein (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 7. (iii) a 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) a 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 an 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; (c) first and second Gardnerella vaginalis (G. vaginalis)-specific amplification oligomers for amplifying a target region of a Gardnerella vaginalis (G. vaginalis) target nucleic acid, wherein (i) the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a first Gardnerella vaginalis (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 Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a second Gardnerella vaginalis (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 in which target nucleic acids of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis), if present in the sample, are used as templates to produce one or more amplification products corresponding to target regions of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis); (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Lactobacillus species, A. vaginae, and Gardnerella vaginalis in the sample.

[0064] Embodiment 2 is the method of embodiment 1, (1) the first Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 10; (2) the second Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 7; (3) the third Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:8; (4) the fourth Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:9; (5) the first A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18; (6) the second A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 17; (7) the first Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence comprises the nucleotide sequence of residues 36 to 52 of SEQ ID NO: 15; and / or (8) The second Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.

[0065] Embodiment 3 is the method of embodiment 1 or 2, (1) the first Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 10; (2) the second Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 7; (3) the third Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:8; (4) the fourth Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:9; (5) the first A. vaginae-specific target hybridizing sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18; (6) the second A. vaginae-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 17; (7) the first Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence consists of the nucleotide sequence of residues 36 to 52 of SEQ ID NO: 15; and / or (8) The second Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:14.

[0066] Embodiment 4 is the method of any one of Embodiments 1 to 3, 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 its respective target hybridizing sequence.

[0067] Embodiment 5 is the method of embodiment 4, wherein the promoter sequence is a T7 promoter sequence.

[0068] Embodiment 6 is the method of embodiment 5, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:10.

[0069] Embodiment 7 is the method of embodiment 4, the first Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10; the first A. vaginae-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 18; and / or The first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises the nucleotide sequence of residues 9 to 52 of SEQ ID NO:15.

[0070] Example 8 is the method of Example 7, wherein the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO:15.

[0071] Embodiment 9 is the method of any one of embodiments 1 to 8, further comprising, prior to step (2), purifying the target nucleic acids of Lactobacillus spp., A. vaginae, and G. vaginalis, if present, from other components in the sample.

[0072] Embodiment 10 is the method of embodiment 9, in which the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe.

[0073] Embodiment 11 is the method of embodiment 10, wherein the sample is contacted with first and second capture probe oligomers; the first capture probe oligomer comprises a target hybridizing sequence that specifically hybridizes to a target sequence within the target nucleic acid of a Lactobacillus species, and the second capture probe hybridizing sequence comprises a target hybridizing sequence that specifically hybridizes to a target sequence within each of the target nucleic acids of A. vaginae and Gardnerella vaginalis; Each of the first and second capture probe target hybridizing sequences is covalently linked to a sequence or moiety that binds to the immobilized probe.

[0074] Embodiment 12 is the method of embodiment 11, wherein the first capture probe target hybridizing sequence specifically hybridizes to a target sequence within each of the target nucleic acids of Lactobacillus crispatus (L. crispatus), Lactobacillus jensenii (L. jensenii), and Lactobacillus gasseri (L. gasseri).

[0075] Embodiment 13 is the method of embodiment 12, The first capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO:6.

[0076] Embodiment 14 is the method of embodiment 12 or 13, wherein the second capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO:13.

[0077] Embodiment 15 is the method of embodiment 14, the first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:6; and / or The second capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0078] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the detecting step (3) comprises contacting one or more amplification products with a first Lactobacillus-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of a Lactobacillus species, a first A. vaginae-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of A. vaginae, and a first Gardnerella vaginalis-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of G. vaginalis; and This includes detecting the presence or absence of a target-hybridized Lactobacillus-specific detection probe, an A. vaginae-specific detection probe, and / or a Gardnerella vaginalis-specific detection probe.

[0079] Embodiment 17 is the method of embodiment 16, 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 Gardnerella vaginalis (G. vaginalis)-specific detection probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO:16.

[0080] Embodiment 18 is the method of embodiment 17, the first A. vaginae-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 6-21 of SEQ ID NO: 19; and / or The first Gardnerella vaginalis (G. vaginalis)-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1-18 of SEQ ID NO:16.

[0081] Embodiment 19 is the method of any one of embodiments 16 to 18, wherein the first Lactobacillus-specific detection probe target hybridizing sequence specifically hybridizes to a target region of each of the target nucleic acids of L. crispatus and L. jensenii; The method further includes contacting one or more amplification products with a second Lactobacillus-specific detection probe that includes a target hybridizing sequence that specifically hybridizes to a target region of a target nucleic acid of L. gasseri.

[0082] Embodiment 20 is the method of embodiment 19, 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 corresponds substantially to the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0083] Embodiment 21 is the method of embodiment 20, the first Lactobacillus-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1-17 of SEQ ID NO: 11; and / or The second Lactobacillus-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0084] Embodiment 22 is the method of any one of embodiments 16 to 18, wherein each of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe comprises a label.

[0085] Embodiment 23 is the method of any one of embodiments 19 to 21, wherein each of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe comprises a label.

[0086] Embodiment 24 is the method of embodiment 22 or 23, wherein the label is a chemiluminescent label or a fluorescent label.

[0087] Embodiment 25 is the method of embodiment 22 or 23, wherein the detecting step (3) occurs during the amplifying step (2).

[0088] Embodiment 26 is the method of embodiment 25, wherein each detection probe comprises a fluorescent label and a quencher.

[0089] Embodiment 27 is the method of embodiment 26, in which each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0090] Embodiment 28 is the method of any one of embodiments 16 to 18, wherein at least one of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridizing sequence.

[0091] Embodiment 29 is the method of embodiment 28, wherein each of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe is a molecular torch or a molecular beacon.

[0092] Embodiment 30 is the method of any one of embodiments 19 to 21, wherein at least one of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridizing sequence.

[0093] Embodiment 31 is the method of embodiment 30, wherein each of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe is a molecular torch or a molecular beacon.

[0094] Embodiment 32 is the method of any one of embodiments 1 to 31, wherein the amplification reaction in step (2) is an isothermal amplification reaction.

[0095] Embodiment 33 is the method of embodiment 32, wherein the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0096] Embodiment 34 is the method of embodiment 32 or 33, wherein the amplification reaction is a real-time amplification reaction.

[0097] Embodiment 35 is a composition or kit for determining the presence or absence of each of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis) in a sample, the composition or kit comprising: (a) first, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying a target region of a target nucleic acid of a Lactobacillus species, wherein (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 7. (iii) a 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) a 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 an 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; (c) first and second Gardnerella vaginalis (G. vaginalis)-specific amplification oligomers for amplifying a target region of a Gardnerella vaginalis (G. vaginalis) target nucleic acid, wherein (i) the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a first Gardnerella vaginalis (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 Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a second Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:14.

[0098] Embodiment 36 is the composition or kit of embodiment 35, the first Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 10; the second Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:7; the third Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:8; The fourth Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:9; the first A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18; the second A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 17; the first Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO: 15; and / or The second Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.

[0099] Embodiment 37 is the composition or kit of embodiment 35 or 36, the first Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 10; the second Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:7; the third Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:8; The fourth Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:9; the first A. vaginae-specific target hybridizing sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18; the second A. vaginae-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 17; the first Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence consists of the nucleotide sequence of residues 36-52 of SEQ ID NO: 15; and / or The second Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:14.

[0100] Embodiment 38 is the composition or kit of any one of embodiments 35 to 37, At least one of the first Lactobacillus-specific amplification oligomer, the first A. vaginae-specific amplification oligomer, and the first Gardnerella vaginalis-specific amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' to its respective target hybridizing sequence.

[0101] Embodiment 39 is the composition or kit of embodiment 38, wherein the promoter sequence is a T7 promoter sequence.

[0102] Embodiment 40 is the composition or kit of embodiment 39, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:10.

[0103] Embodiment 41 is the composition or kit of embodiment 38, the first Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10; the first A. vaginae-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 18; and / or The first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises the nucleotide sequence of residues 9 to 52 of SEQ ID NO:15.

[0104] Embodiment 42 is the composition or kit of embodiment 41, wherein the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

[0105] Embodiment 43 is the composition or kit of any one of embodiments 35 to 42, further comprising at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe.

[0106] Embodiment 44 is the composition or kit of embodiment 43, wherein the composition or kit comprises a first and a second capture probe oligomer;

[0107] the first capture probe oligomer comprises a target hybridizing sequence that specifically hybridizes to a target sequence within the target nucleic acid of a Lactobacillus species, and the second capture probe hybridizing sequence comprises a target hybridizing sequence that specifically hybridizes to a target sequence within each of the target nucleic acids of A. vaginae and Gardnerella vaginalis; Each of the first and second capture probe target hybridizing sequences is covalently linked to a sequence or moiety that binds to the immobilized probe.

[0108] Embodiment 45 is the composition or kit of embodiment 44, wherein the first capture probe target hybridizing sequence specifically hybridizes to a target sequence within each of the target nucleic acids of Lactobacillus crispatus (L. crispatus), Lactobacillus jensenii (L. jensenii), and L. gasseri (L. gasseri).

[0109] Embodiment 46 is the composition or kit of embodiment 45, The first capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO:6.

[0110] Embodiment 47 is the composition or kit of embodiment 45 or 46, wherein the second capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO:13.

[0111] Embodiment 48 is the composition or kit of embodiment 47, the first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:6; and / or The second capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0112] Embodiment 49 is the composition or kit of any one of embodiments 35 to 48, further comprising a first Lactobacillus-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of a Lactobacillus species, a first A. vaginae-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of A. vaginae, and a first Gardnerella vaginalis-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of G. vaginalis.

[0113] Embodiment 50 is the composition or kit of embodiment 49, 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 Gardnerella vaginalis (G. vaginalis)-specific detection probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO:16.

[0114] Embodiment 51 is the composition or kit of embodiment 50, the first A. vaginae-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 6-21 of SEQ ID NO: 19; and / or The first Gardnerella vaginalis (G. vaginalis)-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1-18 of SEQ ID NO:16.

[0115] Embodiment 52 is the composition or kit of any one of embodiments 49 to 51, wherein the first Lactobacillus-specific detection probe target hybridizing sequence specifically hybridizes to a target region of each of the target nucleic acids of L. crispatus and L. jensenii;

[0116] The composition or kit further comprises a second Lactobacillus-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of a target nucleic acid of L. gasseri.

[0117] Embodiment 53 is the composition or kit of embodiment 52, 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 corresponds substantially to the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0118] Embodiment 54 is the composition or kit of embodiment 53, the first Lactobacillus-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1-17 of SEQ ID NO: 11; and / or The second Lactobacillus-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0119] Embodiment 55 is the composition or kit of any one of embodiments 49 to 51, wherein each of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe comprises a label.

[0120] Embodiment 56 is the composition or kit of any one of embodiments 52 to 54, wherein each of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe comprises a label.

[0121] Embodiment 57 is the composition or kit of embodiment 55 or 56, wherein the label is a chemiluminescent label or a fluorescent label.

[0122] Embodiment 58 is the composition or kit of embodiment 55 or 56, wherein each detection probe comprises a fluorescent label and a quencher.

[0123] Embodiment 59 is the composition or kit of embodiment 58, wherein each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0124] Embodiment 60 is the composition or kit of any one of embodiments 49 to 51, wherein at least one of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridizing sequence.

[0125] Embodiment 61 is a composition or kit of embodiment 60, wherein each of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe is a molecular torch or a molecular beacon.

[0126] Embodiment 62 is the composition or kit of any one of embodiments 52 to 54, wherein at least one of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridizing sequence.

[0127] Embodiment 63 is a composition or kit of embodiment 62, wherein each of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe is a molecular torch or a molecular beacon.

[0128] Embodiment 64 is a method for determining the presence or absence of bacterial vaginosis (BV) in a subject, the method comprising: (a) providing a sample from a subject suspected of having BV; (b) conducting an assay to detect Lactobacillus species, A. vaginae, and Gardnerella vaginalis in the sample; (c) assigning a quantitative value to each of Lactobacillus species, A. vaginae, and Gardnerella vaginalis based on the detection assay; (d) subtracting the quantitative value of Lactobacillus species from the greater of the quantitative value of A. vaginae and the quantitative value of Gardnerella vaginalis; (e) assigning a single BV score based on step (d); and (f) determining whether or not a subject has BV based on a comparison of the BV score with a cutoff value;

[0129] Embodiment 65 is the method of embodiment 64, wherein a minimum quantification value is imposed for Lactobacillus spp. in step (c).

[0130] Embodiment 66 is the method of embodiment 64 or 65, wherein minimum quantitative values ​​are imposed in step (c) for A. vaginae and Gardnerella vaginalis.

[0131] Embodiment 67 is the method of any one of embodiments 64 to 66, wherein the quantitative values ​​for each of Lactobacillus spp., A. vaginae, and G. vaginalis are standardized.

[0132] Embodiment 68 is the method of embodiment 67, wherein the quantitative values ​​for each of Lactobacillus spp., A. vaginae, and G. vaginalis are in units of log copies, and normalization comprises subtracting the median population value from the value determined from the detection assay.

[0133] Embodiment 69 is the method of any one of embodiments 64 to 68, wherein the quantitative values ​​of each of Lactobacillus spp., A. vaginae, and G. vaginalis are weighted.

[0134] Embodiment 70 is the method of any one of embodiments 64 to 69, in which step (d) further comprises adding an adjustment constant.

[0135] Embodiment 71 is the method of any one of embodiments 64 to 69, wherein step (d) further comprises adding an internal standard (IC) adjustment factor to compensate for sample inhibition of the detection assay, wherein the IC adjustment factor is based on the ratio of (i) the observed IC value generated from the detection assay to (ii) the expected IC value of the detection assay.

[0136] Embodiment 72 is the method of embodiment 64, wherein the BV score is assigned using the following formula: BV score = C0 + W L Max(L S ,F LS )+W GA Max(A S ,G S ,F GAS )+W IC Log2 (IC ratio), C0 is a tuning constant, W L is a weighting constant for Lactobacillus species, Max(L S ,F LS ) is L S and F LS is the larger of S is the observed normalized quantitative value for Lactobacillus species, and F LS is the minimum standardized quantitative value imposed for Lactobacillus species, W GAis the weighting constant for A. vaginae and G. vaginalis, Max(A S ,G S ,F GAS ) is A S , G S , and F GAS is the larger of S is the observed normalized quantitative value for A. vaginae, and G S is the observed normalized quantitative value for Gardnerella vaginalis (G. vaginalis), and F GAS is the minimum standardized quantitative value calculated for A. vaginae and G. vaginalis, W IC is the weighting constant for the internal standard (IC), The IC ratio is the ratio of (i) the observed internal standard (IC) value generated from the detection assay to (ii) the expected IC value of the detection assay.

[0137] Embodiment 73 is the method of embodiment 72, further comprising: GAS is 0.

[0138] Embodiment 74 is the method of any one of embodiments 64 to 73, wherein the assays for detecting Lactobacillus spp., A. vaginae, and G. vaginalis are nucleic acid-based detection assays.

[0139] Embodiment 75 is the method of embodiment 74, wherein the nucleic acid-based detection assay targets 16S rRNA of Lactobacillus spp., A. vaginae, and G. vaginalis.

[0140] Embodiment 76 is the method of embodiment 75, wherein the nucleic acid-based detection assay targets: (a) the L. crispatus 16S rRNA region corresponding to the region of SEQ ID NO: 1 from about nucleotide position 40 to about nucleotide position 265; (b) Lactobacillus jensenii (L. jensenii) 16S rRNA region corresponding to the region of SEQ ID NO:2 from about nucleotide position 43 to about nucleotide position 247 (c) the 16S rRNA region of Lactobacillus gasseri (L. gasseri) corresponding to the region of SEQ ID NO:3 from about nucleotide position 93 to about nucleotide position 298; (d) a 16S rRNA region of A. vaginae corresponding to the region of SEQ ID NO: 4 from about nucleotide position 540 to about nucleotide position 625, and / or (e) The 16S rRNA region of Gardnerella vaginalis (G. vaginalis) corresponding to the region of SEQ ID NO:5 from about nucleotide position 172 to about nucleotide position 227.

[0141] Embodiment 77 is the method of any one of embodiments 74 to 76, wherein the nucleic acid-based detection assay is an amplification-based assay.

[0142] Embodiment 78 is the method of embodiment 77, in which the amplification-based assay comprises an isothermal amplification reaction.

[0143] Embodiment 79 is the method of embodiment 78, wherein the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0144] Embodiment 80 is the method of embodiment 78 or 79, wherein the amplification reaction is a real-time amplification reaction.

[0145] Embodiment 81 is the method of embodiment 79, in which the nucleic acid-based detection assay is an amplification-based assay comprising: (1) contacting the sample with: and (ii) a second Lactobacillus-specific amplification oligomer that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO:7. (iii) a 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) a fourth Lactobacillus-specific amplification oligomer comprises a fourth Lactobacillus-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 9; and (ii) a second A. vaginae-specific amplification oligomer comprising a first A. vaginae-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:18; and a second A. vaginae-specific amplification oligomer comprising a second A. vaginae-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:17. first and second Gardnerella vaginalis (G. vaginalis)-specific amplification oligomers for amplifying a target region of a Gardnerella vaginalis (G. vaginalis) target nucleic acid, wherein (i) the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a first Gardnerella vaginalis (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 Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a second Gardnerella vaginalis (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 in which target nucleic acids of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis), if present in the sample, are used as templates to produce one or more amplification products corresponding to target regions of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis); (3) Detecting the presence or absence of one or more amplification products.

[0146] Embodiment 82 is the method of embodiment 81, the first Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 10; the second Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:7; the third Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:8; The fourth Lactobacillus-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:9; the first A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18; the second A. vaginae-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO: 17; the first Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO: 15; and / or The second Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.

