Composition and method for detecting bacterial nucleic acids and diagnosing bacterial vaginosis

Abstract

To provide a composition and method for detecting bacterial nucleic acids and diagnosing bacterial vaginosis. [Solution] A method for diagnosing a target bacterial vaginosis is disclosed, comprising performing an assay to detect one or more of the following species in a target sample: Lactobacillus, Atopobium vaginae, and Gardneralla vaginalis. Also disclosed are compositions and methods for detecting the nucleic acids of Lactobacillus, Atopobium vaginae, and / or Gardneralla vaginalis in a sample.

Description

[Technical Field]

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

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

[0003] No single pathogen is known to cause BV. Most researchers and the CDC believe that bacterial vaginosis is a result of a disruption of the normal bacterial 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 microbiome in the body's environment, such as the vagina. See Nibali et al., Journal of Oral Microbiology, 6:22962, 2014.

[0004] BV is diagnosed in clinics using the Amsel Criteria and in laboratories using the Nugent Scoring System. The latter relies on counting bacterial morphologies with the help of Gram staining. Thus, the Nugent score is a visual assessment of dysbiosis, scoring harmful bacteria against beneficial bacteria. See Nugent et al., Journal of Clinical Microbiology 29:297-301, 1991. The Amsel Criteria evaluates a sample for the presence of scutellaria cells, pH, color, and odor, which are key symptoms associated with BV. See Amsel et al., American Journal of Medicine 74:14-22, 1983. Wet mounts of the sample are examined under a microscope to detect scutellaria cells, which are human epithelial cells covered with bacteria thought to consist mainly of Gardnerella vaginalis (G. vaginalis).

[0005] Molecular testing generally targets multiple organisms that have a strong correlation with bacterial vaginosis. The specific organisms targeted vary from test to test. In almost all cases, large quantities of anaerobic bacteria are targeted, such as Atopobium, Gardnerella, and Megasphaera species.

[0006] The FDA-approved BV trial was "BD Affirm TM "VPIII Microbial Identification Test" (2010) and "BD MAX" TM Only the "Vaginal Panel" (2016) is available. Both 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% compared to scoring Gram staining, 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. 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 a combination of Nugent and Amsel results in 323 women (93% African American, 7% white non-Hispanic). They reported a sensitivity of 96.9% and a specificity of 92.6% compared to a combination of Nugent and Amsel scores. Results for this assay compared to Nugent scores alone were not reported. [Overview of the project] [Means for solving the problem]

[0008] In one embodiment, 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 involves the following steps: (a) providing a sample from a subject suspected of having BV; (b) performing an assay to detect Lactobacillus species, Atopovium vaginae and Gardnerella vaginalis in the sample; (c) detecting Lactobacillus species, Atopovium vaginae and Gardnerella vaginalis The process includes (d) assigning a quantitative value to each of the G. vaginalis species based on a detection assay, (e) subtracting the quantitative value of the Lactobacillus species from the larger of the quantitative values ​​of Atopovium vaginae and Gardnerella vaginalis, (f) assigning a single BV score based on step (d), and (g) determining the presence or absence of the BV in question based on a comparison of the BV score with a cutoff value. In some embodiments, in step (c), the minimum quantitative value is assigned to the Lactobacillus species and / or Atopovium vaginae and Gardnerella vaginalis. Typically, the quantitative values ​​for each Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are standardized and / or weighted. In some variations involving standardized quantitative values, the quantitative values ​​for each Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are in log copies, and standardization involves subtracting the median of the population values ​​from the value determined from the detection assay. Step (d) may further include the addition of adjustment constants.In certain embodiments, step (d) further includes adding an internal standard (IC) adjustment factor that compensates for sample inhibition of the detection assay, the IC adjustment factor being based on the ratio of (i) an observed IC value generated from the detection assay to (ii) an 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 a calibration constant, W L is a weighting constant for the Lactobacillus species, Max(L S , F LS ) is the greater of L S and F LS , where L S is the observed normalized quantification value for the Lactobacillus species, F LS is the imposed minimum normalized quantification value for the Lactobacillus species, W GA is a weighting constant for Atopobium vaginae and Gardnerella vaginalis, Max(A S , G S , F GAS ) is the greater of A S , G S , and F GAS , where A S is the observed normalized quantification value for Atopobium vaginae, G S is the observed normalized quantification value for Gardnerella vaginalis, and F GASThis is the smallest standardized quantitative value for Atopobium vaginae and Gardnerella vaginalis (e.g., 0), W IC is the 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 as described above, the assays for detecting Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are nucleic acid-based detection assays. Particularly suitable nucleic acid-based detection assays include amplification-based assays, such as those 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 assay targets the 16S rRNA of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis. In certain modifications, the nucleic acid-based detection assay detects: (i) the 16S rRNA region of Lactobacillus crispatus (L. crispatus) corresponding to the region of SEQ ID NO: 1, from nucleotide position approximately 40 to nucleotide position approximately 265; (ii) the 16S rRNA region of Lactobacillus jensenii (L. jensenii) corresponding to the region of SEQ ID NO: 2, from nucleotide position approximately 43 to nucleotide position approximately 247; (iii) the 16S rRNA region of Lactobacillus gasseri (L. gasseri) corresponding to the region of SEQ ID NO: 3, from nucleotide position approximately 93 to nucleotide position approximately 298; (iv) the 16S rRNA region of Atopobium vaginae (A. vaginae) corresponding to the region of SEQ ID NO: 4, from nucleotide position approximately 540 to nucleotide position approximately 625; and / or (v) the 16S rRNA region of Gardnerella vaginalis (G. vaginalis) corresponding to the region of SEQ ID NO: 5, from nucleotide position approximately 172 to nucleotide position approximately 227. It targets the rRNA region.