[0147] Embodiment 83 is the method of embodiment 81 or 82, the first Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 10; the second Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:7; the third Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:8; The fourth Lactobacillus-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:9; the first A. vaginae-specific target hybridizing sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18; the second A. vaginae-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO: 17; the first Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence consists of the nucleotide sequence of residues 36-52 of SEQ ID NO: 15; and / or The second Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:14.

[0148] Embodiment 84 is the method of any one of embodiments 81 to 83, At least one of the first Lactobacillus-specific amplification oligomer, the first A. vaginae-specific amplification oligomer, and the first Gardnerella vaginalis-specific amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' to its respective target hybridizing sequence.

[0149] Embodiment 85 is the method of embodiment 84, in which the promoter sequence is a T7 promoter sequence.

[0150] Embodiment 86 is the method of embodiment 85, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:10.

[0151] Embodiment 87 is the method of embodiment 84, the first Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10; the first A. vaginae-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 18; and / or The first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises the nucleotide sequence of residues 9 to 52 of SEQ ID NO:15.

[0152] Embodiment 88 is the method of embodiment 87, wherein the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

[0153] Embodiment 89 is the method of any one of embodiments 81 to 88, further comprising, prior to step (2), purifying the target nucleic acids of Lactobacillus spp., A. vaginae, and G. vaginalis, if present, from other components in the sample.

[0154] Embodiment 90 is the method of embodiment 89, in which the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe.

[0155] Embodiment 91 is the method of embodiment 90, wherein the sample is contacted with first and second capture probe oligomers; the first capture probe oligomer comprises a target hybridizing sequence that specifically hybridizes to a target sequence within the target nucleic acid of a Lactobacillus species, and the second capture probe hybridizing sequence comprises a target hybridizing sequence that specifically hybridizes to a target sequence within each of the target nucleic acids of A. vaginae and Gardnerella vaginalis;

[0156] Each of the first and second capture probe target hybridizing sequences is covalently linked to a sequence or moiety that binds to the immobilized probe.

[0157] Embodiment 92 is the method of embodiment 91, wherein the first capture probe target hybridizing sequence specifically hybridizes to a target sequence within each of the target nucleic acids of Lactobacillus crispatus (L. crispatus), Lactobacillus jensenii (L. jensenii), and Lactobacillus gasseri (L. gasseri).

[0158] Embodiment 93 is the method of embodiment 92, The first capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO:6.

[0159] Embodiment 94 is the method of embodiment 92 or 93, wherein the second capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO:13.

[0160] Embodiment 95 is the method of embodiment 94, the first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:6; and / or The second capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0161] Embodiment 96 is the method of any one of embodiments 81 to 95, wherein the detecting step (3) comprises contacting one or more amplification products with a first Lactobacillus-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of a Lactobacillus species, a first A. vaginae-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of A. vaginae, and a first Gardnerella vaginalis-specific detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of G. vaginalis; and This includes detecting the presence or absence of a target-hybridized Lactobacillus-specific detection probe, an A. vaginae-specific detection probe, and / or a Gardnerella vaginalis-specific detection probe.

[0162] Embodiment 97 is the method of embodiment 96, 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 Gardnerella vaginalis (G. vaginalis)-specific detection probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO:16.

[0163] Embodiment 98 is the method of embodiment 97, the first A. vaginae-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 6-21 of SEQ ID NO: 19; and / or The first Gardnerella vaginalis (G. vaginalis)-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1-18 of SEQ ID NO:16.

[0164] Embodiment 99 is the method of any one of embodiments 96 to 98, wherein the first Lactobacillus-specific detection probe target hybridizing sequence specifically hybridizes to a target region of each of the target nucleic acids of L. crispatus and L. jensenii; The method further includes contacting one or more amplification products with a second Lactobacillus-specific detection probe that includes a target hybridizing sequence that specifically hybridizes to a target region of a target nucleic acid of L. gasseri.

[0165] Embodiment 100 is the method of embodiment 99, 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 corresponds substantially to the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0166] Embodiment 101 is the method of embodiment 100, the first Lactobacillus-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1-17 of SEQ ID NO: 11; and / or The second Lactobacillus-specific detection probe target hybridizing sequence comprises the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0167] Embodiment 102 is the method of any one of embodiments 96 to 98, wherein each of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe comprises a label.

[0168] Embodiment 103 is the method of any one of embodiments 99 to 101, wherein each of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe comprises a label.

[0169] Embodiment 104 is the method of embodiment 102 or 103, in which the label is a chemiluminescent label or a fluorescent label.

[0170] Embodiment 105 is the method of embodiment 102 or 103, in which the detecting step (3) occurs during the amplifying step (2).

[0171] Embodiment 106 is the method of embodiment 105, in which each detection probe comprises a fluorescent label and a quencher.

[0172] Embodiment 107 is the method of embodiment 106, in which each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0173] Embodiment 108 is the method of any one of embodiments 96 to 98, wherein at least one of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridizing sequence.

[0174] Embodiment 109 is the method of embodiment 108, wherein each of the first Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe is a molecular torch or a molecular beacon.

[0175] Embodiment 110 is the method of any one of embodiments 99 to 101, wherein at least one of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridizing sequence.

[0176] Embodiment 111 is the method of embodiment 110, wherein each of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first A. vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe is a molecular torch or a molecular beacon.

[0177] Embodiment 112 is the method of any one of embodiments 81 to 111, wherein the amplification reaction in step (2) is an isothermal amplification reaction.

[0178] Embodiment 113 is the method of embodiment 112, wherein the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0179] Embodiment 114 is the method of embodiment 112 or 113, wherein the amplification reaction is a real-time amplification reaction.

[0180] Embodiment 115 is the method of any one of embodiments 64 to 114, wherein the method comprises detecting 10 or fewer bacterial genera associated with BV.

[0181] Embodiment 116 is the method of any one of embodiments 64 to 114, wherein the method comprises detecting no more than five bacterial genera associated with BV.

[0182] Embodiment 117 is the method of any one of embodiments 64 to 114, wherein the method does not include detection of bacterial genera associated with BV other than Lactobacillus, Atopobium, and Gardnerella.

[0183] Embodiment 118 is the method of any one of embodiments 64 to 117, wherein if the presence of BV is indicated in the subject, the method further comprises administering to the subject a treatment regimen for BV.

[0184] Embodiment 119 is the method of any one of embodiments 64 to 117, wherein the method is for monitoring BV in a subject, and the subject is undergoing a treatment regimen for BV prior to step (a).

[0185] Embodiment 120 is the method of embodiment 119, wherein if the presence of BV is indicated in the subject, the method further includes either (i) administering a treatment regimen for BV to the subject, or (ii) administering a different treatment regimen for BV to the subject.

[0186] Embodiment 121 is a method for determining the presence or absence of Lactobacillus spp. in a sample, the method comprising: (1) contacting a sample suspected of containing Lactobacillus spp. with first, second, third, and fourth amplification oligomers for amplifying a target region of a target nucleic acid of Lactobacillus spp., 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 in which a target nucleic acid of Lactobacillus spp., if present in the sample, is used as a template to produce one or more amplification products corresponding to a target region of Lactobacillus spp.; (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Lactobacillus species in the sample.

[0187] Embodiment 122 is the method of embodiment 121, the first target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 10; the second target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:7; the third target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:8; and / or The fourth target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:9.

[0188] Embodiment 123 is the method of embodiment 122, the first target hybridizing sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 10; the second target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:7; the third target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:8; and / or The fourth target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:9.

[0189] Embodiment 124 is the method of any one of embodiments 121 to 123, wherein the first amplification oligomer is a promoter primer or promoter provider, further comprising a promoter sequence located 5' to its respective target hybridizing sequence.

[0190] Embodiment 125 is the method of embodiment 124, wherein the promoter sequence is a T7 promoter sequence.

[0191] Embodiment 126 is the method of embodiment 125, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:10.

[0192] Embodiment 127 is the method of embodiment 124,

[0193] The first amplification oligomer comprises the nucleotide sequence of SEQ ID NO:10.

[0194] Embodiment 128 is the method of any one of embodiments 121 to 127, further comprising, prior to step (2), purifying the target nucleic acid of the Lactobacillus spp., if present, from other components in the sample.

[0195] Embodiment 129 is the method of embodiment 128, wherein the purifying step comprises contacting the sample with at least one capture probe oligomer comprising a target-hybridizing sequence covalently linked to an immobilized probe-binding sequence or moiety, wherein the capture probe target-hybridizing sequence specifically hybridizes to a target sequence within the target nucleic acid of a Lactobacillus species.

[0196] Embodiment 130 is the method of embodiment 129, wherein the capture probe target hybridizing sequence specifically hybridizes to a target sequence within each of the target nucleic acids of Lactobacillus crispatus (L. crispatus), Lactobacillus jensenii (L. jensenii), and Lactobacillus gasseri (L. gasseri).

[0197] Embodiment 131 is the method of embodiment 130, The first capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO:6.

[0198] Embodiment 132 is the method of embodiment 131, in which the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:6.

[0199] Embodiment 133 is the method of any one of embodiments 121 to 132, wherein the detecting step (3) comprises contacting the one or more amplification products with a first detection probe comprising a target-hybridizing sequence that specifically hybridizes to a target region of a Lactobacillus spp.; This involves detecting the presence or absence of a target hybridized detector probe.

[0200] Embodiment 134 is the method of embodiment 133, wherein the first detection probe target hybridizing sequence specifically hybridizes to a target region of each of the target nucleic acids of L. crispatus and L. jensenii; The method further includes contacting one or more amplification products with a second detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of a target nucleic acid of Lactobacillus gasseri (L. gasseri).

[0201] Embodiment 135 is the method of embodiment 134, the first detector probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-17 of SEQ ID NO: 11; and / or The second detector probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0202] Embodiment 136 is the method of embodiment 135, the first detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1-17 of SEQ ID NO: 11; and / or The second detector probe target hybridizing sequence comprises the nucleotide sequence of residues 7-23 of SEQ ID NO:12.

[0203] Embodiment 137 is the method of embodiment 133, in which the first detection probe comprises a label.

[0204] Embodiment 138 is the method of embodiment 137, in which the label is a chemiluminescent label or a fluorescent label.

[0205] Embodiment 139 is the method of embodiment 137, in which the detecting step (3) occurs during the amplifying step (2).

[0206] Embodiment 140 is the method of embodiment 139, in which the detection probe comprises a fluorescent label and a quencher.

[0207] Embodiment 141 is the method of embodiment 140, in which the detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0208] Embodiment 142 is the method of any one of embodiments 134 to 136, wherein each of the first and second Lactobacillus-specific detection probes comprises a label.

[0209] Embodiment 143 is the method of embodiment 142, in which the label is a chemiluminescent label or a fluorescent label.

[0210] Embodiment 144 is the method of embodiment 142, in which the detecting step (3) occurs during the amplifying step (2).

[0211] Embodiment 145 is the method of embodiment 144, in which each detection probe comprises a fluorescent label and a quencher.

[0212] Embodiment 146 is the method of embodiment 145, in which each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0213] Embodiment 147 is the method of embodiment 133, in which the first detection probe further comprises a non-target hybridizing sequence.

[0214] Embodiment 148 is the method of embodiment 147, in which the first detection probe is a molecular torch or a molecular beacon.

[0215] Embodiment 149 is the method of any one of embodiments 134 to 136, wherein at least one of the first and second detection probes further comprises a non-target hybridizing sequence.

[0216] Embodiment 150 is the method of embodiment 149, in which each of the first and second detection probes is a molecular torch or a molecular beacon.

[0217] Embodiment 151 is a method for determining the presence or absence of Atopobium vaginae (A. vaginae) in a sample, the method comprising: (1) contacting a sample suspected of containing A. vaginae with first and second amplification oligomers for amplifying a target region of an 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) conducting an in vitro nucleic acid amplification reaction in which A. vaginae target nucleic acid, if present in the sample, is used as a template to produce one or more amplification products corresponding to target regions of A. vaginae; (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of A. vaginae in the sample. Embodiment 152 is the method of embodiment 151, the first target hybridizing sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 18; and / or The second target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:17.

[0218] Embodiment 153 is the method of embodiment 151 or 152, the first target hybridizing sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18; and / or The second target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:17.

[0219] Embodiment 154 is the method of any one of embodiments 151 to 153, wherein the first amplification oligomer is a promoter primer or promoter provider, further comprising a promoter sequence located 5' to its respective target hybridizing sequence.

[0220] Embodiment 155 is the method of embodiment 154, in which the promoter sequence is a T7 promoter sequence.

[0221] Embodiment 156 is the method of embodiment 155, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:18.

[0222] Embodiment 157 is the method of embodiment 154, in which the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO:18.

[0223] Embodiment 158 ​​is the method of any one of embodiments 151 to 157, further comprising, prior to step (2), purifying the A. vaginae target nucleic acid, if present, from other components in the sample.

[0224] Embodiment 159 is the method of embodiment 158, wherein the purification step comprises contacting the sample with at least one capture probe oligomer comprising a target hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe, wherein the capture probe target hybridizing sequence specifically hybridizes to a target sequence within the target nucleic acid of A. vaginae.

[0225] Embodiment 160 is the method of embodiment 159, in which the capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO:13.

[0226] Embodiment 161 is the method of embodiment 160, in which the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0227] Embodiment 162 is the method of any one of embodiments 151 to 161, wherein the detecting step (3) comprises contacting the one or more amplification products with a detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of A. vaginae; This involves detecting the presence or absence of a target hybridized detector probe.

[0228] Embodiment 163 is the method of embodiment 162, wherein the detection probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 6 to 21 of SEQ ID NO:19.

[0229] Embodiment 164 is the method of embodiment 163, in which the detection probe target hybridizing sequence comprises the nucleotide sequence of residues 6 to 21 of SEQ ID NO:19.

[0230] Embodiment 165 is the method of any one of embodiments 162 to 164, wherein the detection probe comprises a label.

[0231] Embodiment 166 is the method of embodiment 165, in which the label is a chemiluminescent label or a fluorescent label.

[0232] Embodiment 167 is the method of embodiment 165, in which the detecting step (3) occurs during the amplifying step (2).

[0233] Embodiment 168 is the method of embodiment 167, in which the detection probe comprises a fluorescent label and a quencher.

[0234] Embodiment 169 is the method of embodiment 168, in which the detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0235] Embodiment 170 is the method of any one of embodiments 162 to 164, wherein the detection probe further comprises a non-target hybridizing sequence.

[0236] Embodiment 171 is the method of embodiment 170, in which the detection probe is a molecular torch or a molecular beacon.

[0237] Embodiment 172 is a method for determining the presence or absence of Gardnerella vaginalis (G. vaginalis) in a sample, the method comprising: (1) contacting a sample suspected of containing Gardnerella vaginalis (G. vaginalis) with first and second amplification oligomers for amplifying a target region of a Gardnerella vaginalis (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 in which a Gardnerella vaginalis (G. vaginalis) target nucleic acid, if present in the sample, is used as a template to produce one or more amplification products corresponding to a target region of Gardnerella vaginalis (G. vaginalis); (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Gardnerella vaginalis (G. vaginalis) in the sample. Embodiment 173 is the method of embodiment 172, the first target hybridizing sequence comprises the nucleotide sequence of residues 36-52 of SEQ ID NO: 15; and / or The second target hybridizing sequence comprises the nucleotide sequence of SEQ ID NO:14.

[0238] Embodiment 174 is the method of embodiment 172 or 173, the first target hybridizing sequence consists of the nucleotide sequence of residues 36-52 of SEQ ID NO: 15; and / or The second target hybridizing sequence consists of the nucleotide sequence of SEQ ID NO:14.

[0239] Embodiment 175 is the method of any one of embodiments 172 to 174, wherein the first amplification oligomer is a promoter primer or promoter provider, further comprising a promoter sequence located 5' to the respective target hybridizing sequence.

[0240] Embodiment 176 is the method of embodiment 175, in which the promoter sequence is a T7 promoter sequence.

[0241] Embodiment 177 is the method of embodiment 176, wherein the promoter sequence has the nucleotide sequence of residues 9 to 36 of SEQ ID NO:15.

[0242] Embodiment 178 is the method of embodiment 175, wherein the first amplification oligomer comprises the nucleotide sequence of residues 9 to 52 of SEQ ID NO:15.

[0243] Embodiment 179 is the method of embodiment 178, in which the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

[0244] Embodiment 180 is the method of any one of embodiments 172 to 179, further comprising, prior to step (2), purifying the Gardnerella vaginalis (G. vaginalis) target nucleic acid, if present, from other components in the sample.

[0245] Embodiment 181 is the method of embodiment 180, wherein the purification step includes contacting the sample with at least one capture probe oligomer comprising a target hybridizing sequence covalently linked to a sequence or moiety that binds to the immobilized probe, wherein the capture probe target hybridizing sequence specifically hybridizes to a target sequence within a target nucleic acid of Gardnerella vaginalis (G. vaginalis).

[0246] Embodiment 182 is the method of embodiment 181, in which the capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO:13.

[0247] Embodiment 183 is the method of embodiment 182, in which the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO:13.

[0248] Embodiment 184 is the method of any one of embodiments 172 to 183, wherein the detecting step (3) comprises contacting the one or more amplification products with a detection probe comprising a target hybridizing sequence that specifically hybridizes to a target region of Gardnerella vaginalis (G. vaginalis); This involves detecting the presence or absence of a target hybridized detector probe.

[0249] Embodiment 185 is the method of embodiment 184, wherein the detection probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO:16.

[0250] Embodiment 186 is the method of embodiment 185, in which the detection probe target hybridizing sequence comprises the nucleotide sequence of residues 1 to 18 of SEQ ID NO:16.

[0251] Embodiment 187 is the method of any one of embodiments 184 to 186, wherein the detection probe comprises a label.

[0252] Embodiment 188 is the method of embodiment 187, in which the label is a chemiluminescent label or a fluorescent label.

[0253] Embodiment 189 is the method of embodiment 187, in which the detecting step (3) occurs during the amplifying step (2).

[0254] Embodiment 190 is the method of embodiment 189, wherein each detection probe comprises a fluorescent label and a quencher.

[0255] Embodiment 191 is the method of embodiment 190, in which each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0256] Embodiment 192 is the method of any one of embodiments 184 to 186, wherein the detection probe further comprises a non-target hybridizing sequence.

[0257] Embodiment 193 is the method of embodiment 192, in which the detection probe is a molecular torch or a molecular beacon.

[0258] Embodiment 194 is the method of any one of embodiments 121 to 193, wherein the amplification reaction in step (2) is an isothermal amplification reaction.

[0259] Embodiment 195 is the method of embodiment 194, in which the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0260] Embodiment 196 is the method of embodiment 194 or 195, wherein the amplification reaction is a real-time amplification reaction.

[0261] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art related to the methods and compositions being described. As used herein, the following terms and phrases have the meanings ascribed to them unless specified otherwise.

[0262] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0263] The conjunction "or" should be construed in an inclusive sense, ie, equivalent to "and / or," unless an inclusive sense is unwarranted in the context.

[0264] The term "about" refers to slight variations in the amount of a component of a composition that do not significantly affect the activity or stability of the composition. In some embodiments, "about" encompasses variations within 10%, 5%, 2%, 1%, or 0.5% of the stated value.

[0265] All ranges should be interpreted as inclusive of the endpoints unless expressly excluded, such as "excluding the endpoints," so "in the range of 10 to 15" includes the values ​​10 and 15, and all integer and (where possible) non-integer values ​​between the endpoints, for example, 11, 11.5, 12 and 1 / 3, 4π, etc.

[0266] Additionally, the terms "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" are open-ended and not intended to be limiting.

[0267] A "sample" includes any specimen that may contain Lactobacillus spp., Atopobium vaginae, or Gardnerella vaginalis, or components thereof, such as nucleic acids or fragments of nucleic acids. A sample may contain Lactobacillus spp., Atopobium veginae, or Gardnerella vaginalis, or components thereof, such as nucleic acids or fragments of nucleic acids. "Biological samples" include any tissue or material from a living or deceased human that may contain Gardnerella vaginae or Gardnerella vaginalis or components thereof (e.g., target nucleic acids derived therefrom), including, for example, vaginal swab samples, cervical brush samples, respiratory tissue or exudates such as from bronchoscopy, bronchoalveolar lavage (BAL), or lung biopsy, sputum, saliva, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, feces, urine, semen, or other bodily fluids or materials. Biological samples may be treated to physically or mechanically disrupt tissue or cellular structures, thus releasing intracellular components into a solution that may further contain enzymes, buffers, salts, detergents, etc., used to prepare the biological sample for analysis using standard methods. Samples may also include processed samples, such as those obtained by passing the sample over or through a filtration device, or following centrifugation, or by attachment to a medium, matrix, or support.

[0268] "Nucleic acid" refers to a polymeric compound containing two or more covalently linked nucleosides or nucleoside analogs with nitrogenous heterocyclic bases or base analogs, where the nucleosides are linked together by phosphodiester or other bonds to form a polynucleotide. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and their analogs. The nucleic acid "backbone" can be composed of various linkages, including one or more of sugar-phosphodiester linkages, peptide-nucleic acid linkages (as in "peptide nucleic acids" or PNAs; see PCT Publication WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar moiety of a nucleic acid can be either ribose or deoxyribose, or similar compounds with known substitutions, such as 2'-methoxy and 2'-halide substitutions (e.g., 2'-F). Nitrogenous bases include the conventional bases (A, G, C, T, U), their analogs (e.g., inosine, 5-methylisocytosine, isoguanine, The Biochemistry of the Nucleic Acids Acids) 5-36, edited by Adams et al. 第 11th ed., 1992; Abraham et al., 2007, BioTechniques 43:617-24), which may be a derivative of a purine or pyrimidine base (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituents at the 5- or 6-position, purine bases with modified or replaced substituents at the 2-, 6-, and / or 8-positions, e.g., 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4These include -alkyl-pyrimidines, and pyrazolo-compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines (U.S. Pat. Nos. 5,378,825, 6,949,367, and PCT WO 93 / 13121). Nucleic acids can contain "abasic" residues, in which the backbone does not contain a nitrogenous base for one or more residues (U.S. Pat. No. 5,585,481). Nucleic acids can contain only conventional sugars, bases, and linkages as found in RNA and DNA, or can contain conventional components and substitutions (e.g., conventional bases linked by a 2' methoxy backbone, or nucleic acids containing a mixture of conventional bases and one or more base analogs). Nucleic acids may include "locked nucleic acids" (LNAs), in which one or more nucleotide monomers have a bicyclic furanose unit locked into an RNA-mimetic sugar conformation, which enhances hybridization affinity to complementary sequences in single-stranded RNA (ssRNA), single-stranded DNA (ssDNA), or double-stranded DNA (dsDNA) (Vester et al., Biochemistry 43:13233-41, 2004). Nucleic acids may contain modified bases to alter the function or behavior of the nucleic acid, for example, the addition of 3'-terminal dideoxynucleotides to block the addition of additional nucleotides to the nucleic acid. Synthetic methods for making nucleic acids in vitro are well known in the art, but nucleic acids can also be purified from natural sources using routine techniques.

[0269] As used herein, the term "polynucleotide" refers to a nucleic acid strand. Throughout this application, nucleic acids are designated from the 5' to the 3' end. Standard nucleic acids, such as DNA and RNA, are typically synthesized "5' to 3'," i.e., by the addition of nucleotides to the 3' end of the elongating nucleic acid.

[0270] As used herein, a "nucleotide" is a nucleic acid subunit consisting of a phosphate group, a five-carbon sugar, and a nitrogenous base. The five-carbon sugar found in RNA is ribose. In DNA, the five-carbon sugar is 2'-deoxyribose. The term also includes analogs of such subunits, such as a methoxy group at the 2' position of ribose (2'-O-Me).

[0271] As used herein, a "nucleic acid-based detection assay" is an assay for the detection of a target sequence within a target nucleic acid, which utilizes another oligonucleotide that specifically hybridizes to the target sequence.

[0272] In certain embodiments according to the present invention, nucleic acid-based detection assay is "amplification-based assay", that is, an assay that utilizes one or more steps to amplify nucleic acid target sequence.A variety of amplification methods for use in detection assays are well known in the art, some of which are further summarized herein.For clarity, amplification-based assay may include one or more steps that do not amplify target sequence, such as the steps used in non-amplification-based assay methods (e.g., hybridization assay or cleavage-based assay).

[0273] In other embodiments, the nucleic acid-based detection assay is a "non-amplification-based assay," i.e., an assay that does not rely on any step to amplify the nucleic acid target sequence. For clarity, a nucleic acid-based detection assay that includes a primer extension reaction in the absence of a corresponding downstream amplification oligomer (e.g., primer extension with reverse transcriptase to produce an RNA:DNA duplex, followed by RNase digestion of the RNA to obtain a single-stranded cDNA complementary to the RNA target, but no cDNA copy is produced) is understood to be a non-amplification-based assay.

[0274] An exemplary non-amplification-based assay is a "cleavage-based assay," which relies on the specific cleavage by a flap endonuclease of a linear double-stranded break structure formed by specific hybridization of overlapping oligonucleotides to a target nucleic acid. In these assays, a probe oligonucleotide containing a non-target hybridizing flap region is cleaved by the flap endonuclease in an overlap-dependent manner, releasing a cleavage product that is then detected. The principles of cleavage-based assays are well known in the art, and exemplary assays are described, for example, in Lyamichev et al. (Nat. Biotechnol. 17:292-296, 1999), Ryan et al. (Mol. Diagn. 4:135-144, 1999), Allawi et al. (J. Clin. Microbiol. 44:3443-3447, 2006), U.S. Patent Nos. 5,846,717 and 6,706,471 to Brow et al., and U.S. Patent No. 5,614,402 to Dahlberg et al. Cleavage-based assays include, for example, the commercially available Invader® assay (Hologic, Madison, Wis.).

[0275] As used herein, "target nucleic acid" refers to a nucleic acid containing a target sequence to be detected. The target nucleic acid may be DNA or RNA as described herein, and may be either single-stranded or double-stranded. The target nucleic acid may also contain other sequences other than the target sequence.

[0276] By "isolated" it is meant that the sample containing the target nucleic acid is removed from its natural environment, but the term does not imply any degree of purification.

[0277] As used herein, the term "target sequence" refers to a specific nucleotide sequence of a target nucleic acid to be detected. "Target sequence" includes a complex sequence to which an oligonucleotide (e.g., a probe oligonucleotide, a priming oligonucleotide, and / or a promoter oligonucleotide) is complexed during the detection process (e.g., an amplification-based detection assay such as TMA or PCR, or a non-amplification-based detection assay such as a cleavage-based assay). If the target nucleic acid is originally single-stranded, the term "target sequence" also refers to a sequence complementary to the "target sequence" present in the target nucleic acid. If the target nucleic acid is originally double-stranded, the term "target sequence" refers to both the sense (+) and antisense (-) strands. When selecting a target sequence, those skilled in the art will understand that a "unique" sequence should be selected to distinguish between unrelated and closely related target nucleic acids.

[0278] "Target hybridization sequence" is used herein to refer to the oligomer portion that is configured to hybridize with a target nucleic acid sequence.Preferably, the target hybridization sequence is configured to specifically hybridize with a target nucleic acid sequence.The target hybridization sequence may be, but is not necessarily, 100% complementary to the portion of the target sequence that it is configured to hybridize with.The target hybridization sequence may also contain inserted, deleted, and / or substituted nucleotide residues relative to the target sequence.The target hybridization sequence may be less than 100% complementary to the target sequence when the target nucleic acid is a multiple strain within a species, for example, in the case of an oligomer that is configured to hybridize with various strains of Lactobacillus.It is understood that there are other reasons for configuring the target hybridization sequence to have less than 100% complementarity with the target nucleic acid.

[0279] As used herein, the term "targeting a sequence" with respect to a region of a nucleic acid of Lactobacillus spp., A. vaginae, or G. vaginalis refers to the process by which an oligonucleotide hybridizes to a target sequence in a manner that allows for detection as described herein. In one embodiment, the oligonucleotide is complementary to the nucleic acid sequence of the targeted Lactobacillus spp., A. vaginae, or G. vaginalis and contains no mismatches. In another embodiment, the oligonucleotide is complementary to but contains one, two, three, four, or five mismatches with the nucleic acid sequence of the targeted Lactobacillus spp., A. vaginae, or G. vaginalis. Preferably, an oligonucleotide that hybridizes to a target nucleic acid sequence contains at least 10 and up to 50 nucleotides complementary to the target sequence. At least 10 and up to 50 are inclusive ranges, and are understood to include 10, 50, and every integer therebetween. Preferably, the oligomer specifically hybridizes to the target sequence.

[0280] The term "configured to" refers to the actual arrangement of the polynucleotide sequence makeup of a referenced oligonucleotide target hybridizing sequence. For example, an oligonucleotide configured to specifically hybridize to a target sequence has a polynucleotide sequence that specifically hybridizes to the referenced sequence under stringent hybridization conditions.

[0281] As used herein, the term "configured to specifically hybridize" means that the target hybridizing region of an oligonucleotide is designed to have a polynucleotide sequence capable of targeting a sequence in a referenced Lactobacillus species, A. vaginae, or G. vaginalis target region. Such oligonucleotides are not limited to targeting only that sequence, but rather are useful as compositions, in kits, or in methods for targeting target nucleic acids in Lactobacillus species, A. vaginae, or G. vaginalis. The oligonucleotides are designed to function as components of assays for the detection of Lactobacillus spp., A. vaginae, or G. vaginalis from a sample, and are therefore designed to target Lactobacillus spp., A. vaginae, or G. vaginalis in the presence of other nucleic acids typically found in test samples. "Specifically hybridize" does not mean exclusively hybridize, as is understood in the art, since some small level of hybridization to non-target nucleic acids may occur. Rather, "specifically hybridize" means that the oligonucleotide is configured to function in an assay that primarily hybridizes to the target, such that accurate detection of the target nucleic acid in a sample can be determined. The term "configured to" refers to the actual arrangement of the polynucleotide sequence configuration of the oligonucleotide target hybridizing sequence.

[0282] As used herein with respect to a targeting nucleic acid of Lactobacillus spp., A. vaginae, or G. vaginalis, the term "fragment" refers to a single contiguous nucleic acid. In certain embodiments, the fragment comprises contiguous nucleotides from the 16S ribosomal RNA of Lactobacillus spp., A. vaginae, or G. vaginalis, and the number of 16S contiguous nucleotides in the fragment is less than the number of entire 16S ribosomal nucleotides.

[0283] As used herein, the term "region" refers to a portion of a nucleic acid, the portion being smaller than the entire nucleic acid. For example, if the referenced nucleic acid is an oligonucleotide promoter primer, the term "region" can be used to refer to a smaller promoter portion of the entire oligonucleotide. Similarly, and by way of example only, if the nucleic acid is 16S ribosomal RNA, the term "region" can be used to refer to a smaller region of the nucleic acid, the smaller region being targeted by one or more oligonucleotides of the present invention. As another non-limiting example, if the referenced nucleic acid is an amplicon, the term region can be used to refer to a smaller nucleotide sequence identified for hybridization by the target hybridizing sequence of a probe.

[0284] The interchangeable terms "oligomer," "oligo," and "oligonucleotide" generally refer to nucleic acids having fewer than 1,000 nucleotide (nt) residues, including polymers within a range having a lower limit of about 5 nt residues and an upper limit of about 500-900 nt residues. In some embodiments, oligonucleotides fall within a size range having a lower limit of about 12-15 nt and an upper limit of about 50-600 nt, while other embodiments fall within a range having a lower limit of about 15-20 nt and an upper limit of about 22-100 nt. Oligonucleotides may be purified from naturally occurring sources or synthesized using any of a variety of well-known enzymatic or chemical methods. The term "oligonucleotide" does not denote any specific function for the reagent, but rather is used generically to cover all such reagents described herein. Oligonucleotides may serve a variety of different functions. For example, an oligonucleotide can function as a primer if it is capable of specific hybridization to a complementary strand and further extension in the presence of a nucleic acid polymerase; an oligonucleotide can function as a primer and provide a promoter if it contains a sequence recognized by an RNA polymerase and allows transcription (e.g., a T7 primer); an oligonucleotide can function to detect a target nucleic acid if it is capable of hybridizing to a target nucleic acid, or an amplicon thereof, and further provides a detectable moiety (e.g., an acridinium ester compound).

[0285] As used herein, an oligonucleotide "substantially corresponds" to a particular reference nucleic acid sequence means that the oligonucleotide is sufficiently similar to the reference nucleic acid sequence such that the oligonucleotide has similar hybridization properties to the reference nucleic acid sequence, such that the oligonucleotide hybridizes to the same target nucleic acid sequence under stringent hybridization conditions. Those skilled in the art will understand that a "substantially corresponding oligonucleotide" may differ from the reference sequence and still hybridize to the same target nucleic acid sequence. It is also understood that a first nucleic acid corresponding to a second nucleic acid includes its RNA and DNA counterparts, as well as its complements, unless the context clearly indicates otherwise. This variation from the nucleic acid can be described in terms of the percentage of identical bases within the sequence or the percentage of perfectly complementary bases between a probe or primer and its target sequence. Thus, in certain embodiments, an oligonucleotide "substantially corresponds" to a reference nucleic acid sequence if the percentage of base identity or complementarity is between 100% and about 80%. In a preferred embodiment, the percentage is between 100% and about 85%. In more preferred embodiments, the percentage is 100% to about 90%, and in other preferred embodiments, the percentage is 100% to about 95%. Similarly, a region of a nucleic acid or amplified nucleic acid can be considered herein as corresponding to a reference nucleic acid sequence. Those skilled in the art will understand the various modifications to hybridization conditions that may be required at various percentages of complementarity to allow hybridization to specific target sequences without causing unacceptable levels of nonspecific hybridization.

[0286] An "amplification oligomer" is an oligomer, or its complement, that has at least its 3' end complementary to a target nucleic acid, hybridizes to the target nucleic acid or its complement, and participates in a nucleic acid amplification reaction. An example of an amplification oligomer is a "primer" that hybridizes to the target nucleic acid and contains a 3' OH end that is extended by a polymerase during the amplification process. Another example of an amplification oligomer is an oligomer that is not extended by a polymerase (e.g., because it has a 3' blocked end), but participates in or facilitates amplification. For example, the 5' region of an amplification oligonucleotide may contain a promoter sequence that is non-complementary to the target nucleic acid (which may be referred to as a "promoter primer" or "promoter provider"). Those skilled in the art will understand that an amplification oligomer that functions as a primer can be modified to include a 5' promoter sequence and thus function as a promoter primer. The incorporation of a 3' blocked end further modifies the promoter primer, allowing it to hybridize to the target nucleic acid and provide an upstream promoter sequence that helps initiate transcription, but does not provide a primer for oligonucleotide extension. Such modified oligos are referred to herein as "promoter provider" oligomers. Amplification oligonucleotides range in size from about 10 nt to about 70 nt in length (not including any promoter sequence or poly-A tail) and include those containing at least about 10 contiguous bases, or at least 12 contiguous bases, that are complementary to a region of the target nucleic acid sequence (or its complementary strand). The contiguous bases are at least 80%, or at least 90%, or fully complementary to the target sequence to which the amplification oligomer binds. Amplification oligomers may contain modified nucleotides or analogs, or additional nucleotides that participate in the amplification reaction but are not complementary to or contained in the target nucleic acid or template sequence. For example, an amplification oligomer may contain additional nucleotides at its 5' end complementary to its 3' end for the purpose of forming a hairpin to modulate priming efficiency (e.g., a promoter primer may contain additional nucleotides upstream of the promoter sequence for this purpose).When referring to a range of lengths of oligonucleotides, amplicons, or other nucleic acids, it is understood that the range includes all integers (e.g., a length of 19 to 25 contiguous nucleotides includes 19, 20, 21, 22, 23, 24, and 25).