[0010] In a particular embodiment of the method for diagnosing BV as described above, which includes a nucleic acid-based detection assay, the assay is an amplification-based assay comprising the following steps: (1) Bring the sample into contact with the following: First, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 7 (iii) The third Lactobacillus-specific amplified oligomer comprises a substantially corresponding second Lactobacillus-specific target hybridize sequence, (iv) The fourth Lactobacillus-specific amplified oligomer comprises a fourth Lactobacillus-specific target hybridize sequence, which substantially corresponds to the nucleotide sequence of SEQ ID NO: 8. First and second Atopobium vaginae (A. vaginae) specific amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae (A. vaginae), wherein (i) the first Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a first Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and (ii) the second Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a second Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. First and second Gardnerella vaginalis-specific amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first Gardnerella vaginalis-specific amplification oligomer comprises a first Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and (ii) the second Gardnerella vaginalis-specific amplification oligomer comprises a second Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14. (2) If target nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are present in the sample, perform an in vitro nucleic acid amplification reaction using them as templates to produce one or more amplification products corresponding to the target regions of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis. (3) Detect the presence or absence of one or more amplification products.

[0011] In some variations of the method for diagnosing BV, including the amplification-based detection assay described above, the first Lactobacillus-specific target hybridize sequence includes the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridize sequence includes the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridize sequence includes the nucleotide sequence of SEQ ID NO: 8, and the fourth Lactobacillus-specific target hybridize sequence includes the nucleotide sequence of SEQ ID NO: 9. The first Atopobium vaginae-specific target hybridization sequence includes the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, the second Atopobium vaginae-specific target hybridization sequence includes the nucleotide sequence of SEQ ID NO: 17, the first Gardnerella vaginalis-specific target hybridization sequence includes the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or the second Gardnerella vaginalis-specific target hybridization sequence includes the nucleotide sequence of SEQ ID NO: 14.In some such embodiments, the first Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 8, the fourth Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 9, and the first At The Atopovium vaginae-specific target hybridization sequence consists of the nucleotide sequence from residues 28-45 of SEQ ID NO: 18, the second Atopovium vaginae-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 17, the first Gardnerella vaginalis-specific target hybridization sequence consists of the nucleotide sequence from residues 36-52 of SEQ ID NO: 15, and / or the second Gardnerella vaginalis-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 14.

[0012] In some embodiments of methods for diagnosing BV, including amplification-based detection assays as described above, at least one of a first Lactobacillus-specific amplification oligomer, a first Atopovium vaginae-specific amplification oligomer, and a first Gardnerella vaginalis-specific amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' relative to the respective target hybridize sequence. Particularly suitable promoter sequences are T7 promoter sequences, such as a promoter sequence having the nucleotide sequence of residues 1-27 in SEQ ID NO: 10 (or, for example, residues 1-27 in SEQ ID NO: 18 or residues 9-35 in SEQ ID NO: 15). In certain modifications, the first Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10, the first Atopobium vaginae-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 18, and / or the first Gardnerella vaginalis-specific amplification oligomer comprises the nucleotide sequence of residues 9-52 of SEQ ID NO: 15. In some embodiments, the first Gardnerella vaginalis-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

[0013] In certain embodiments of a method for diagnosing BV, including amplification-based detection assays as described above, the method further comprises, prior to step (2), purifying, if present, target nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis from other components in the sample. In some such embodiments, the purification step involves contacting the sample with at least one capture probe oligomer containing a target hybridized sequence covalently bound to a sequence or portion bound to an immobilized probe. For example, a sample can be brought into contact with a first capture probe oligomer containing a target hybridize sequence that specifically hybridizes to a target sequence within the target nucleic acid of a Lactobacillus species, and a second capture probe target hybridize sequence containing a target hybridize sequence that specifically hybridizes to a target sequence within the target nucleic acids of Atopovium vaginae and Gardnerella vaginalis, respectively, with each of the first and second capture probe target hybridize sequences covalently bound to a sequence or portion that binds to the immobilized probe, and in some such embodiments, the first capture probe target hybridize sequence specifically hybridizes to a target sequence within the target nucleic acids of Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri, respectively. In certain modifications including the first and second target capture probes as described above, the first capture probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO: 6, and / or the second capture probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO: 13. In some embodiments, the first capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 6, and / or the second capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 13.