[0287] As used herein, a "promoter" is a specific nucleic acid sequence that binds to a nucleic acid and is recognized by a DNA-dependent RNA polymerase ("transcriptase") as a signal to initiate transcription of RNA at a specific site.

[0288] As used herein, "promoter provider" or "provider" refers to an oligonucleotide that contains a first and a second region and is modified to prevent initiation of DNA synthesis from its 3' end. The "first region" of a promoter provider oligonucleotide contains a base sequence that hybridizes to a DNA template; the hybridizing sequence is located 3' to the promoter region, but is not necessarily adjacent to it. The hybridizing portion of a promoter oligonucleotide is typically at least 10 nucleotides in length and may extend up to 50 nucleotides or more in length. The "second region" contains a promoter sequence for an RNA polymerase. The promoter oligonucleotide is preferably engineered to prevent it from being elongated by an RNA- or DNA-dependent DNA polymerase, such as reverse transcriptase, and preferably contains a blocking moiety at its 3' end, as described above. As referred to herein, a "T7 provider" is a blocked promoter provider oligonucleotide that provides an oligonucleotide sequence recognized by T7 RNA polymerase.

[0289] "Amplification" refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence, its complement, or its fragments. The multiple copies may be referred to as amplicons or amplification products. Known amplification methods include both thermal cycling and isothermal amplification. In some embodiments, isothermal amplification is preferred. Replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification are non-limiting examples of nucleic acid amplification methods. Replicase-mediated amplification uses self-replicating RNA molecules and a replicase such as QB replicase (e.g., U.S. Pat. No. 4,786,600). PCR amplification uses DNA polymerase, primer pairs, and thermal cycling to synthesize multiple copies of two complementary strands of dsDNA or multiple copies from cDNA (e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,800,159). LCR amplification uses four or more different oligonucleotides to amplify a target and its complementary strand through multiple cycles of hybridization, ligation, and denaturation (e.g., U.S. Pat. Nos. 5,427,930 and 5,516,663). SDA uses primers containing recognition sites for restriction endonucleases and an endonuclease that nicks one strand of a semi-modified DNA duplex containing the target sequence, thereby resulting in amplification in a series of primer extension and strand displacement steps (e.g., U.S. Pat. Nos. 5,422,252, 5,547,861, and 5,648,211). While preferred embodiments use amplification methods suitable for amplifying RNA target nucleic acids, such as transcription-mediated amplification (TMA) or NASBA, it will be apparent to those skilled in the art that the oligomers disclosed herein can readily be used as primers in other amplification methods.

[0290] "Transcription-associated amplification," also referred to herein as "transcription-mediated amplification" (TMA), refers to nucleic acid amplification that uses an RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods generally employ an RNA polymerase, a DNA polymerase, deoxyribonucleoside triphosphates, ribonucleoside triphosphates, and a template-complementary oligonucleotide containing a promoter sequence, and may optionally include one or more other oligonucleotides. The TMA method is an embodiment of the amplification method used to amplify and detect target sequences described herein. Variations of transcription-associated amplification are well known in the art, as previously disclosed in detail (e.g., U.S. Patent Nos. 4,868,105, 5,124,246, 5,130,238, 5,399,491, 5,437,990, 5,554,516, and 7,374,885, as well as PCT Nos. WO88 / 01302, WO88 / 10315, and WO95 / 03430). Those skilled in the art will understand that the disclosed compositions can be used in amplification methods based on the extension of oligomeric sequences by polymerases.

[0291] As used herein, the term "real-time TMA" refers to transcription-mediated amplification ("TMA") of a target nucleic acid monitored by a real-time detection means.

[0292] The term "amplicon," used interchangeably with "amplification product," refers to a nucleic acid molecule produced during an amplification procedure that is complementary to or homologous to a sequence contained within a target sequence. These terms can be used to refer to a single-stranded amplification product, a double-stranded amplification product, or one of the strands of a double-stranded amplification product.

[0293] "Probe," "detection probe," "detection oligonucleotide," and "detection probe oligomer" are used interchangeably to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid or amplified nucleic acid under conditions that promote hybridization to enable detection of the target sequence or amplified nucleic acid. Detection can be direct (e.g., a probe directly hybridized to its target sequence) or indirect (e.g., a probe linked to its target via an intermediate molecular structure). Probes can be DNA, RNA, analogs thereof, or combinations thereof, and can be labeled or unlabeled. The "target sequence" of a probe generally refers to a smaller nucleic acid sequence within a larger nucleic acid sequence that specifically hybridizes to at least a portion of the probe oligomer through standard base pairing. The probes may contain target-specific sequences and other sequences that contribute to the three-dimensional conformation of the probe (e.g., U.S. Patent Nos. 5,118,801, 5,312,728, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, and U.S. Patent Application Publication No. 20060068417). In a preferred embodiment, the detection probe contains a 2' methoxy backbone, which may result in a higher signal.

[0294] "TaqMan (登録商標) The term "probe" refers to a detection oligonucleotide that typically contains a fluorescent dye at the 5' base and a non-fluorescent quencher dye (quencher) at the 3' base. When illuminated, the excited fluorescent dye transfers energy to a nearby quencher dye molecule rather than fluorescing, resulting in a non-fluorescent substrate. During amplification, the exonuclease activity of the polymerase cleaves the TaqMan probe, separating the fluorophore from the quencher, thereby releasing an unquenched signal from the fluorophore as an indication of amplification.

[0295] As used herein, "label" refers to a moiety or compound directly or indirectly attached to a probe that is detected or provides a detectable signal. Direct labeling can occur through a bond or interaction that links the label to the probe, including covalent or non-covalent interactions, such as hydrogen bonding, hydrophobic and ionic interactions, or the formation of chelate or coordination complexes. Indirect labeling can occur through the use of a bridging moiety or "linker," such as a binding pair member, antibody, or additional oligomer, that is either directly or indirectly labeled and can amplify the detectable signal. Labels include radionuclides, ligands (e.g., biotin, avidin), enzymes or enzyme substrates, reactive groups, or chromophores (e.g., dyes, particles, or beads that impart a detectable color), light-emitting compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent labels), or fluorophores. Labels can be detectable in homogeneous assays in which bound, labeled probes in a mixture exhibit a detectable change, e.g., instability or different degradation characteristics, that differ from unbound, labeled probes. "Homogeneous detectable labels" can be detected without physically removing the label in its unbound form or bound from the labeled probe (e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Labels include chemiluminescent compounds, such as standard acridinium esters ("AE") and AE compounds, including derivatives (e.g., U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,639,604). Methods for synthesis, attachment of labels to nucleic acids, and detection of labels are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) Chapter 10, and U.S. Pat. Nos. 5,658,737, 5,656,207, 5,547,842, 5,283,174, and 4,581,333).Multiple labels and multiple types of labels may be present on a particular probe, or detection may use a mixture of probes where each probe is labeled with a compound that produces a detectable signal (see, e.g., U.S. Pat. Nos. 6,180,340 and 6,350,579).

[0296] As used herein, the structure referred to as a "molecular torch" is designed to contain distinct self-complementary regions ("closing domains") connected by binding regions ("target binding domains") and hybridizing to each other under predetermined hybridization assay conditions. All or part of the nucleotide sequence comprising the target closing domain can also function as the target binding domain. Thus, the target closing sequence can include target binding sequences, non-target binding sequences, and combinations thereof.

[0297] The terms "capture probe," "capture oligonucleotide," "target capture oligonucleotide," and "capture probe oligomer" are used interchangeably herein to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a target nucleic acid by standard base pairing and binds to a binding partner on an immobilized probe, capturing the target nucleic acid to a support. One example of a capture oligomer is an oligonucleotide that includes two binding regions: a target hybridizing sequence and an immobilized probe binding region. In a variation of this example, the two regions may reside on two different oligomers linked to each other by one or more linkers. In another embodiment of a capture oligomer, the target hybridizing sequence is a sequence that includes a random or nonrandom poly-GU, poly-GT, or poly-U sequence for nonspecifically binding to the target nucleic acid and binding it to the immobilized probe on the support (see, e.g., PCT Publication WO 2008 / 016988). The immobilized probe binding region may be a nucleic acid sequence referred to as a tail. The tail is a sequence of approximately 10 to 40 nucleotides (e.g., A) that binds to a complementary immobilized sequence attached to a support particle or support matrix. 10 ~A 40 ) or about 14-33 nt (e.g., T3A14 ~T3A 30 ) in some embodiments. 0~4 A 10~40 Another example of a capture oligomer contains two regions, a target hybridizing sequence and a binding pair member that is not a nucleic acid sequence.

[0298] As used herein, "immobilized oligonucleotide," "immobilized probe," or "immobilized nucleic acid" refers to a nucleic acid binding partner that directly or indirectly binds a capture oligomer to a support. Support-bound immobilized probes facilitate separation of the capture probe-bound target from unbound materials in a sample. One embodiment of an immobilized probe is an oligomer bound to a support, facilitating separation of the bound target sequence from unbound materials in a sample. Supports can include known materials such as solution-free matrices and particles made of nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal, or other compositions, one embodiment of which is magnetically attractable particles. The support can be monodisperse magnetic spheres (e.g., uniform size ±5%) to which the immobilized probe is directly (via covalent bonding, chelation, or ionic interaction) or indirectly (via one or more linkers), and the bond or interaction between the probe and the support is stable under hybridization conditions. DETAILED DESCRIPTION OF THE INVENTION

[0299] The present invention generally relates to methods and compositions for determining the presence or absence of selected bacterial organisms in a sample. In some embodiments, the present invention provides methods and compositions for diagnosing bacterial vaginosis (BV) in a subject. In other, non-mutually exclusive embodiments, the present invention provides methods for detecting one or more of Lactobacillus species, A. vaginae, and Gardnerella vaginalis (G. vaginalis) in a sample, the methods comprising performing amplification-based detection of 16S rRNA target nucleic acids from one or more of Lactobacillus species, A. vaginae, and Gardnerella vaginalis (G. vaginalis). The present invention further provides compositions (including reaction mixtures) and kits containing oligomer combinations for detecting one or more of Lactobacillus spp., A. vaginae, and G. vaginalis. The oligomer combinations generally include at least two amplification oligomers for detecting one or more of Lactobacillus spp., A. vaginae, and G. vaginalis in a sample, and may further include one or more additional oligomers described herein, such as capture probes and / or detection probes, for amplification-based detection of Lactobacillus spp., A. vaginae, and G. vaginalis.

[0300] Methods for diagnosing BV generally involve detecting the presence or absence of one or more of Lactobacillus species, A. vaginae, and Gardnerella vaginalis in a sample from a subject suspected of having BV. Specifically, assays are performed to specifically detect Lactobacillus species, A. vaginae, and G. vaginalis in each sample. Based on the results of the detection assays, quantitative values ​​are assigned to each of Lactobacillus species, A. vaginae, and G. vaginalis, and these quantitative values ​​are used to determine a single BV score. The presence or absence of BV is then determined based on a comparison of the BV score to a cutoff value, which is usually predetermined. Generally, a BV score above a cutoff value indicates a BV-positive status, and the determination of the BV score involves subtracting the Lactobacillus quantification value from the greater of the A. vaginae and G. vaginalis quantification values, where the quantification value is in units of log copies. The determination of the cutoff value can be based on predictive modeling using results from a cohort of clinical specimens. The cutoff value can be selected to maximize agreement or to target clinical sensitivity or specificity rates for the infection status of BV patients established by a clinical reference method (e.g., the Nugent score, including intermediate scores resolved by the Amsel Criteria). In some embodiments, a minimum quantitative value is imposed on Lactobacillus species and / or A. vaginae and G. vaginalis, and if the qualitative value measured by the detection assay is less than the respective imposed minimum value, the minimum value is used as the quantitative value for determining the BV score.

[0301] The quantitative values ​​of Lactobacillus species, A. vaginae, and / or G. vaginalis may also be normalized and / or weighted. For example, the BV score determination utilizes only the maximum of these two values. Because observed concentrations of G. vaginalis tend to be higher than those of A. vaginae in clinical sample populations, normalization can be used to balance the contribution of each quantitative value of A. vaginae and G. vaginalis to the BV score calculation. In some variations, normalization involves adjusting the quantitative value measured by the detection assay by a population statistic, such as the population median (e.g., dividing the concentration in copies by the population median, or, if the quantitative data is in log copies, subtracting the median log copies). Weighting can be used to influence the effect that a change in a term's value (e.g., the quantitative value of a Lactobacillus species, the quantitative value of A. vaginae or G. vaginalis, or the quantitative value of another term in the prediction equation) has on the score value. The higher the weight, the greater the potential effect of a change in the term's value on the score; the lower the weight, the smaller the potential effect.

[0302] The BV score calculation formula may further include additional terms. In some variations, the BV score calculation further includes adding or subtracting an adjustment constant to adjust the desired cutoff value (e.g., a cutoff value of zero). In some variations, the BV score calculation further includes adding an internal standard (IC) adjustment factor that can be used to compensate for sample inhibition in the detection assay. A particularly suitable IC adjustment factor is based on the ratio of the observed IC value generated from the detection assay to the expected IC value of the detection assay.

[0303] In certain variations of the method for diagnosing BV, the BV score is assigned using the following formula: BV score = C0 + W L Max(L S ,F LS )+W GA Max(A S ,G S ,F GAS )+W IC Log2 (IC ratio), where the elements of the formula are as explained in Table 1. [Table 1]

[0304] In some particular embodiments of the method for diagnosing BV, in which the BV score is determined by the above formula, the A. vaginalis / Gardnerella vaginalis (F GA ) is set to zero (0) before normalization.

[0305] While Lactobacillus spp., A. vaginae, and G. vaginalis can be detected using any suitable method, it is currently preferred to detect these bacteria using nucleic acid-based detection assays. Nucleic acid-based detection assays according to the present invention generally utilize oligonucleotides that specifically hybridize to target nucleic acids of Lactobacillus spp., A. vaginae, or G. vaginalis with minimal cross-reactivity with other nucleic acids suspected to be present in the sample.Thus, oligonucleotides for nucleic acid-based detection of selected species of Lactobacillus spp., A. vaginae, or G. vaginalis, e.g., Trichomonas spp., Trichomonas vaginalis, Candida spp., bacteria from the order Clostridiales, Clostridium-like species, Eggerthella spp., Enterobacteriaceae, Peptostreptococcus micros, Aerococcus christensenii, Leptotrichia amniotic fluid, and the like, can be used to detect bacterial infections such as ulcers, ulcerative colitis, and bacterial infections. amnionii, Peptoniphilus spp., Dialister spp., Mycoplasma hominis, Sneathia sanguinegens, Anaerococcus tetradius, Mobiluncus spp., Mobiluncus hominis, Megasphaera spp., Prevotella spp., Leptotrichia sanguinegens, and Finegoldia magna. In one aspect, a nucleic acid-based detection assay according to the invention further comprises components for detecting one or more of these organisms, or other bacterial genera associated with BV.

[0306] In certain embodiments, the nucleic acid-based detection assay targets the 16S rRNA, or the gene encoding the 16S rRNA, of Lactobacillus species, A. vaginae, and / or Gardnerella vaginalis. Particularly suitable target regions of the 16S rRNA or encoding gene are (i) the 16S rRNA region of L. crispatus corresponding to the region of SEQ ID NO: 1 from about nucleotide position 40 to about nucleotide position 265, (ii) the 16S rRNA region of L. jensenii corresponding to the region of SEQ ID NO: 2 from about nucleotide position 43 to about nucleotide position 247, (iii) the 16S rRNA region of L. gasseri corresponding to the region of SEQ ID NO: 3 from about nucleotide position 93 to about nucleotide position 298, (iv) the 16S rRNA region of A. vaginae corresponding to the region of SEQ ID NO: 4 from about nucleotide position 540 to about nucleotide position 625, and (v) the 16S rRNA region of Gardnerella vaginalis corresponding to the region of SEQ ID NO: 5 from about nucleotide position 172 to about nucleotide position 227. This is the rRNA region.In particular variations of the nucleic acid-based detection assays targeting the 16S rRNA region as described above, (a) the Lactobacillus-specific oligonucleotide comprises a target hybridizing region comprising a sequence substantially corresponding to the sequence of SEQ ID NO:7, a sequence substantially corresponding to the sequence of SEQ ID NO:8, a sequence substantially corresponding to the sequence of SEQ ID NO:9, a sequence substantially corresponding to the sequence of residues 28-45 of SEQ ID NO:10, a sequence substantially corresponding to the sequence of residues 1-17 of SEQ ID NO:11, or a sequence substantially corresponding to the sequence of residues 7-23 of SEQ ID NO:12; and (b) the A. vaginae e) the specific oligonucleotide comprises a target hybridizing region comprising a sequence substantially corresponding to the sequence of SEQ ID NO:17, a sequence substantially corresponding to the sequence of residues 28-45 of SEQ ID NO:18, or a sequence substantially corresponding to the sequence of residues 6-21 of SEQ ID NO:19, and / or (c) the Gardnerella vaginalis (G. vaginalis)-specific oligonucleotide comprises a target hybridizing region comprising a sequence substantially corresponding to the sequence of SEQ ID NO:14, a sequence substantially corresponding to the sequence of residues 36-52 of SEQ ID NO:15, or a sequence substantially corresponding to the sequence of residues 1-18 of SEQ ID NO:16. In some such embodiments, (a) the Lactobacillus-specific oligonucleotide comprises a target hybridizing region comprising or consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, residues 28-45 of SEQ ID NO:10, residues 1-17 of SEQ ID NO:11, or residues 7-23 of SEQ ID NO:12; (b) the A. vaginae-specific oligonucleotide comprises a target hybridizing region comprising or consisting of SEQ ID NO:17, residues 28-45 of SEQ ID NO:18, or residues 6-21 of SEQ ID NO:19; and / or (c) the Gardnerella vaginalis-specific oligonucleotide comprises a target hybridizing region comprising or consisting of SEQ ID NO:14, residues 36-52 of SEQ ID NO:15, or residues 1-18 of SEQ ID NO:16.In certain embodiments, the nucleic acid-based detection assay utilizes at least two or three Lactobacillus-specific oligonucleotides, at least two or three A. vaginae-specific oligonucleotides, and / or at least two or three Gardnerella vaginalis-specific oligonucleotides, which may be oligonucleotides selected from those specified above.

[0307] In some embodiments of methods involving the use of nucleic acid-based detection assays, amplification-based assays are used to detect Lactobacillus species, A. vaginae, and / or Gardnerella vaginalis. Such variations generally involve amplifying a target sequence within a bacterial target nucleic acid using an in vitro nucleic acid amplification reaction and detecting the amplification product, e.g., by specifically hybridizing the amplification product with a nucleic acid detection probe that provides a signal indicative of the presence of the bacterial target in the sample. The amplification step involves contacting the sample with two or more amplification oligomers specific for a target sequence in the target nucleic acid (e.g., a target sequence in 16S rRNA) to produce an amplification product if the target nucleic acid is present in the sample. Amplification involves, for example, using at least one nucleic acid polymerase to synthesize additional copies of the target sequence or its complement by extending sequences from amplification oligomers (primers) using a template strand. One embodiment for detecting amplification products uses a hybridization step that involves contacting the amplification products with at least one probe specific to a sequence amplified by the selected amplification oligomers, e.g., a sequence contained in the target sequence flanking the selected amplification oligomer pair. Suitable amplification methods include, for example, replicase-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA). Such amplification methods are well known in the art (see, e.g., the description of amplification methods in the definitions section above) and are easily used in the methods of the present invention.

[0308] For example, some amplification methods using TMA amplification include the following steps: Briefly, a target nucleic acid containing a sequence to be amplified is provided as a single-stranded nucleic acid (e.g., ssRNA or ssDNA). Those skilled in the art will understand that conventional melting of double-stranded nucleic acids (e.g., dsDNA) can be used to provide a single-stranded target nucleic acid. A promoter primer specifically binds to the target nucleic acid at its target sequence, and reverse transcriptase (RT) extends the 3' end of the promoter primer using the target strand as a template to produce a cDNA copy of the target sequence strand, resulting in an RNA:DNA duplex. RNase digests the RNA strand of the RNA:DNA duplex, and a second primer specifically binds to its target sequence located on the cDNA strand downstream from the promoter primer end. RT uses the first cDNA template to extend the 3' end of the second primer to synthesize a new DNA strand, generating a dsDNA containing a functional promoter sequence. RNA polymerase specific to the promoter sequence then initiates transcription, producing RNA transcripts that are approximately 100 to 1000 amplified copies ("amplicons") of the initial target strand in the reaction. Amplification continues when a second primer specifically binds to its target sequence in each amplicon, and RT generates DNA copies from the amplicon RNA template, producing an RNA:DNA duplex. RNase in the reaction mixture digests the amplicon RNA from the RNA:DNA duplex, and the promoter primer specifically binds to its complementary sequence in the newly synthesized DNA. RT extends the 3' end of the promoter primer, producing dsDNA containing a functional promoter to which RNA polymerase binds and transcribes additional amplicons complementary to the target strand. This autocatalytic cycle of generating more amplicon copies is repeated throughout the reaction, resulting in approximately one billion-fold amplification of the target nucleic acid present in the sample. Amplification products can be detected in real time during amplification or at the end of the amplification reaction by using probes that specifically bind to the target sequence contained in the amplification product. Detection of a signal resulting from the bound probe indicates the presence of the target nucleic acid in the sample.

[0309] In some embodiments, the method utilizes a "reverse" TMA reaction. In such variations, the initiating or "forward" amplification oligomer is a priming oligonucleotide that hybridizes to the target nucleic acid near the 3' end of the target region. Reverse transcriptase (RT) synthesizes a cDNA strand by extending the 3' end of the primer using the target nucleic acid as a template. The second or "reverse" amplification oligomer is a promoter primer or promoter provider having a target hybridizing sequence configured to hybridize to the target sequence contained within the synthesized cDNA strand. If the second amplification oligomer is a promoter primer, RT extends the 3' end of the promoter primer using the cDNA strand as a template to generate a second cDNA copy of the target sequence strand, thereby generating a dsDNA containing a functional promoter sequence. Amplification then continues essentially as described above, with initiation of transcription from the promoter sequence using RNA polymerase. Alternatively, when the second amplification oligomer is a promoter provider, a terminating oligonucleotide hybridizing to the target sequence near the 5' end of the target region is typically used to terminate the extension of the priming oligomer at the 3' end of the terminating oligonucleotide, thereby providing a defined 3' end for the initial cDNA strand synthesized by extension from the priming oligomer.The target hybridizing sequence of the promoter provider then hybridizes to the defined 3' end of the initial cDNA strand, and the 3' end of the cDNA strand is extended to add a sequence complementary to the promoter sequence of the promoter provider, resulting in the formation of a double-stranded promoter sequence.The initial cDNA strand is then used as a template to transcribe multiple RNA transcripts complementary to the initial cDNA strand without the promoter portion, using an RNA polymerase that recognizes the double-stranded promoter and then initiates transcription.Each of these RNA transcripts can then be used to serve as a template for further amplification from the first priming amplification oligomer.

[0310] Thus, in certain embodiments involving amplification-based detection assays, a combination of at least two amplification oligomers is utilized to detect the 16S rRNA of Lactobacillus species or the gene encoding the 16S rRNA of Lactobacillus species. The oligomer combination can include first and second amplification oligomers for amplifying the 16S rRNA region of Lactobacillus crispatus corresponding to the region of SEQ ID NO:1 from about nucleotide position 40 to about nucleotide position 265, the 16S rRNA region of Lactobacillus jensenii corresponding to the region of SEQ ID NO:2 from about nucleotide position 43 to about nucleotide position 247, and / or the 16S rRNA region of Lactobacillus gasseri corresponding to the region of SEQ ID NO:3 from about nucleotide position 93 to about nucleotide position 298. For example, in some embodiments, a first amplification oligomer comprises a target hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:10, and a second amplification oligomer comprises a target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9; in some such variations, a first amplification oligomer comprises a target hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:10, and a second amplification oligomer comprises a target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:7, and the oligomer combination further comprises a third and a fourth amplification oligomer, wherein the third amplification oligomer comprises a target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:8, and the fourth amplification oligomer comprises a target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:9.In more specific variations, the first amplification oligomer comprises a target hybridizing sequence comprising or consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:10, and the second amplification oligomer comprises a target hybridizing sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9; in some such embodiments, the first amplification oligomer comprises a target hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:10, the second amplification oligomer comprises a target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:7, and the oligomer combination further comprises a third and a fourth amplification oligomer, wherein the third amplification oligomer comprises a target hybridizing sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:8, and the fourth amplification oligomer comprises a target hybridizing sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:9. In some embodiments, such as those described above, at least one amplification oligomer is a promoter primer or promoter provider that further comprises a promoter sequence located 5' of its respective target hybridizing sequence (e.g., a T7 promoter sequence, such as the nucleotide sequence of residues 1-27 of SEQ ID NO:10); in some such embodiments, the first amplification oligomer is a promoter primer or promoter provider. In more specific variations, the first amplification oligomer consists of the nucleotide sequence of SEQ ID NO:10, and the second amplification oligomer consists of the nucleotide sequence of SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9; in some such embodiments, the first amplification oligomer consists of the nucleotide sequence of SEQ ID NO:10, the second amplification oligomer consists of the nucleotide sequence of SEQ ID NO:7, and the oligomer combination further comprises a third and a fourth amplification oligomer, wherein the third amplification oligomer consists of the nucleotide sequence of SEQ ID NO:8, and the fourth amplification oligomer consists of the nucleotide sequence of SEQ ID NO:9.

[0311] In some embodiments involving amplification-based detection assays, a combination of at least two amplification oligomers is utilized to detect A. vaginae 16S rRNA or the gene encoding A. vaginae 16S rRNA. The oligomer combination can include first and second amplification oligomers for amplifying a nucleic acid target region of A. vaginae corresponding to the region of SEQ ID NO:4 from about nucleotide position 540 to about nucleotide position 625. For example, in some embodiments, the first amplification oligomer includes a target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:17, and / or the second amplification oligomer includes a target hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO:18. In more specific variations, the first amplification oligomer comprises a target hybridizing sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:17, and / or the second amplification oligomer comprises a target hybridizing sequence comprising or consisting of the nucleotide sequence of residues 28-45 of SEQ ID NO:18. In some embodiments as described above, at least one amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located 5' of the respective target hybridizing sequence (e.g., a T7 promoter sequence, such as the nucleotide sequence of residues 1-27 of SEQ ID NO:18), and in some such embodiments, the first amplification oligomer is a promoter primer or promoter provider. In more specific variations, the first amplification oligomer consists of the nucleotide sequence of SEQ ID NO:17, and / or the second amplification oligomer consists of the nucleotide sequence of SEQ ID NO:18.

[0312] In some embodiments involving amplification-based detection assays, a combination of at least two amplification oligomers is utilized to detect Gardnerella vaginalis (G. vaginalis) 16S rRNA or the gene encoding Gardnerella vaginalis (G. vaginalis) 16S rRNA. The oligomer combination can include first and second amplification oligomers for amplifying a nucleic acid target region of Gardnerella vaginalis (G. vaginalis) corresponding to the region of SEQ ID NO:5 from about nucleotide position 172 to about nucleotide position 227. For example, in some embodiments, the first amplification oligomer includes a target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:14, and / or the second amplification oligomer includes a target hybridizing sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO:15. In more specific variations, the first amplification oligomer comprises a target hybridizing sequence comprising or consisting of the nucleotide sequence of SEQ ID NO:14, and / or the second amplification oligomer comprises a target hybridizing sequence comprising or consisting of the nucleotide sequence of residues 36-52 of SEQ ID NO:15. In some embodiments as described above, at least one amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located 5' of the respective target hybridizing sequence (e.g., a T7 promoter sequence, such as the nucleotide sequence of residues 9-35 of SEQ ID NO:15), and in some such embodiments, the first amplification oligomer is a promoter primer or promoter provider. In more specific variations, the first amplification oligomer consists of the nucleotide sequence of SEQ ID NO:14, and / or the second amplification oligomer comprises the nucleotide sequence of residues 9-52 of SEQ ID NO:15 (e.g., the second amplification oligomer consists of the nucleotide sequence of SEQ ID NO:15).

[0313] Detection of amplification products can be achieved by various methods for detecting signals specifically associated with the amplified target sequence. Nucleic acids may be associated with a surface that produces a detectable physical change, such as an electrical change. Amplified nucleic acids can be detected by concentrating them in or on a matrix and detecting the nucleic acid or a dye associated with the nucleic acid (e.g., an intercalating agent such as ethidium bromide or SYBR Green), or by detecting an increase in a dye associated with the nucleic acid in the solution phase. Other detection methods may use nucleic acid detection probes that specifically hybridize to sequences in the amplification product and are configured to detect the presence of a probe:product complex, or may use a complex of probes that can amplify a detectable signal associated with the amplification product (e.g., U.S. Pat. Nos. 5,424,413, 5,451,503, and 5,849,481). Directly or indirectly labeled probes that specifically associate with the amplification product provide a detectable signal indicating the presence of the target nucleic acid in the sample. For example, if the target nucleic acid is 16S rRNA of Lactobacillus spp., A. vaginae, and / or Gardnerella vaginalis (G. vaginalis), the amplification product will contain a target sequence within the 16S rRNA or a sequence complementary to a sequence within the 16S rRNA, and the probe will bind directly or indirectly to a sequence contained in the amplification product to indicate the presence of 16S rRNA of Lactobacillus spp., A. vaginae, and / or Gardnerella vaginalis (G. vaginalis) in the test sample.

[0314] The detection probe that hybridizes to the complementary amplified sequence may be a DNA or RNA oligomer, or an oligomer containing a combination of DNA and RNA nucleotides, or an oligomer synthesized with a modified backbone, such as an oligomer containing one or more 2'-methoxy-substituted ribonucleotides. The probe used to detect the amplified sequence may be unlabeled and indirectly detected (e.g., by binding of another binding partner to a moiety on the probe), or may be labeled with various detectable labels. In some embodiments of methods for diagnosing BV, such as certain embodiments using transcription-mediated amplification (TMA), the detection probe is a linear chemiluminescent-labeled probe, such as a linear acridinium ester (AE)-labeled probe.

[0315] The detection step can also provide further information about the amplified sequence, such as, for example, all or part of its nucleic acid base sequence. Detection can be performed after the amplification reaction is completed, or can be performed simultaneously with the amplification of the target region, for example, in real time. In one embodiment, the detection step allows for homogeneous detection, for example, detection of hybridized probes without removing unhybridized probes from the mixture. (See, for example, U.S. Patent Nos. 5,639,604 and 5,283,174).

[0316] In embodiments in which amplification products are detected near or at the end of the amplification step, a linear detection probe can be used to provide a signal indicating hybridization of the probe to the amplification product. One example of such detection uses a luminescently labeled probe that hybridizes to the target nucleic acid. The luminescent label is then hydrolyzed from the unhybridized probe. Detection is performed by chemiluminescence using a luminometer. (See, e.g., International Patent Application Publication No. WO 89 / 002476, incorporated herein by reference.) In other embodiments using real-time detection, the detection probe can be, for example, a hairpin probe, such as a molecular beacon, a molecular torch, or a hybridization switch probe, labeled with a reporter moiety that is detected when bound to the amplified product. Such a probe can include a target-hybridizing sequence and a non-target-hybridizing sequence. Various forms of such probes have been previously described (see, e.g., U.S. Patent Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, U.S. Patent Application Publication Nos. 20060068417A1 and 20060194240A1, each of which is incorporated herein by reference).

[0317] In certain embodiments involving amplification-based detection assays targeting 16S rRNA of Lactobacillus spp., A. vaginae, and / or Gardnerella vaginalis (G. vaginalis), or genes encoding 16S rRNA of Lactobacillus spp., A. vaginae, and / or Gardnerella vaginalis (G. vaginalis), the methods utilize one or more detection probes that specifically hybridize to the 16S rRNA amplification product of Lactobacillus spp., A. vaginae, and / or Gardnerella vaginalis (G. vaginalis). In certain variations, (A) a Lactobacillus-specific detection probe specifically hybridizes to (1) a nucleic acid target region corresponding to the region of SEQ ID NO: 1 from about nucleotide position 40 to about nucleotide position 265, (2) a nucleic acid target region corresponding to the region of SEQ ID NO: 2 from about nucleotide position 43 to about nucleotide position 247, and / or (3) a nucleic acid target region corresponding to the region of SEQ ID NO: 3 from about nucleotide position 93 to about nucleotide position 298; (B) an A. vaginae-specific detection probe specifically hybridizes to a nucleic acid target region corresponding to the region of SEQ ID NO: 4 from about nucleotide position 540 to about nucleotide position 625; and / or (C) a Gardnerella vaginalis-specific detection probe specifically hybridizes to a nucleic acid target region corresponding to the region of SEQ ID NO: 5 from about nucleotide position 172 to about nucleotide position 227.For example, in some variations, a first probe for detecting amplification products of Lactobacillus species includes a target hybridizing sequence that specifically hybridizes to a target region of each of the target nucleic acids of L. crispatus and L. jensenii, and a second probe for detecting amplification products of Lactobacillus species includes a target hybridizing sequence that specifically hybridizes to a target region of the target nucleic acid of L. gasseri. In some such embodiments, a first Lactobacillus-specific detection probe comprises a target hybridizing sequence substantially corresponding to the sequence of residues 1-17 of SEQ ID NO:11, and / or a second Lactobacillus-specific detection probe comprises a target hybridizing sequence substantially corresponding to the sequence of residues 7-23 of SEQ ID NO:12 (e.g., a first probe comprising the target hybridizing sequence of residues 1-17 of SEQ ID NO:11 and / or a second probe comprising the target hybridizing sequence of residues 7-23 of SEQ ID NO:12). In some variations, a probe for detecting an A. vaginae amplification product comprises a target hybridizing sequence substantially corresponding to the sequence of residues 6-21 of SEQ ID NO:19 (e.g., a probe comprising the target hybridizing sequence of residues 6-21 of SEQ ID NO:19). In some variations, a probe for detecting Gardnerella vaginalis (G. vaginalis) amplification products comprises a target hybridizing sequence substantially corresponding to the sequence of residues 1-18 of SEQ ID NO: 16 (e.g., a probe comprising the target hybridizing sequence of residues 1-18 of SEQ ID NO: 16).In certain embodiments, a first probe for detecting an amplification product of Lactobacillus species comprises or consists of the sequence of SEQ ID NO: 11, a second probe for detecting an amplification product of Lactobacillus species comprises or consists of the sequence of SEQ ID NO: 12, a probe for detecting an amplification product of A. vaginae comprises or consists of the sequence of SEQ ID NO: 19, and / or a probe for detecting an amplification product of Gardnerella vaginalis comprises or consists of the sequence of SEQ ID NO: 16.