[0014] In some embodiments of a method for diagnosing BV, including the amplification-based detection assay described above, the detection step (3) includes (i) a first Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of a Lactobacillus species, a first Atopovium vaginae-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of Atopovium vaginae, and Gardnerella (ii) contacting a first Gardnerella vaginalis-specific detection probe containing a target hybridize sequence that specifically hybridizes to the target region of G. vaginalis with one or more amplification products, and (ii) detecting the presence or absence of a target hybridized Lactobacillus-specific detection probe, an Atopovium vaginae-specific detection probe, and / or a Gardnerella vaginalis-specific detection probe. In some embodiments, the first Atopovium vaginae-specific detection probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 6-21 of SEQ ID NO: 19, and / or the first Gardnerella vaginalis-specific detection probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO: 16. In more specific modifications, the first Atopovium vaginae-specific detection probe target hybridize sequence includes the nucleotides of residues 6-21 of SEQ ID NO: 19, and / or the first Gardnerella vaginalis-specific detection probe target hybridize sequence includes the nucleotide sequence of residues 1-18 of SEQ ID NO: 16.In some embodiments, a first Lactobacillus-specific detection probe target hybridize sequence specifically hybridizes to the target regions of the target nucleic acids of Lactobacillus crispatus and Lactobacillus jensenii, respectively, and the method further comprises contacting one or more amplification products with a second Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to the target region of the target nucleic acid of Lactobacillus gasseri, and in some such embodiments, the first Lactobacillus The LUS-specific detection probe target hybridize 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 hybridize sequence substantially corresponds to the nucleotide sequence of residues 7-23 of SEQ ID NO: 12. In a more specific variation, the first Lactobacillus-specific detection probe target hybridize sequence includes the nucleotide sequence of residues 1-17 of SEQ ID NO: 11, and / or the second Lactobacillus-specific detection probe target hybridize sequence includes the nucleotide sequence of residues 7-23 of SEQ ID NO: 12.

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

[0016] In some embodiments of the method for diagnosing BV, which involves 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 includes a fluorescent label and a quencher. Particularly suitable detection probes including a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0017] In certain embodiments of a method for diagnosing BV, including the use of detection probes as described above, at least one of a first Lactobacillus-specific detection probe, a first Atopovium vaginae-specific detection probe, and a first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridize sequence. In some embodiments further including the use of a second Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe further comprises a non-target hybridize sequence. In some such variations, each of the first Lactobacillus-specific detection probe, the first Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe (or each of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe) is a molecular torch or molecular beacon.

[0018] In some embodiments of the methods for diagnosing BV as described above, the method includes the detection of 10 or fewer bacterial genera associated with BV. For example, in certain modifications, the method includes the detection of 5 or fewer bacterial genera associated with BV. In certain modifications, the method does not include the detection of 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 a subject, the method further includes administering a treatment plan for BV to the subject.

[0020] In some embodiments of the methods for diagnosing BV as described above, the method is a method for monitoring BV in a subject, the subject having received a treatment plan 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 plan for BV to the subject, or (ii) administering a different treatment plan for BV to the subject.

[0021] In one embodiment, the present invention provides a multiplex method for determining the presence or absence of each of the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in a sample. This method typically includes the following steps. (1) Contact a sample suspected to contain at least one of the following species: Lactobacillus, Atopovium vaginae, and Gardnerella vaginalis, (a) First, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 7 (iii) The third Lactobacillus-specific amplified oligomer comprises a second Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 8, and (iv) The fourth Lactobacillus-specific amplified oligomer comprises a fourth Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 9. (b) First and second Atopobium vaginae (A. vaginae) specific amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae (A. vaginae), wherein (i) the first Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a first Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and (ii) the second Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a second Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. (c) First and second Gardnerella vaginalis-specific amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first Gardnerella vaginalis-specific amplification oligomer comprises a first Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and (ii) the second Gardnerella vaginalis-specific amplification oligomer comprises a second Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14. (2) If target nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are present in the sample, perform an in vitro nucleic acid amplification reaction using them as templates to produce one or more amplification products corresponding to the target regions of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis. (3) Detect the presence or absence of one or more amplification products to determine the presence or absence of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in the sample.

[0022] In some variations of the multiplex method described above, the first Lactobacillus-specific target hybridization sequence includes the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridization sequence includes the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridization sequence includes the nucleotide sequence of SEQ ID NO: 8, and the fourth Lactobacillus-specific target hybridization sequence includes the nucleotide sequence of SEQ ID NO: 9. The first Atopobium vaginae-specific target hybridization sequence includes the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, the second Atopobium vaginae-specific target hybridization sequence includes the nucleotide sequence of SEQ ID NO: 17, the first Gardnerella vaginalis-specific target hybridization sequence includes the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or the second Gardnerella vaginalis-specific target hybridization sequence includes the nucleotide sequence of SEQ ID NO: 14.In some such embodiments, the first Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 8, the fourth Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 9, and the first At The Atopovium vaginae-specific target hybridization sequence consists of the nucleotide sequence from residues 28-45 of SEQ ID NO: 18, the second Atopovium vaginae-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 17, the first Gardnerella vaginalis-specific target hybridization sequence consists of the nucleotide sequence from residues 36-52 of SEQ ID NO: 15, and / or the second Gardnerella vaginalis-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 14.