[0318] In some embodiments of methods involving the use of nucleic acid-based detection assays, a non-amplification-based assay is used to detect Lactobacillus species, A. vaginae, and / or Gardnerella vaginalis. In some such embodiments, the non-amplification-based assay is a hybridization assay involving hybridization of a specific detection probe to a target nucleic acid. Methods for performing polynucleotide hybridization assays have been well developed in the art. Hybridization assay procedures and conditions vary depending on the application and are selected according to known general binding methods, including those described in, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor, NY, 2002) and Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, San Diego, CA, 1987). Typically, probes and samples are mixed under conditions that allow specific nucleic acid hybridization, and then specific hybridization of the probes to their respective targets is detected. Nucleic acid hybridization can be adapted to a variety of assay formats. One suitable format is the sandwich assay format, which is particularly adaptable to hybridization under non-denaturing conditions. The key component of a sandwich assay is a solid support to which an unlabeled, immobilized nucleic acid probe complementary to a portion of the DNA sequence is adsorbed or covalently attached. The target nucleic acid hybridizes to the immobilized probe, and a second, labeled detection probe complementary to a second, different region of the same DNA strand to which the unlabeled, immobilized nucleic acid probe hybridizes hybridizes to the [target nucleic acid]:[immobilized probe] duplex, detecting the target nucleic acid.Another exemplary format utilizes electrochemical detection of target nucleic acids hybridized to unlabeled detection probes immobilized on a suitable electrode surface as a signal transducer (see, e.g., Drummond et al., Nat. Biotechnol. 21:1192, 2003; Gooding, Electroanalysis 14:1149, 2002; Wang, Anal. Chim. Acta 469:63, 2002; Cagnin et al., Sensors 9:3122, 2009; Katz and Willner, Electroanalysis 15:913, 2003; Daniels and Pourmand, Electroanalysis 19:1239, 2007).

[0319] In certain embodiments involving hybridization assays, detection probes are utilized to detect 16S rRNA of Lactobacillus spp., A. vaginae, and / or Gardnerella vaginalis (G. vaginalis), or genes encoding 16S rRNA of Lactobacillus spp., A. vaginae, and / or Gardnerella vaginalis (G. vaginalis). In some such embodiments, (A) a probe for detecting 16S rRNA of Lactobacillus spp., or a gene encoding 16S rRNA of Lactobacillus spp., specifically hybridizes to (1) a nucleic acid target region corresponding to the region of SEQ ID NO: 1 from about nucleotide position 40 to about nucleotide position 265, (2) a nucleic acid target region corresponding to the region of SEQ ID NO: 2 from about nucleotide position 43 to about nucleotide position 247, and / or (3) a nucleic acid target region corresponding to the region of SEQ ID NO: 3 from about nucleotide position 93 to about nucleotide position 298, and (B) a probe for detecting 16S rRNA of A. vaginae, or a gene encoding 16S rRNA of A. vaginae. (C) a probe for detecting the 16S rRNA of Gardnerella vaginalis (G. vaginalis) or a gene encoding the 16S rRNA of Gardnerella vaginalis (G. vaginalis) specifically hybridizes to a nucleic acid target region corresponding to a region of SEQ ID NO: 5 from about nucleotide position 540 to about nucleotide position 625; and / or (D) a probe for detecting the 16S rRNA of Gardnerella vaginalis (G. vaginalis) specifically hybridizes to a nucleic acid target region corresponding to a region of SEQ ID NO: 5 from about nucleotide position 172 to about nucleotide position 227.For example, in some variations, a first probe for detecting 16S rRNA of Lactobacillus species or a gene encoding 16S rRNA of Lactobacillus species comprises a target hybridizing sequence that specifically hybridizes to a target region of each of Lactobacillus crispatus and L. jensenii, and a second probe for detecting 16S rRNA of Lactobacillus species or a gene encoding 16S rRNA of Lactobacillus species comprises a target hybridizing sequence that specifically hybridizes to a target region of each of Lactobacillus crispatus and L. jensenii, and a second probe for detecting 16S rRNA of Lactobacillus species or a gene encoding 16S rRNA of Lactobacillus species comprises a target hybridizing sequence that specifically hybridizes to a target region of each of Lactobacillus crispatus and L. jensenii, and a second probe for detecting 16S rRNA of Lactobacillus species or a gene encoding 16S rRNA of Lactobacillus species comprises a target hybridizing sequence that specifically hybridizes to a target region of Lactobacillus crispatus and L. jensenii, and a second probe for detecting ... The second probe for detecting the rRNA-encoding gene comprises a target hybridizing sequence that specifically hybridizes to a target region of a L. gasseri target nucleic acid; in some such embodiments, the first Lactobacillus-specific detection probe comprises a target hybridizing sequence substantially corresponding to the sequence of residues 1-17 of SEQ ID NO:11, and / or the second Lactobacillus-specific detection probe comprises a target hybridizing sequence substantially corresponding to the sequence of residues 7-23 of SEQ ID NO:12 (e.g., a first probe comprising the target hybridizing sequence of residues 1-17 of SEQ ID NO:11, and / or a second probe comprising the target hybridizing sequence of residues 7-23 of SEQ ID NO:12). In some variations, a probe for detecting A. vaginae 16S rRNA, or the gene encoding A. vaginae 16S rRNA, comprises a target hybridizing sequence substantially corresponding to the sequence of residues 6-21 of SEQ ID NO: 19 (e.g., a probe comprising the target hybridizing sequence of residues 6-21 of SEQ ID NO: 19). In some variations, a probe for detecting G. vaginalis 16S rRNA, or the gene encoding G. vaginalis 16S rRNA, comprises a target hybridizing sequence substantially corresponding to the sequence of residues 1-18 of SEQ ID NO: 16 (e.g., a probe comprising the target hybridizing sequence of residues 1-18 of SEQ ID NO: 16).In certain embodiments, the first probe for detecting 16S rRNA of Lactobacillus spp. or the gene encoding 16S rRNA of Lactobacillus spp. comprises or consists of the sequence of SEQ ID NO: 11, the second probe for detecting 16S rRNA of Lactobacillus spp. or the gene encoding 16S rRNA of Lactobacillus spp. comprises or consists of the sequence of SEQ ID NO: 12, the probe for detecting 16S rRNA of A. vaginae or the gene encoding 16S rRNA of A. vaginae comprises or consists of the sequence of SEQ ID NO: 19, and / or the 16S rRNA of G. vaginalis. A probe for detecting rRNA or the gene encoding 16S rRNA of Gardnerella vaginalis (G. vaginalis) comprises or consists of the sequence of SEQ ID NO:16.

[0320] In some embodiments, the non-amplification-based assay for detecting Lactobacillus spp., A. vaginae, and / or Gardnerella vaginalis (G. vaginalis) is a cleavage-based assay, in which a probe oligonucleotide comprising a non-target hybridizing flap region is cleaved by a flap endonuclease in an overlap-dependent manner to release cleavage products that are then detected. Exemplary cleavage-based assay reagents are described, for example, by Lyamichev et al. (Nat. Biotechnol. 17:292-296, 1999), Ryan et al. (Mol. Diagn. 4:135-144, 1999), and Allawi et al. (J. Clin. Microbiol. 44:3443-3447, 2006). Appropriate conditions for flap endonuclease reactions are known or can be readily determined using methods well known in the art (see, e.g., Kaiser et al., J. Biol. Chem. 274:2138-721394, 1999). Exemplary flap endonucleases that can be used in the method include Thermus aquaticus DNA polymerase I, Thermus thermophilus DNA polymerase I, mammalian FEN-1, Archaeoglobus fulgidus FEN-1, Methanococcus jannaschii FEN-1, Pyrococcus furiosus FEN-1, Methanobacterium thermoautotrophicum FEN-1, Thermus thermophilus FEN-1, and the like. thermophilus FEN-1, CLEAVASE® (Hologic, Madison, Wisconsin), S. cerevisiae RTH1, S. cerevisiae RAD27, Schizosaccharomyces pombe rad2, bacteriophage T5 5'-3' exonuclease, Pyrococcus horikoshii FEN-1, human endonuclease 1, calf thymus 5'-3' exonuclease, and their homologs in eubacteria, eukaryotes, and archaea, including, for example, members of the class II family of structure-specific enzymes, and enzymatically active mutants or variants thereof. Descriptions of flap endonucleases are found, for example, in Lyamichev et al., Science 260:778-783, 1993; Eis et al., Nat. Biotechnol. 19:673-676, 2001; Shen et al., Trends in Bio. Sci. 23:171-173, 1998; Kaiser et al., J. Biol. Chem. 274:21387-21394, 1999; Ma et al., J. Biol. Chem. 275:24693-24700, 2000; Allawi et al., J. Molecular Biology ( J. Mol. Biol. 328:537-554, 2003, Sharma et al., J. Biol. Chem. 278:23487-23496, 2003, and Feng et al., Nat. Struct. Mol. Biol. 11:450-456, 2004.

[0321] In certain variations, the cleavage-based assay detects RNA target nucleic acids of Lactobacillus spp., A. vaginae, and / or G. vaginalis, and the cleavage-based assay utilizes a flap endonuclease capable of cleaving an RNA:DNA linear duplex structure. In some alternative embodiments, the cleavage-based assay detects DNA target nucleic acids of Lactobacillus spp., A. vaginae, and / or G. vaginalis, and the cleavage-based assay utilizes a flap endonuclease capable of cleaving a DNA:DNA linear duplex structure. Exemplary flap endonucleases capable of cleaving RNA:DNA duplexes include polymerase-deficient 5' nucleases from the genus Thermus, as well as certain CLEAVASE® enzymes (Hologic, Madison, Wis.), such as, for example, CLEAVASE® BN (a BstX-NotI deletion of Taq polymerase; see U.S. Pat. No. 5,614,402), CLEAVASE® II (an "AG" mutant of full-length Taq polymerase; see U.S. Pat. No. 5,614,402), CLEAVASE® VII (a synthetic deficiency mutation of full-length Thermus thermophilus polymerase), CLEAVASE® IX (a polymerase-deficient mutant of Tth DNA polymerase), and CLEAVASE® XII (a polymerase-deficient chimeric polymerase constructed from fragments of taq DNA polymerase and Tth DNA polymerase).Exemplary flap endonucleases capable of cleaving DNA:DNA double strands include the flap endonucleases described above, as well as CLEAVASE® 2.0 (Archaeoglobus fulgidus FEN-1), CLEAVASE® 2.1 (Archaeoglobus fulgidus FEN-1 with six C-terminal histidines), CLEAVASE® 3.0 (Archaeoglobus veneficus FEN-1), and CLEAVASE® 3.1 (Archaeoglobus veneficus FEN-1 with six C-terminal histidines).

[0322] In some embodiments, the cleavage-based assay detects RNA target nucleic acids of Lactobacillus species, A. vaginae, and / or G. vaginalis, and the assay includes synthesizing a DNA complement of the RNA target region, where this cDNA strand hybridizes to overlapping first and second probes to form a linear double-stranded break structure for cleavage by a flap endonuclease. Reaction conditions for synthesizing cDNA from an RNA template using an RNA-dependent DNA polymerase (reverse transcriptase) are well known in the art.

[0323] In certain embodiments utilizing nucleic acid-based detection assays, the methods further include purifying the target nucleic acids of Lactobacillus spp., A. vaginae, and G. vaginalis from other components in the sample. Such purification may include methods for separating and / or concentrating the organisms contained in the sample from other sample components. In certain embodiments, purifying the target nucleic acids includes capturing the target nucleic acids and specifically or nonspecifically separating the target nucleic acids from other sample components. Nonspecific target capture methods may involve selective precipitation of the nucleic acids from a substantially aqueous mixture, attachment of the nucleic acids to a support that is washed to remove other sample components, or other means of physically separating the nucleic acids from a mixture containing Lactobacillus spp., A. vaginae, and G. vaginalis nucleic acids and other sample components.

[0324] In some embodiments, target nucleic acids (e.g., 16S rRNA target nucleic acids or genes encoding 16S rRNA) of Lactobacillus species, A. vaginae, and G. vaginalis are separated from other sample components by hybridizing the target nucleic acid to a capture probe oligomer. The capture probe oligomer contains a target-hybridizing sequence configured to specifically or nonspecifically hybridize to the target nucleic acid to form a [target nucleic acid]:[capture probe] complex separated from other sample components. Capture probes containing target-hybridizing sequences suitable for nonspecific capture of target nucleic acids are described, for example, in International PCT Publication No. WO 2008 / 016988, which is incorporated herein by reference. In some variations, the step of separating the target nucleic acid from other sample components includes contacting the sample with (i) a first capture probe oligomer comprising a target hybridizing sequence that specifically hybridizes to a target sequence within the target nucleic acid of a Lactobacillus species (e.g., a target hybridizing sequence that specifically hybridizes to a target sequence within each of the target nucleic acids of Lactobacillus crispatus (L. crispatus), L. jensenii (L. jensenii), and L. gasseri), and (ii) a second capture probe oligomer comprising a target hybridizing sequence that specifically hybridizes to a target sequence within each of the target nucleic acids of A. vaginae and Gardnerella vaginalis (G. vaginalis).In some particular variations involving the first and second capture probe oligomers of (i) and (ii) above, the first capture probe oligomer comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of residues 1-19 of SEQ ID NO:6 (e.g., a capture probe comprising the target-hybridizing sequence of residues 1-19 of SEQ ID NO:6), and / or the second capture probe oligomer comprises a target-hybridizing sequence substantially corresponding to the nucleotide sequence of residues 1-20 of SEQ ID NO:13 (e.g., a capture probe comprising the target-hybridizing sequence of residues 1-20 of SEQ ID NO:13). In preferred variations, the capture probe binds the [target nucleic acid]:[capture probe] complex to the immobilized probe to form a [target nucleic acid]:[capture probe]:[immobilized probe] complex, which is separated from the sample and optionally washed to remove non-target sample components (see, e.g., U.S. Pat. Nos. 6,110,678, 6,280,952, and 6,534,273). In such variations, the capture probe oligomer further comprises a sequence or moiety that allows the capture probe, along with its bound target sequence, to bind to an immobilized probe attached to a solid support, thereby allowing the hybridized target nucleic acid to be separated from other sample components.

[0325] In more specific embodiments, the capture probe oligomer includes a tail portion (e.g., a 3' tail) that is not complementary to the target nucleic acid but functions as a moiety that specifically hybridizes to a sequence on the immobilized probe, thereby allowing the target nucleic acid to be separated from other sample components, as previously described, for example, in U.S. Patent No. 6,110,678, which is incorporated herein by reference. Any sequence can be used in the tail region, which is generally about 5 nt to 50 nt in length, and in preferred embodiments, about 10 nt to about 40 nt (e.g., A 10 ~A 40 ), more preferably about 14 nt to about 33 nt (e.g., A 14 ~A 30 or T3A 14 ~T3A 30), which binds to a complementary immobilized sequence (e.g., poly-T) attached to a solid support, e.g., a matrix or particle. In some such embodiments, including (i) a first capture probe oligomer comprising a target hybridizing sequence that specifically hybridizes to a target sequence within each of the 16S rRNA target nucleic acids of L. crispatus, L. jensenii, and L. gasseri, and / or (ii) a second capture probe oligomer comprising a target hybridizing sequence that specifically hybridizes to a target sequence within each of the 16S rRNA target nucleic acids of A. vaginae and G. vaginalis, the first capture probe oligomer comprises or consists of the nucleotide sequence of SEQ ID NO:6, and / or the second capture probe oligomer comprises or consists of the nucleotide sequence of SEQ ID NO:13.

[0326] Target capture typically occurs under hybridization conditions, usually by forming a double-stranded T tail sequence:immobilized probe sequence. mThis occurs in a liquid-phase mixture containing one or more capture probe oligomers hybridized to the target nucleic acid at a temperature greater than 100°C. In embodiments including a capture probe tail, the [target nucleic acid]:[capture probe] complex is captured by adjusting the hybridization conditions so that the capture probe tail hybridizes to the immobilized probe, and the entire complex on the solid support is then separated from other sample components. The support with the bound [immobilized probe]:[capture probe]:[target nucleic acid] may be washed one or more times to further remove sample components. A preferred embodiment uses a particulate solid support, such as paramagnetic beads, whereby the particles with the bound [target nucleic acid]:[capture probe]:[immobilized probe] complex can be suspended in a wash solution and recovered from the wash solution, preferably by using magnetic attraction. In method embodiments including the use of an amplification-based detection assay, to limit the number of handling steps, the target nucleic acid can be amplified by simply mixing the target nucleic acid in the complex on the support with amplification oligomers and proceeding with the amplification step.

[0327] In some embodiments of the method for diagnosing BV, in which detection of Lactobacillus species, A. vaginae, and / or Gardnerella vaginalis indicates BV in the subject, the method further comprises treating BV in the subject. Treatment regimens for BV are generally known in the art and include, for example, administration of antibiotics such as metronidazole (e.g., Flagyl, METROGEL-VAGINAL), clindamycin (e.g., Cleocin, CLINDESSE), and tinidazole (e.g., TINDAMAX). In certain variations, the subject has not previously been diagnosed with BV. In other embodiments, the subject has previously been diagnosed with BV and is undergoing treatment for BV at the time the diagnostic method of the present disclosure is performed. Such variations are particularly useful for monitoring the subject's treatment for BV. For example, if the method indicates that babesiosis is still present in the subject, the subject can continue treatment. In some embodiments, the same treatment regimen (i.e., the same treatment the subject is receiving at the time the diagnostic method is performed) is readministered to the subject. Alternatively, the continued presence of BV in a subject receiving treatment may indicate a need for a change in ongoing treatment, and a different treatment regimen (e.g., a different drug therapy, or an increased dose and / or frequency of the drug) is administered to the subject.

[0328] According to the present invention, detection of the presence or absence of Lactobacillus species, A. vaginae, and / or Gardnerella vaginalis can be performed separately for each target (e.g., in separate reaction vessels, sequentially or in parallel) or together as a multiplex reaction system. Thus, in some embodiments, the methods described herein (e.g., methods for diagnosing BV) utilize multiplex reactions, in which the reaction mixture includes reagents for assaying multiple (e.g., at least two, three, four, or more) different target sequences in parallel. In these cases, the reaction mixture may include multiple different target-specific oligonucleotides for conducting the detection assay. For example, in methods utilizing amplification-based detection assays, the multiplex reaction may include multiple sets (e.g., multiple pairs) of amplification oligomers (e.g., multiple pairs of PCR primers or multiple pairs of TMA amplification oligomers (e.g., multiple pairs of promoter and non-promoter primers for TMAs, or multiple pairs of promoter and non-promoter primers)). In other embodiments utilizing cleavage-based detection assays, the multiplex reaction may include multiple probe oligonucleotides with different flaps, multiple different overlapping probe oligonucleotides, and multiple different FRET cassettes for detecting the different flaps once the different flaps are cleaved.