[0023] In some embodiments of the multiplex method described above, at least one of the first Lactobacillus-specific amplification oligomer, the first Atopobium 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 at 5' relative to the respective target hybridize sequence. Particularly suitable promoter sequences are T7 promoter sequences, such as a promoter sequence having the nucleotide sequence of residues 1-27 in SEQ ID NO: 10 (or, for example, residues 1-27 in SEQ ID NO: 18 or residues 9-35 in SEQ ID NO: 15). In certain modifications, the first Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10, the first Atopobium vaginae-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 18, and / or the first Gardnerella vaginalis-specific amplification oligomer comprises the nucleotide sequence of residues 9-52 of SEQ ID NO: 15. In some embodiments, the first Gardnerella vaginalis-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

[0024] In certain embodiments of the multiplex method as described above, the method further comprises, prior to step (2), purifying, if present, the target nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis from other components in the sample. In some such embodiments, the purification step comprises contacting the sample with at least one capture probe oligomer containing a target hybridized sequence covalently bound to a sequence or portion bound to the immobilized probe. For example, a sample can be brought into contact with a first capture probe oligomer containing a target hybridize sequence that specifically hybridizes to a target sequence within the target nucleic acid of a Lactobacillus species, and a second capture probe target hybridize sequence containing a target hybridize sequence that specifically hybridizes to a target sequence within the target nucleic acids of Atopovium vaginae and Gardnerella vaginalis, respectively, with each of the first and second capture probe target hybridize sequences covalently bound to a sequence or portion that binds to the immobilized probe, and in some such embodiments, the first capture probe target hybridize sequence specifically hybridizes to a target sequence within the target nucleic acids of Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri, respectively. In certain modifications including the first and second target capture probes as described above, the first capture probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO: 6, and / or the second capture probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO: 13. In some embodiments, the first capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 6, and / or the second capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 13.

[0025] In some embodiments of the multiplex method described above, the detection step (3) includes (i) a first Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to the target region of a Lactobacillus species, a first Atopovium vaginae-specific detection probe containing a target hybridize sequence that specifically hybridizes to the target region of Atopovium vaginae, and Gardnerella vaginalis (G (ii) contacting a first Gardnerella vaginalis (G. vaginalis) specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of G. vaginalis with one or more amplification products, and (ii) detecting the presence or absence of a target hybridized Lactobacillus (Lactobacillus) specific detection probe, an Atopovium vaginae (A. vaginae) specific detection probe, and / or a Gardnerella vaginalis (G. vaginalis) specific detection probe. In some embodiments, the first Atopovium vaginae-specific detection probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 6-21 of SEQ ID NO: 19, and / or the first Gardnerella vaginalis-specific detection probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO: 16. In more specific modifications, the first Atopovium vaginae-specific detection probe target hybridize sequence includes the nucleotides of residues 6-21 of SEQ ID NO: 19, and / or the first Gardnerella vaginalis-specific detection probe target hybridize sequence includes the nucleotide sequence of residues 1-18 of SEQ ID NO: 16.In some embodiments, a first Lactobacillus-specific detection probe target hybridize sequence specifically hybridizes to the target regions of the target nucleic acids of Lactobacillus crispatus and Lactobacillus jensenii, respectively, and the method further comprises contacting one or more amplification products with a second Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to the target region of the target nucleic acid of Lactobacillus gasseri, and in some such embodiments, the first Lactobacillus The LUS-specific detection probe target hybridize 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 hybridize sequence substantially corresponds to the nucleotide sequence of residues 7-23 of SEQ ID NO: 12. In a more specific variation, the first Lactobacillus-specific detection probe target hybridize sequence includes the nucleotide sequence of residues 1-17 of SEQ ID NO: 11, and / or the second Lactobacillus-specific detection probe target hybridize sequence includes the nucleotide sequence of residues 7-23 of SEQ ID NO: 12.

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

[0027] In some embodiments of the multiplex method, including 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 includes a fluorescent label and a quencher. Particularly suitable detection probes including a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0028] In certain embodiments of the multiplex method, including the use of the detection probes described above, at least one of the first Lactobacillus-specific detection probe, the first Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridize sequence. In some embodiments, further including the use of a second Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe further comprises a non-target hybridize sequence. In some such variations, each of the first Lactobacillus-specific detection probe, the first Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe (or each of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe) is a molecular torch or 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 modifications, 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 embodiment, the present invention provides a composition or kit for determining the presence or absence of each of the Lactobacillus species, Atopovium 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 the target region of the target nucleic acid of the Lactobacillus species, where (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 7 (iii) The third Lactobacillus-specific amplified oligomer comprises a second Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 8, and (iv) The fourth Lactobacillus-specific amplified oligomer comprises a fourth Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 9. (b) First and second Atopobium vaginae (A. vaginae) specific amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae (A. vaginae), wherein (i) the first Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a first Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and (ii) the second Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a second Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. (c) First and second Gardnerella vaginalis-specific amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first Gardnerella vaginalis-specific amplification oligomer comprises a first Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and (ii) the second Gardnerella vaginalis-specific amplification oligomer comprises a second Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14.

[0031] In some variations of the above composition or kit, the first Lactobacillus-specific target hybridization sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridization sequence comprises the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridization sequence comprises the nucleotide sequence of SEQ ID NO: 8, and the fourth Lactobacillus-specific target hybridization sequence comprises the nucleotide sequence of SEQ ID NO: 9. The first Atopobium vaginae-specific target hybridization sequence includes the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, the second Atopobium vaginae-specific target hybridization sequence includes the nucleotide sequence of SEQ ID NO: 17, the first Gardnerella vaginalis-specific target hybridization sequence includes the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or the second Gardnerella vaginalis-specific target hybridization sequence includes the nucleotide sequence of SEQ ID NO: 14.In some such embodiments, the first Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 8, the fourth Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 9, and the first At The Atopovium vaginae-specific target hybridization sequence consists of the nucleotide sequence from residues 28-45 of SEQ ID NO: 18, the second Atopovium vaginae-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 17, the first Gardnerella vaginalis-specific target hybridization sequence consists of the nucleotide sequence from residues 36-52 of SEQ ID NO: 15, and / or the second Gardnerella vaginalis-specific target hybridization sequence consists of the nucleotide sequence of SEQ ID NO: 14.

[0032] In some embodiments of the above composition or kit, at least one of the first Lactobacillus-specific amplification oligomer, the first Atopobium 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 at 5' relative to the respective target hybridize sequence. Particularly suitable promoter sequences are T7 promoter sequences, such as a promoter sequence having the nucleotide sequence of residues 1-27 in SEQ ID NO: 10 (or, for example, residues 1-27 in SEQ ID NO: 18 or residues 9-35 in SEQ ID NO: 15). In certain modifications, the first Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 10, the first Atopobium vaginae-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 18, and / or the first Gardnerella vaginalis-specific amplification oligomer comprises the nucleotide sequence of residues 9-52 of SEQ ID NO: 15. In some embodiments, the first Gardnerella 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 composition or kit further comprises at least one capture probe oligomer containing a target hybridized sequence covalently bound to a sequence or portion that binds to the immobilized probe. For example, the composition or kit may include a first capture probe oligomer containing a target hybridize sequence that specifically hybridizes to a target sequence within a target nucleic acid of a Lactobacillus species, and a second capture probe target hybridize sequence containing a target hybridize sequence that specifically hybridizes to a target sequence within the target nucleic acids of Atopovium vaginae and Gardnerella vaginalis, respectively, wherein each of the first and second capture probe target hybridize sequences is covalently bound to a sequence or portion that binds to the immobilized probe, and in some such embodiments, the first capture probe target hybridize sequence specifically hybridizes to a target sequence within the target nucleic acids of Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri, respectively. In certain modifications including the first and second target capture probes as described above, the first capture probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO: 6, and / or the second capture probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO: 13. In some embodiments, the first capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 6, and / or the second capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 13.

[0034] In some embodiments of the compositions or kits described above, the composition or kit further comprises a first Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of a Lactobacillus species, a first Atopovium vaginae-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of Atopovium vaginae, and a first Gardnerella vaginalis-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of G. vaginalis. In some embodiments, the first Atopovium vaginae-specific detection probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 6-21 of SEQ ID NO: 19, and / or the first Gardnerella vaginalis-specific detection probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-18 of SEQ ID NO: 16. In more specific modifications, the first Atopovium vaginae-specific detection probe target hybridize sequence includes the nucleotides of residues 6-21 of SEQ ID NO: 19, and / or the first Gardnerella vaginalis-specific detection probe target hybridize sequence includes the nucleotide sequence of residues 1-18 of SEQ ID NO: 16.In some embodiments, the first Lactobacillus-specific detection probe target hybridize sequence specifically hybridizes to the target regions of the target nucleic acids of Lactobacillus crispatus and Lactobacillus jensenii, respectively, and the composition or kit further comprises a second Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to the target region of the target nucleic acid of Lactobacillus gasseri, and in some such embodiments, the first Lactobacillus-specific detection probe The first probe target hybridize 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 hybridize sequence substantially corresponds to the nucleotide sequence of residues 7-23 of SEQ ID NO: 12. In a more specific variation, the first Lactobacillus-specific detection probe target hybridize sequence includes the nucleotide sequence of residues 1-17 of SEQ ID NO: 11, and / or the second Lactobacillus-specific detection probe target hybridize sequence includes the nucleotide sequence of residues 7-23 of SEQ ID NO: 12.