[0329] Additional microbial detection assays can similarly be performed to determine the presence and / or relative abundance of multiple microorganisms associated with BV, including, by way of example only, one or more anaerobic gram-positive cocci, Eggerthella spp., bacteria from the order Clostridiales, Clostridium-like species, Enterobacteriaceae, Peptostreptococcus micros, Aerococcus christensenii, Leptotrichia amnionii, Peptoniphilus spp., Dialister spp., Mycoplasma hominis, Sneathia sanguinegens, Anaerococcus tetrazius, and the like. tetradius, Mobiluncus spp., Mobiluncus hominis, Eggerthella hongkongensis, Prevotella spp., Megasphaera spp., Leptotrichia sanguinegens, and Finegoldia magna. Assays can be performed individually or multiplexed. Thus, diagnosing BV can involve identifying multiple microorganisms and, optionally, determining their relative abundance in a sample.

[0330] In certain embodiments, the method for diagnosing BV involves the detection of 10 or fewer bacterial genera associated with BV. In other embodiments, the method involves the detection of 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, or 4 or fewer bacterial genera associated with BV. In some variations, the method does not involve the detection of bacterial genera associated with BV other than Lactobacillus, Atopobium, and Gardnerella.

[0331] Also provided by the present invention are oligomers or combinations thereof for determining the presence or absence of one or more of Lactobacillus spp., A. vaginae, and G. vaginalis in a sample. In various embodiments, the oligomers or combinations thereof include oligomers as described herein for methods for determining the presence or absence of Lactobacillus spp., A. vaginae, and / or G. vaginalis in a sample. In some variations, the oligomer combination includes at least one Lactobacillus-specific oligonucleotide (e.g., at least two or three Lactobacillus-specific oligonucleotides, each binding to a different target sequence), at least one A. vaginae-specific oligonucleotide (e.g., at least two or three A. vaginae-specific oligonucleotides, each binding to a different target sequence), and / or at least one Gardnerella vaginalis-specific oligonucleotide (e.g., at least two or three G. vaginalis-specific oligonucleotides, each binding to a different target sequence).In some variations, the oligomer combination includes at least two Lactobacillus-specific oligonucleotides (e.g., at least three Lactobacillus-specific oligonucleotides, each binding to a different target sequence), at least two A. vaginae-specific oligonucleotides (e.g., at least three A. vaginae-specific oligonucleotides, each binding to a different target sequence), and / or at least two Gardnerella vaginalis-specific oligonucleotides (e.g., at least three G. vaginalis-specific oligonucleotides, each binding to a different target sequence). In some embodiments, the oligomer combination comprises at least two Lactobacillus-specific amplification oligonucleotides for amplifying nucleic acid target regions of Lactobacillus species, at least two A. vaginae-specific amplification oligonucleotides for amplifying nucleic acid target regions of A. vaginae, and / or at least two Gardnerella vaginalis-specific amplification oligonucleotides for amplifying nucleic acid target regions of G. vaginalis. In some embodiments, the compositions of the present invention comprise one or more detection probe oligomers for detecting Lactobacillus species target nucleic acids, A. vaginae target nucleic acids, and / or G. vaginalis target nucleic acids. The oligomers or combinations thereof may be in the form of a reaction mixture or kit containing the oligomers. The reaction mixture or kit may further include a number of optional components, such as, for example, a capture probe nucleic acid or an array of capture probe nucleic acids.For amplification reaction mixtures, the reaction mixture typically includes other reagents suitable for performing in vitro amplification, such as, for example, buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase), and typically includes test sample components in which target nucleic acids of Lactobacillus species, A. vaginae, and / or Gardnerella vaginalis may or may not be present. Kits containing oligomer combinations for amplifying one or more target nucleic acid regions of Lactobacillus species, A. vaginae, and / or G. vaginalis may also include other reagents suitable for performing in vitro amplification, such as buffers, salt solutions, appropriate triphosphate nucleotides (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP), and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase). For oligomer combinations (e.g., reaction mixtures or kits) that include a combination of amplification oligomers targeting a common target nucleic acid along with a detection probe, the selection of amplification oligomers and detection probe oligomers are related by the common target region (i.e., the combination includes a probe that binds to a sequence amplifiable by the amplification oligomer combination).

[0332] The present invention is further illustrated by the following non-limiting examples. Example 1: BV multivariate algorithm 1. Summary of result interpretation

[0333] To determine BV-positive or BV-negative status, quantitative information on Lactobacillus species ("L. spp," negatively correlated with BV) and Gardnerella vaginalis and Atopobium vaginae ("Gvag" and "Avag," respectively, positively correlated with BV) is combined into a single numerical BV score using a multivariate linear prediction algorithm. This score is compared to a single cutoff value, with negative calls below the cutoff and positive calls above the cutoff.

[0334] In a simplified version of this algorithm, the quantitative value of L. spp (in units of log copies) is subtracted from the Gvag or Avag value (whichever is greater). Simplified score = max(Gvag,Avag)·L.spp

[0335] As practically applied to the interpretation of APTIMA BV results, the formula becomes more complex: a negative constant is added (to zero out scores at clinical decision points), an additional coefficient (IC term) is added (to compensate for observed inhibition), a minimum value is imposed on L. spp (to improve reproducibility), the concentrations of the three analytes are standardized (to balance the contribution of Gvag and Avag in the algorithm), and each term is weighted.

[0336] The formula applied is: Score = C0+W L *Max(LM L ,F L -M L )+W AG *Max(GM G ,AM A ,F GA -M G )+WIC*log2(IC / calIC)

[0337] 2. Learn more about interpreting results Sample interpretation consists of assessing validity criteria, calculating a BV score, and interpreting the score. This example begins with the TTime value of the internal standard (IC) signal and the log copies / mL quantitative values ​​of L. spp., Gvag, and Avag (L, G, A) for each sample being evaluated.

[0338] 2.1 Calculating the BV score For specimens that meet the validity criteria (not described here), a BV score is calculated. Calculation of the BV score consists of three steps: 1) analyte normalization, 2) calculation of the IC ratio, and 3) application of the general equation. 2.1.1 Analyte Standardization

[0339] In a population of clinical samples, observed Gvag concentrations tend to be higher than Avag concentrations. Because the linear prediction equation uses only the maximum value (Gvag LogC or Avag LogC), terms are smoothed to balance the likelihood of each analyte participating in the algorithm. A robust method of smoothing is to adjust the terms by the population statistic (median). If concentrations were in units of copies, they would be divided by the median, and relative concentrations would be given as normalized values. Because the quantitative data used here are in units of log copies, an equivalent operation would be to subtract the median LogC value.

[0340] Standardization serves two main functions. 1) smooth the cardinality numbers (as above), 2) Improve the confidence interval of the trained constant.

[0341] This form of normalization applies to all terms that exist in units of log copies, including the three sample analyte values ​​(L, G, A) and the basal value F. L and F GA Each of these is calculated by multiplying it by the population median (M L , M G , M A ) by subtracting . Normalized values ​​are indicated by the "S" subscript in the general equation. [Table 2]

[0342] 2.1.2 Calculation of IC ratio The IC ratio is used in the general equation as part of a term that compensates for analyte inhibition to reduce the chance of false negative calls. The IC ratio is calculated by multiplying the observed value of the internal standard (IC) TTime by the IC It is calculated as the average observed IC TTime of valid KitCalibrator replicates.

[0343] For samples that do not generate an IC TTime, the IC ratio is defined as the constant ICd. A default IC TTime can be calculated as the mean observed IC TTime of valid KitCalibrator replicates multiplied by the ICd. Any IC TTime that is not less than the default IC TTime will use the ICd as the IC ratio. IC ratio= Observed IC TTime Maximum IC ratio = ICd Expected IC TTime

[0344] 2.1.3 General equations The formula coded into the PANTHER® software for interpreting APTIMA® BV results is called the general equation, where the subscript "S" refers to values ​​that have been standardized by subtracting the population median value for the relevant analyte. BV score = C0 + W L Max(L S ,F LS )+W A Max(A S ,F AS )+W G Max(G S ,F GS )+W GA Max(A S ,G S ,FGAS )+W IC Log2 (IC ratio)

[0345] This general equation was implemented before completing the algorithm. A and W G We set both to zero, so the (applied) equation can be simplified to: BV score = C0 + W L Max(L S ,F LS )+W GA Max(A S ,G S ,F GAS )+W IC Log2 (IC ratio)

[0346] This formula adds four terms together to create a score. [Table 3]

[0347] 2.2 Interpretation of BV score To determine BV-positive or BV-negative status (reported call), the numeric BV score is compared to a single cutoff value (currently set to zero). If the specimen's BV score is less than the cutoff value, the specimen is reported as BV-negative. If the score is equal to or greater than the cutoff value, the specimen is reported as BV-positive.

[0348] 3. Application of multivariate algorithms to the interpretation of clinical specimen results To interpret the clinical outcomes of BV, the “true result” was determined by the Nugent score using the intermediate grade determined by the Amsel Criteria, as shown in Table 4 . [Table 4]

[0349] The constants used in the algorithm are listed in Table 5, along with the values ​​used in this analysis. [Table 5]

[0350] Two sets of data were collected from BV RT-TMA PANTHER test results for the APTIMA product under development. The training set consisted of 1,204 test results, one test per subject. This data set was used to derive the constant values ​​shown in Table 5. The validation set consisted of 1,076 test results, one test per subject. Although the subject populations were identical, there were no common test results between the training and validation sets. (The terms "training set" and "validation set" herein refer to sets of clinical specimens tested with the same assay design to optimize an algorithm for making clinical calls (positive or negative). The training set is a group of specimens used to design a preliminary algorithm, and the validation set is a separate set of specimens used to test the accuracy of the algorithm's performance.)

[0351] Using the constants in Table 5 in the general equations, we obtain the estimates of clinical performance (sensitivity / specificity) for the training and validation sets shown in Table 6. [Table 6]

[0352] BV may manifest differently among different races and ethnicities. Therefore, the training and validation sets are analyzed and displayed by race / ethnicity using the following categories: Black or African American regardless of ethnicity (Black), Non-Hispanic White (White-NH), Hispanic White (White-Hisp), Asian regardless of ethnicity (Asian), and Other. The Other category consists of Islander, Indian, Middle Eastern, Native American, and other, unknown, or mixed race people. [Table 7] [Table 8]

[0353] Example 2: Amplified interference It is desirable for amplification oligos to specifically detect the 16S rRNA of the target species while minimizing interference from other organisms that may be present at high titers in the desired specimen type. Alignment of the 16S rRNA sequences of G. vag with those of other vaginal organisms showed similarity between G. vag and Bifidobacteria sequences, suggesting that interference from Bifidobacteria or other related organisms may affect G. vag detection in some specimens. Additional G. vag amplification oligos were designed to reduce the potential for interference.

[0354] The initial design targeted amplification of SEQ ID NO:5 from about bases 964 to 1033 (region 1). Additional oligos were screened targeting a site with potentially higher specificity (SEQ ID NO:5, bases about 160 to 277 (region 2)).

[0355] To test for interference, primer sets were tested in a manual RT-TMA format on samples containing 1E4 CFU / mL G. vag, with or without higher concentrations of Bifidobacterium breve present in the samples. Each set was tested in five replicates, and the mean values ​​(RFU range, non-normalized Ttime, and non-normalized Tslope) for each condition are shown relative to the 1E4 CFU / mL G. vag condition in the absence of B. breve. [Table 9]

[0356] Test results for G. vag amplification interference. Both sets in Region 2 demonstrated relatively stable G. vag detection in the presence of high titers of B. breve. In contrast, Set 1 (Region 1) showed a decrease in RFU range, a decrease in Tslope, and an increase in Ttime in the presence of increasing concentrations of B. breve, indicating G. vag amplification interference.

[0357] Initial oligo combinations for detecting Lactobacillus species (L. gasseri, L. jensenii, and L. crispatus) showed reduced detection of low concentrations of L. gasseri in the presence of high concentrations of several other organisms. To reduce interference and improve specificity, separate forward primers were designed for each of the three target Lactobacillus species. The original and modified oligo sets were used in a manual RT-TMA format to detect L. gasseri at 1E4 CFU / mL alone or in the presence of 1E6 CFU / mL Neisseria gonorrhoeae, 1E6 CFU / mL Clostridium difficile, 1E6 CFU / mL Acinetobacter lwoffii, 5E9 copies / mL Mycoplasma hominis, or 1E6 CFU / mL Lactobacillus inerus. The two conditions used the same promoter primer and torch combinations (SEQ ID NO: 10, SEQ ID NO: 259, and SEQ ID NO: 11). The forward primers used for each condition are listed in the table below. To assess interference, the mean TTime for each sample was divided by the mean TTime for 1E4 CFU / mL of L. gasseri. A TTime ratio greater than 100% indicates a delayed TTime (amplification interference). [Table 10]

[0358] Test results for amplification interference of Lactobacillus species. The initial conditions (using a single forward primer for L. gasseri, L. jensenii, and L. crispatus) showed amplification interference by Clostridium difficile, Acinetobacter lwoffi, and Mycoplasma hominis, whereas the modified conditions (using separate forward primers for each target species) showed no amplification interference.

[0359] Example 3: Lactobacillus target capture oligo screening To improve on the initial Lactobacillus species target capture oligo design (SEQ ID NO: 257), additional designs were developed and screened in parallel with the existing design for samples containing 1E4 CFU / mL of L. crispatus, L. gasseri, or L. jensenii. The amplification and detection oligos (common to all conditions) were SEQ ID NO: 258, SEQ ID NO: 10, SEQ ID NO: 259, and SEQ ID NO: 11. Screening was performed in manual RT-TMA format with three replicates per condition, and the average normalized Ttime values ​​are shown. [Table 11]

[0360] Because the conditions varied only with the target capture oligo, slower amplification (high Ttime) could be due to reduced target capture efficiency. In condition 1, no target capture oligo was used, resulting in delayed or absent Ttime. The use of target capture oligos is expected to improve this by increasing capture efficiency. Each of the other conditions utilized a single TCO for capture of three Lactobacillus species. In conditions 4 through 8, the TCO was designed to specifically capture Lactobacillus 16S rRNA, prioritizing these sequences over sequences from other genera. In conditions 2 and 3, the TCO was designed to capture rRNA from not only Lactobacillus but also other bacteria.

[0361] Results Conditions 5, 6, and 7 have faster Ttime (more efficient capture) of all three target Lactobacillus species than both the previous Lactobacillus TCO design (condition 4) and the less specific TCOs (conditions 2 and 3). Of the three preferred Lactobacillus TCO designs, condition 7 (TCO SEQ ID NO: 6) demonstrated the highest efficiency (lowest Ttime) in this experiment.

[0362] Example 4: Cross-reactivity in early multiplexing A multiplexed RT-TMA reagent configuration for detecting G. vag, A. vag, and Lactobacillus species was constructed utilizing the oligos in Table 12. [Table 12]

[0363] The reagents were tested on the PANTHER instrument against pools containing one to five non-target organisms at high titers (most often 1E6 CFU / mL), with five replicates per sample. An unexpected signal in the FAM channel was observed in pool #7, which consisted of four organisms: Mobiluncus curtisii (5E9 copies rRNA / mL), Mycoplasma genitalium (1E6 CFU / mL), Mycoplasma hominis (5E9 copies rRNA / mL), and Neisseria gonorrhoeae (1E6 CFU / mL). Follow-up tests were performed on samples containing individual organisms from pool #7. The reagents were tested twice per control condition and four times per test condition on the PANTHER instrument. The average normalized T times are shown in Table 13 below. [Table 13]

[0364] An unexpected FAM signal was observed only for Mycoplasma genitalium (M. genitalium). To determine which oligomer was responsible for the unexpected signal, experiments were performed in which individual oligomers were omitted from the multiplex mixture for each condition. The multiplex utilized the oligomers in Table 12, omitting SEQ ID NO: 23. Each of the eight reagent sets (a control condition containing all oligomers and seven conditions in which a single oligomer was omitted) was tested in manual RT-TMA format against specificity pool #7, with triplicates per condition. Normalized FAM Ttimes are shown below in Table 14. [Table 14]

[0365] This experiment demonstrated that SEQ ID NO:259, SEQ ID NO:10, and SEQ ID NO:45 were required for unexpected FAM signal generation. To test the sufficiency of these oligos for detecting M. genitalium on FAM, an RT-TMA reagent was constructed in which these three oligomers (SEQ ID NO:259, SEQ ID NO:10, and SEQ ID NO:45) represented the only torch, promoter primer, and primer. M. genitalium was detected on FAM in all four replicates tested, while A. vag, G. vag, L. gas, and the negative control were not detected with this oligomer combination (see Table 15). Therefore, this set of oligomers is sufficient. [Table 15]

[0366] The risk of M. genitalium cross-reactivity for the multiplexes listed in Table 12 was not flagged in the initial bioinformatics assessment. SEQ ID NO: 10 and SEQ ID NO: 259 did not meet the initial criteria for expected hybridization to M. genitalium. Therefore, the observed cross-reactivity was unexpected.

[0367] Example 5 To increase sensitivity for A.vag, multiplex testing was performed in parallel with two A.vag systems: (1) a Lacto-A.vag-G.vag multiplex ("LAG-5") using a biphasic A.vag system with a long amplicon, and (2) a Lacto-A.vag-G.vag multiplex ("LAG-6") using a non-biphasic A.vag system with a short amplicon. These A.vag systems were initiated with the oligos shown in Table 16. [Table 16]

[0368] The A.vag biphasic system was too fast to provide a TTime from clinically relevant high concentrations of A.vag (≥1E11 cp / mL) equivalent to IVT. We rescreened previously designed A.vag T7 oligos. One T7 with a predicted secondary structure slowed the TTime.