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

[0036] In certain embodiments of the composition or kit containing the detection probes described above, at least one of the first Lactobacillus-specific detection probe, the first Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridize sequence. In some embodiments further comprising a second Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe further comprises a non-target hybridize sequence. In some such variations, each of the first Lactobacillus-specific detection probe, the first Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe (or each of the first Lactobacillus-specific detection probe, the second Lactobacillus-specific detection probe, the first Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe) is a molecular torch or molecular beacon.

[0037] In yet another aspect, the present invention provides a method for determining the presence or absence of Lactobacillus species in a sample. This method typically includes the following steps: (1) Contact a sample suspected of containing the Lactobacillus species with first, second, third, and fourth amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first amplification oligomer comprises a first target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, (ii) the amplification oligomer comprises a second target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 7, (iii) the third amplification oligomer comprises a third target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 8, and (iv) the fourth amplification oligomer comprises a fourth target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 9. (2) If the target nucleic acid of a Lactobacillus species is present in the sample, an in vitro nucleic acid amplification reaction is performed, which is used as a template to produce one or more amplification products corresponding to the target region of the Lactobacillus species. (3) Detect 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 method for detecting Lactobacillus species as described above, the first Lactobacillus-specific target hybridize sequence comprises the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second Lactobacillus-specific target hybridize sequence comprises the nucleotide sequence of SEQ ID NO: 7, the third Lactobacillus-specific target hybridize sequence comprises the nucleotide sequence of SEQ ID NO: 8, and / or the fourth target hybridize sequence comprises the nucleotide sequence of SEQ ID NO: 9. In some such embodiments, the first target hybridize sequence consists of the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, the second target hybridize sequence consists of the nucleotide sequence of SEQ ID NO: 7, the third target hybridize sequence consists of the nucleotide sequence of SEQ ID NO: 8, and / or the fourth target hybridize sequence consists of the nucleotide sequence of SEQ ID NO: 9.

[0039] In some embodiments of the method for detecting Lactobacillus species as described above, the first amplified oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' relative to each target hybridize sequence. Particularly suitable promoter sequences are T7 promoter sequences, such as the promoter sequence having the nucleotide sequence of residues 1-27 of SEQ ID NO: 10. In certain modifications, the first amplified oligomer comprises the nucleotide sequence of SEQ ID NO: 10.

[0040] In certain embodiments of the methods for detecting Lactobacillus species as described above, the method further comprises, prior to step (2), purifying the target nucleic acid of the Lactobacillus species, 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 containing a target hybridize sequence covalently bound to a sequence or portion bound to an immobilized probe, wherein the capture probe target hybridize sequence specifically hybridizes to a target sequence in the target nucleic acid of the Lactobacillus species. In some embodiments, the capture probe target hybridizing sequence specifically hybridizes to the target sequence within each of the target nucleic acids of Lactobacillus crispatus (L. crispatus), Lactobacillus jensenii (L. jensenii), and Lactobacillus gasseri (L. gasseri). In some such modifications, the first capture probe target hybridizing sequence substantially corresponds to the nucleotide sequence of residues 1-19 of SEQ ID NO: 6, and in a more specific embodiment, the capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 6.

[0041] In some embodiments for detecting Lactobacillus species as described above, the detection step (3) includes (i) contacting a first detection probe containing a target hybridize sequence that specifically hybridizes to a target region of a Lactobacillus species with one or more amplification products to detect the presence or absence of the target hybridize detection probe. In some embodiments, the first detection probe target hybridize sequence specifically hybridizes to the respective target regions of the target nucleic acids of Lactobacillus crispatus and Lactobacillus jensenii, and the method further comprises contacting one or more amplification products with a second detection probe containing a target hybridize sequence that specifically hybridizes to the target region of the target nucleic acid of Lactobacillus gasseri, in some such embodiments the first detection probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-17 of SEQ ID NO: 11, and / or the second detection probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 7-23 of SEQ ID NO: 12, and in a more specific variation, the first detection probe target hybridize sequence contains the nucleotide sequence of residues 1-17 of SEQ ID NO: 11, and / or the second detection probe target hybridize sequence contains 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 includes the use of a first detection probe, the first detection probe including a label. In some embodiments, the method further includes contacting a second probe with one or more amplified oligomers, the second probe including a label. Particularly suitable labels include chemiluminescent and fluorescent labels.

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

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

[0045] In yet another aspect, the present invention provides a method for determining the presence or absence of Atopobium vaginae in a sample. This method typically includes the following steps: (1) Contact a sample suspected of containing Atopobium vaginae with first and second amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae, where (i) the first amplification oligomer comprises a first target hybridize 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 hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. (2) If the target nucleic acid of Atopobium vaginae is present in the sample, perform an in vitro nucleic acid amplification reaction using it as a template to produce one or more amplification products corresponding to the target region of Atopobium vaginae. (3) To detect the presence or absence of one or more amplification products, thereby determining the presence or absence of Atopobium vaginae in the sample.