[0369] Improvements to the A.vag non-biphasic system focused on improving the slope of the A.vag curve. Multiple A.vag torch repeats were designed within the same region and screened in singleplex. To improve sensitivity in multiplex situations, the A.vag torch with the nucleotide sequence of SEQ ID NO: 19 was selected.

[0370] Analytical specificity results were similar between the two A. vag systems (LAG5 and LAG6).

[0371] The improved A.vag oligo set is shown in Table 17 below. [Table 17]

[0372] The clinical performance of LAG5 and LAG6 multiplex oligo combinations (with different A. vag oligo designs as shown in Table 17 above) was compared in two small sets of vaginal clinical swab samples. Data were interpreted using two algorithms: a decision tree algorithm based on quantification from standard curves for L. crisp, G. vag, and A. vag, as described in Example 6, and an uncalibrated Ttime ratio algorithm. For the uncalibrated Ttime ratio algorithm, a specimen was called positive if both of the following criteria were met (otherwise negative): 1) the ratio of the internal standard Ttime to either G. vag Ttime or A. vag Ttime exceeded a cutoff value, and 2) the ratio of the L. spp Ttime to either G. vag Ttime or A. vag Ttime exceeded a different cutoff value. Absent Ttimes were assigned a fixed value. Both LAG5 and LAG6 had similar clinical performance, although LAG6 performed slightly better at pre-set log c / mL cutoff values ​​(Lacto 8.3, G.vag 10.5, and A.vag 7.4). The project proceeded using the LAG6 multiplex design, with all systems being non-biphasic and with shorter A.vag amplicons.

[0373] Example 6 Oligomers having the nucleotide sequences shown in Table 18 were evaluated in the multiplex APTIMA® Bacterial Vaginosis (ABV) Assay for amplification and detection of L. crispatus, L. jensenii, L. gasseri, Gardnerella vaginalis, and Atopobium veginae, as well as a generic internal standard (GIC). [Table 18]

[0374] The oligomers designed and screened for the bacterial vaginosis assay are shown in Table 34. Based on this screening, the oligomers shown in Table 16 were selected for further evaluation on the PANTHER® System for linearity, cross-reactivity, clinical performance, and strain comprehensiveness.

[0375] The following bulk liquid reagents were constructed: 1) TCR: HEPES free acid dihydrate (250 mM), lithium hydroxide monohydrate (310 mM), lithium chloride (1.883 M), EDTA free acid (100 mM), poly dT14 Mag particles (0.03% w / v), lithium hydroxide and hydrochloric acid for pH, and water for volume; 2) Enzymes: HEPES free acid (57.46 mM), EDTA free acid (0.98 mM), Triton X-100 (0.10 v / v), potassium chloride (49.61 mM), anhydrous glycerol (0.2 v / v), disodium EDTA dihydrate (0.39 mM), N-acetyl-L-cysteine ​​(49.58 mM), anhydrous D(+) trehalose (0.03 w / v), cloned MMLV enzyme (224 MR / L), T7 RNA polymerase enzyme (140 MU / L), water for volume, 3) Amplification and promoter reagent: Potassium chloride (23.3 mM), anhydrous glycerol (3.33%), zinc acetate dihydrate (0.05 mM), Pro clin 300 preservative (0.02%), Tween-20 (1%), Trizma base (11.61 mM), Trizma hydrochloride (14.94 mM), magnesium chloride (31 mM), dATP (0.83 mM), dCTP (0.83 mM), dGTP (0.83 mM), dTTP (0.83 mM), ATP (7 mM), CTP (7 mM), GTP (7 mM), UTP (8 mM), water (for volume).

[0376] The amplification and promoter reagents in Table 19 were made by adding oligonucleotides to an aliquot of the amplification and promoter bulk reagent. The TCR reagents in Table 19 were made by adding oligonucleotides and internal standard in vitro transcripts to the TCR bulk reagent. No oligos were added to the enzyme reagent.

[0377] The detergent Tween-20 was added to the amplification / promoter buffer to a final concentration of 1% to reduce aggregation and improve Torch stability.

[0378] Tests were performed on the PANTHER system and the results were analyzed with in-house developed software to interpret real-time curve characteristics. [Table 19]

[0379] Linearity / Dynamic Range Test / Analytical Sensitivity Linearity tests were performed for each of the five assay targets (A. vag, G. vag, L. crisp, L. gas, and L. jen). Two sets of panels were prepared for each analyte. One panel was prepared by adding culture lysates to analyte transport medium, STM (sodium phosphate monobasic monohydrate (2.07 g / L), lithium lauryl sulfate (30 g / L), EDTA disodium dihydrate (0.372 g / L), EGTA free acid (0.38 g / L), disodium hydrogen phosphate (2.13 g / L)). The second panel was prepared by adding in vitro transcripts (IVT) to STM. Each panel was tested in five replicates on the PANTHER system. Panel concentrations are shown in Table 20. [Table 20]

[0380] Table 21 shows a summary of the positivity rates for all five analytes. A. vag and all three Lacto species are 100% IVT positive up to 1E+07 c / mL. G. vag is 100% IVT positive at 1E+08 c / mL. All lysates are 100% positive at 1E+03 CFU / mL.

[0381] Detailed linearity results for all analytes are shown in Tables 22-26. Results shown include positivity, mean RFU range, mean Ttime, mean Tslope, and standard deviation. All reactions were valid as indicated by a common internal standard. [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26]

[0382] Cross-reactivity against microorganisms - pure (analytical specificity) and spiked (inhibition) tests To support cross-reactivity testing, 17 pools of bacterial or yeast material representing normal urogenital flora or organisms closely related to the APTIMA BV targets were constructed (see Table 27). The concentration of each organism was targeted at 1.00E+06 CFU / mL or cell equivalents / mL, which is above the upper limit of expected concentrations.

[0383] Pools were tested purely (without added assay target) to confirm assay specificity. Pools were also tested with added lysates representing each of the assay targets (A. vag, G. vag, and all three Lacto lysates) to test for assay inhibition by other microorganisms. Each condition was tested in quintuplicate. [Table 27-1] [Table 27-2]

[0384] Pure cross-reactivity results The FAM channel (Lacto species) showed a positive signal for pool #16 (Lacto acidophilus). This positive result was expected due to the substantial sequence identity of the test organism to L. crisp. This cross-reactivity is acceptable because L. acidophilus is found in the upper gastrointestinal tract and is likely absent from the vaginal flora.

[0385] The HEX channel (G.vag) gave negative results for all 17 panels.

[0386] The ROX channel (A. vag) showed a positive signal in pool #13 (Atopobium species). The three Atopobium species that comprise pool #13 (A. rimae, A. minimum, and A. parvulum) were separated and retested. The component tests showed that all three Atopobium species gave a positive signal in the ROX channel.

[0387] The Cy5.5 channel (GIC) showed positive results for all 17 pools.

[0388] Results of added cross-reactivity The target assay analyte (L. crisp, L. jen, L. gas, A. vag, or G. vag) was added to each of the 17 pools in Table 27. Positive analytes were added at approximately 3x LOD concentrations and tested in five replicates per panel per pool. The analyte spike concentrations are shown in Table 28. Spiked STM (Pool 0) was tested as a control for comparison. [Table 28]

[0389] A set of bulk reagents was prepared for spiked cross-reactivity testing. The bulk reagents were aliquoted into three 100-test reagent kits for testing. Individual bottles of enzyme were reconstituted separately for each of the three kits. When plotting the Ttime results, Reagent Kit #2 was found to have slower Ttimes across all four channels compared to Reagent Kits #1 and #3. This may have been due to enzyme variability and therefore represents potential variance due to kit-to-kit variability. The analyte Ttime was divided by the GIC Ttime to normalize the data to account for kit-to-kit variability. The Ttime results are shown in Table 29.

[0390] The FAM channel (Lactobacillus species) showed faster Ttime results from pool #16 (Lactobacillus acidophilus) for L. crisp, L. jen, and L. gas in the presence of L. acidophilus, and slightly slower Ttime results for pool #06 (Leptotrichia bucalis, Mobiluncus curtisii, M. genitalium, and M. hominis) when compared to the control (pool #0). Pool #16 also exhibited cross-reactivity during pure testing, which was expected given the close relationship of the test organisms to L. crisp in the 16S design region. Pool #06 was split into subcomponents and retested. Retesting showed a 1.6-minute delay for L. gas in the presence of M. hominis.

[0391] The HEX channel (G. vag) showed a decrease in sensitivity in four out of five replicates when tested in the presence of Pool #12 (Candida tropicalis, Candida krusei, and Candida lusitaniae). The three Candida species comprising Pool #12 were isolated and retested. Component testing indicated that C. krusei caused the decrease in sensitivity. Because C. krusei is found in vaginal flora but is not common, this potential interference should be noted. (BD Max TM In a US clinical trial of a vaginal panel, the C. krusei rate was 4 / 1647, or 0.2%, see BD PI 443710.

[0392] The ROX channel (A. vag) showed a slightly faster Ttime for pool #13 (Atopobium spp.) when compared to the control (pool 0). This was the same pool that caused cross-reactivity in the pure test, and was expected given the close relationship between the nature of the test organism and A. vag, the target of APTIMA BV.

[0393] The Cy5.5 channel (GIC) showed a slightly delayed T time for pool #6 (Leptotrichia bucalis, Mobiluncus curtisii, Mycoplasma genitalium, and Mycoplasma hominis). Constituent testing of these four microorganisms indicates that M. hominis delayed the GIC T time by just over one minute and slightly altered the shape of the GIC appearance curve. Mycoplasma hominis is commonly found in subjects with BV. Using the BV multivariate algorithm described in Example 1, the delayed GIC T time increases the BV score via the IC ratio term, thereby increasing the probability of a BV-positive readout. This can mitigate the effects of reaction inhibition caused by the presence of organisms associated with BV, including M. hominis. [Table 29]

[0394] Evaluation of clinical samples Clinical specimen testing was performed using a set of 100 clinical vaginal swab specimens collected at STM. Samples from symptomatic women from 10 clinical sites were tested with the APTIMA BV assay on a PANTHER instrument. One replicate per sample was tested.

[0395] Clinical samples were run along with 20 standards (five single-analyte standard levels for L. crisp, A. vag, and G. vag, and five mixed-analyte standard levels including all three analytes). Each single-analyte standard was tested in four replicates, and each mixed-analyte standard was tested in five replicates.

[0396] Of 100 clinical specimen replicates tested, 93 were valid, while the remaining 7 replicates were invalid due to insufficient sample volume.

[0397] Quantification of clinical samples was calculated based on standard curves of L. crisp, G. vag, and A. vag.

[0398] Diagnosis of BV status was based on quantification and a decision tree algorithm. The decision tree algorithm can be summarized as follows: (L, A, and G are the measured log copy values ​​of L. crisp, A. vag, and G. vag, respectively; X, Y, and Z are the cutoff values ​​for L. crisp, A. vag, and G. vag, respectively). If L > X, BV is negative; otherwise, if A > Y or G > Z, BV is positive; otherwise, BV is negative. In this analysis, the following cutoff values ​​were used for L. crisp, G. vag, and A. vag: 8.3, 10.4, and 7.4 log c / mL.

[0399] The clinical reference standard used in this example is the Nugent score, including intermediate, as determined by three of the four Amsel Criteria.

[0400] The single-analyte and mixed-analyte standards showed the same sensitivity results (90.2%) and similar specificity results (88.5% and 86.5%, respectively), with one sample altering the call due to differences in quantitation (see Table 30). [Table 30]

[0401] Comprehensive Testing Comprehensive testing was performed using one control strain and all available polymorphic IVT strains for each of the five assay targets. Testing was performed at low and medium concentrations of each strain. Results from the low panel were used to determine the effect of polymorphisms on analyte sensitivity, and results from the medium panel were analyzed to determine the effect of polymorphisms on analyte Ttime.

[0402] The number of strains tested and details of the low and high concentrations targeted for each analyte are shown in Table 31. The results are shown in Table 32.

[0403] A. vagus showed decreased sensitivity at low copy levels for three of the four polymorphic strains tested. Intermediate copy levels showed a Ttime delay of 2 to 3.5 minutes for two of the four strains. The two strains that showed a Ttime delay had two mismatches to the A. vagus T7 oligo. The prevalence of the two strains that showed a Ttime delay was 21.95% and <4.87%.

[0404] G. vag showed no reduction in sensitivity among the polymorphic strains tested. At intermediate copy levels, the polymorphic strain showed a Ttime delay of slightly more than 2 minutes. This polymorphism contains one mismatch in TCO and one mismatch in T7. The prevalence of this G. vag strain is very low (2.04%).

[0405] L. crisp showed a slight decrease in sensitivity at low copy levels for one of the two polymorphic strains tested. Both polymorphic strains showed differences in Ttime. When compared to the control, one strain produced a Ttime that was 3.5 minutes faster, while the other produced a Ttime that was 2 minutes slower. Both of these strains had two mismatches to T7, both with a prevalence of less than 2%.

[0406] L. gas showed 100% sensitivity against all strains tested. Intermediate copy levels showed minimal changes in Ttime for polymorphic strains compared to controls.

[0407] L. jen showed 100% sensitivity against all strains tested. Intermediate copy levels showed minimal changes in Ttime for polymorphic strains compared to controls. [Table 31] [Table 32] array [Table 33-1] [Table 33-2] [Table 33-3] [Table 34-1] [Table 34-2] [Table 34-3] [Table 34-4]

[0408] From the foregoing, it will be understood that, although specific embodiments of the invention have been described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope of the invention. Accordingly, the present invention is not limited except as by the appended claims. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes. To the extent that the scope of any material incorporated by reference conflicts with the explicit content of this disclosure, the explicit content shall control.

[0409] This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy created on August 20, 2019, is named 2019-08-20_01159-0039-60PCT_Seq List_ST25.txt and is 66.8 kilobytes in size. In certain embodiments, for example, the following items are provided: (Item 1) 1. A multiplex method for determining the presence or absence of each of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis) in a sample, said method comprising: (1) contacting a sample suspected of containing at least one of Lactobacillus species, A. vaginae, and Gardnerella vaginalis with: (a) first, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying a target region of a target nucleic acid of a Lactobacillus species, wherein (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 7. (iii) a 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) a 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 an 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; (c) first and second Gardnerella vaginalis (G. vaginalis)-specific amplification oligomers for amplifying a target region of a Gardnerella vaginalis (G. vaginalis) target nucleic acid, wherein (i) the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a first Gardnerella vaginalis (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 Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a second Gardnerella vaginalis (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 in which target nucleic acids of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis), if present in the sample, are used as templates to produce one or more amplification products corresponding to target regions of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis); (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Lactobacillus species, A. vaginae, and Gardnerella vaginalis in the sample; A method comprising: (Item 2) 1. A composition or kit for determining the presence or absence of each of Lactobacillus spp., A. vaginae, and Gardnerella vaginalis (G. vaginalis) in a sample, said composition or kit comprising: (a) first, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying a target region of a target nucleic acid of a Lactobacillus species, wherein (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target-hybridizing sequence that substantially corresponds to the nucleotide sequence of residues 28-45 of SEQ ID NO: 7. (iii) a 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) a 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 an 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; (c) a composition or kit comprising first and second Gardnerella vaginalis (G. vaginalis)-specific amplification oligomers for amplifying a target region of a Gardnerella vaginalis (G. vaginalis) target nucleic acid, wherein (i) the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a first Gardnerella vaginalis (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 Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a second Gardnerella vaginalis (G. vaginalis)-specific target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO:14. (Item 3) 1. A method for determining the presence or absence of bacterial vaginosis (BV) in a subject, said method comprising: (a) providing a sample from a subject suspected of having BV; (b) conducting an assay to detect Lactobacillus species, A. vaginae, and Gardnerella vaginalis in the sample; (c) assigning a quantitative value to each of Lactobacillus species, A. vaginae, and Gardnerella vaginalis based on the detection assay; (d) subtracting the quantitative value of Lactobacillus species from the greater of the quantitative value of A. vaginae and the quantitative value of Gardnerella vaginalis; (e) assigning a single BV score based on step (d); and (f) determining whether or not a subject has BV based on a comparison of the BV score with the cutoff value; A method comprising: (Item 4) 1. A method for determining the presence or absence of Lactobacillus spp. in a sample, said method comprising: (1) contacting a sample suspected of containing Lactobacillus spp. with first, second, third, and fourth amplification oligomers for amplifying a target region of a target nucleic acid of Lactobacillus spp., 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 in which a target nucleic acid of Lactobacillus spp., if present in the sample, is used as a template to produce one or more amplification products corresponding to a target region of Lactobacillus spp.; (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Lactobacillus species in the sample; A method comprising: (Item 5) 1. A method for determining the presence or absence of A. vaginae in a sample, said method comprising: (1) contacting a sample suspected of containing A. vaginae with first and second amplification oligomers for amplifying a target region of an 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) conducting an in vitro nucleic acid amplification reaction in which A. vaginae target nucleic acid, if present in the sample, is used as a template to produce one or more amplification products corresponding to target regions of A. vaginae; (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of A. vaginae in the sample. A method comprising: (Item 6) 1. A method for determining the presence or absence of Gardnerella vaginalis (G. vaginalis) in a sample, said method comprising: (1) contacting a sample suspected of containing Gardnerella vaginalis (G. vaginalis) with first and second amplification oligomers for amplifying a target region of a Gardnerella vaginalis (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 in which a Gardnerella vaginalis (G. vaginalis) target nucleic acid, if present in the sample, is used as a template to produce one or more amplification products corresponding to a target region of Gardnerella vaginalis (G. vaginalis); (3) detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Gardnerella vaginalis (G. vaginalis) in the sample.

Claims

[Claim 1] The invention described in this specification.

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