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

[0047] In some embodiments of the methods for detecting Atopobium vaginae as described above, the first amplified oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' relative to each target hybridize sequence. Particularly suitable promoter sequences are T7 promoter sequences, such as the promoter sequence having the nucleotide sequence of residues 1-27 of SEQ ID NO: 18. In certain modifications, the first amplified oligomer comprises the nucleotide sequence of SEQ ID NO: 18.

[0048] In certain embodiments of the method for detecting Atopobium vaginae (A. vaginae) as described above, the method further comprises, prior to step (2), purifying the target nucleic acid of Atopobium vaginae (A. vaginae), 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 containing a target hybridize sequence covalently bound to a sequence or portion bound to an immobilized probe, the capture probe target hybridize sequence specifically hybridizes to a target sequence in the target nucleic acid of Atopobium vaginae (A. vaginae). In some embodiments, the capture probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO: 13. In a more specific variation, the capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 13.

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

[0050] In a particular embodiment of the method for detecting Atopobium vaginae as described above, the method includes the use of a detection probe, the detection probe including a label. Particularly suitable labels include chemiluminescent and fluorescent labels and chemiluminescent labels.

[0051] In some embodiments of the method for detecting Atopobium vaginae as described above, which involves the use of a labeled detection probe, the detection step (3) occurs during the amplification step (2). In some such modifications, the detection probe includes a fluorescent label and a quencher. Particularly suitable detection probes including a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0052] In certain embodiments of the method for detecting Atopobium vaginae as described above, which involves the use of a detection probe, the detection probe further comprises a non-target hybridized sequence. In some such modifications, 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 in a sample. This method typically includes the following steps: (1) Contact a sample suspected of containing Gardnerella vaginalis with first and second amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first amplification oligomer comprises a first target hybridize 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 hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14. (2) If the target nucleic acid of Gardnerella vaginalis is present in the sample, an in vitro nucleic acid amplification reaction is performed using it as a template to produce one or more amplification products corresponding to the target region of Gardnerella vaginalis. (3) To detect the presence or absence of one or more amplification products, thereby determining the presence or absence of Gardnerella vaginalis in the sample.

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

[0055] In some embodiments of the method for detecting Gardnerella vaginalis (G. vaginalis) as described above, the first amplified oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' relative to each target hybridize sequence. Particularly suitable promoter sequences are T7 promoter sequences, such as the promoter sequence having the nucleotide sequence of residues 9-36 of SEQ ID NO: 15. In certain modifications, the first amplified oligomer comprises the nucleotide sequence of residues 9-52 of SEQ ID NO: 15, and in some such embodiments, the first amplified oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

[0056] In certain embodiments of the method for detecting Gardnerella vaginalis (G. vaginalis) as described above, the method further comprises, prior to step (2), purifying the target nucleic acid of Gardnerella vaginalis (G. vaginalis), 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 containing a target hybridize sequence covalently bound to a sequence or portion thereof that binds to an immobilized probe, the capture probe target hybridize sequence specifically hybridizes to a target sequence within the target nucleic acid of Gardnerella vaginalis (G. vaginalis). In some embodiments, the capture probe target hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO: 13. In a more specific modification, the capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 13.

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

[0058] In certain embodiments of the method for detecting Gardnerella vaginalis as described above, the method includes the use of a detection probe, the detection probe including a label. Particularly suitable labels include chemiluminescent and fluorescent labels and chemiluminescent labels.

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

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

[0061] In some embodiments of the method for detecting Lactobacillus species such as Atopovium vaginae or Gardnerella vaginalis, the amplification reaction in step (2) is an isothermal amplification reaction. In certain modifications, 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 embodiments of the present invention will become apparent from 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 the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in a sample, and the method comprises the following: (1) Contact a sample suspected to contain at least one of the following species: Lactobacillus, Atopovium vaginae, and Gardnerella vaginalis, (a) First, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 7 (iii) The third Lactobacillus-specific amplified oligomer comprises a second Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 8, and (iv) The fourth Lactobacillus-specific amplified oligomer comprises a fourth Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 9. (b) First and second Atopobium vaginae (A. vaginae) specific amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae (A. vaginae), wherein (i) the first Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a first Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and (ii) the second Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a second Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. (c) First and second Gardnerella vaginalis-specific amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first Gardnerella vaginalis-specific amplification oligomer comprises a first Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and (ii) the second Gardnerella vaginalis-specific amplification oligomer comprises a second Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14. (2) If target nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are present in the sample, perform an in vitro nucleic acid amplification reaction using them as templates to produce one or more amplification products corresponding to the target regions of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis. (3) Detect the presence or absence of one or more amplification products to determine the presence or absence of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in the sample.

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

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

[0066] Embodiment 4 is a method according to any one of Embodiments 1 to 3, wherein at least one of the first Lactobacillus-specific amplification oligomer, the first Atopobium 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 at 5' relative to the respective target hybridize sequence.

[0067] Embodiment 5 is the method of Embodiment 4, and the promoter sequence is the T7 promoter sequence.

[0068] Embodiment 6 is the method of Embodiment 5, and 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 amplified oligomer contains the nucleotide sequence of SEQ ID NO: 10. The first Atopobium vaginae-specific amplified oligomer contains the nucleotide sequence of SEQ ID NO: 18, and / or The first Gardnerella vaginalis-specific amplified oligomer contains the nucleotide sequence of residues 9-52 of SEQ ID NO: 15.

[0070] Embodiment 8 is the method of Embodiment 7, wherein the first Gardnerella vaginalis-specific amplified oligomer comprises the nucleotide sequence of SEQ ID NO: 15.

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

[0072] Embodiment 10 is the method of Embodiment 9, wherein the purification step includes contacting the sample with at least one capture probe oligomer containing a target hybridized sequence covalently bound to a sequence or portion bound to an immobilized probe.

[0073] Embodiment 11 is the method of Embodiment 10, wherein the sample is in contact with the first and second capture probe oligomers. The first capture probe oligomer contains 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 contains a target hybridizing sequence that specifically hybridizes to a target sequence within the target nucleic acid of Atopovium vaginae and Gardnerella vaginalis, respectively. Each of the first and second capture probe target hybridized sequences is covalently bound to a sequence or portion 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 the target sequence within 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 hybridize 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 hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO: 13.

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

[0078] Embodiment 16 is a method according to any one of Embodiments 1 to 15, wherein detection step (3) is to contact one or more amplification products with a first Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of a Lactobacillus species, a first Atopovium vaginae-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of Atopovium vaginae, and a first Gardnerella vaginalis-specific detection probe containing a target hybridize 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 Atopovium vaginae-specific detection probe, and / or a Gardnerella vaginalis-specific detection probe.

[0079] Embodiment 17 is the method of Embodiment 16, The first Atopobium vaginae-specific detection probe target hybridized sequence substantially corresponds to the nucleotide sequence of residues 6-21 of SEQ ID NO: 19, and / or The first Gardnerella vaginalis-specific detection probe target hybridize 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 Atopobium vaginae-specific detection probe target hybridized sequence includes the nucleotide sequence of residues 6-21 of SEQ ID NO: 19, and / or The first Gardnerella vaginalis-specific detection probe target hybridize sequence contains the nucleotide sequence of residues 1-18 of SEQ ID NO: 16.

[0081] Embodiment 19 is a method according to any one of Embodiments 16 to 18, wherein the first Lactobacillus-specific detection probe target hybridize sequence to the respective target regions of the target nucleic acids of Lactobacillus crispatus and Lactobacillus jensenii. The method further comprises contacting one or more amplification products with a second Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of a target nucleic acid of Lactobacillus gasseri.

[0082] Embodiment 20 is the method of Embodiment 19, The first Lactobacillus-specific detection probe target hybridized 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 hybridized sequence substantially corresponds 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 hybridized sequence includes the nucleotide sequence of residues 1-17 of SEQ ID NO: 11, and / or The second Lactobacillus-specific detection probe target hybridized sequence contains the nucleotide sequence from residues 7 to 23 of sequence number 12.

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

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

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

[0087] Embodiment 25 is the method of Embodiment 22 or 23, in which the detection step (3) occurs during the amplification step (2).

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

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

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

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

[0092] Embodiment 30 is a method according to 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 Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridized 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 Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe is a molecular torch or molecular beacon.

[0094] Embodiment 32 is the method according to 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, and the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0096] Embodiment 34 is the method of Embodiment 32 or 33, and 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 the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in a sample, the composition or kit comprising: (a) First, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 7 (iii) The third Lactobacillus-specific amplified oligomer comprises a second Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 8, and (iv) The fourth Lactobacillus-specific amplified oligomer comprises a fourth Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 9. (b) First and second Atopobium vaginae (A. vaginae) specific amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae (A. vaginae), wherein (i) the first Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a first Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and (ii) the second Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a second Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. (c) First and second Gardnerella vaginalis-specific amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first Gardnerella vaginalis-specific amplification oligomer comprises a first Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and (ii) the second Gardnerella vaginalis-specific amplification oligomer comprises a second Gardnerella vaginalis-specific target hybridize 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 hybridize sequence includes the nucleotide sequence from residues 28-45 of SEQ ID NO: 10. The second Lactobacillus-specific target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 7. The third Lactobacillus-specific target hybridize sequence contains the nucleotide sequence of Sequence ID No. 8. The fourth Lactobacillus-specific target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 9. The first Atopobium vaginae-specific target hybridize sequence includes the nucleotide sequence of residues 28-45 of SEQ ID NO: 18. The second Atopobium vaginae-specific target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 17. The first Gardnerella vaginalis-specific target hybridization sequence includes the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or The second Gardnerella vaginalis-specific target hybridize sequence contains the nucleotide sequence of Sequence ID No. 14.

[0099] Embodiment 37 is a composition or kit of Embodiment 35 or 36, The first Lactobacillus-specific target hybridize sequence consists of the nucleotide sequence from residues 28-45 of SEQ ID NO: 10. The second Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of Sequence ID No. 7. The third Lactobacillus-specific target hybridize sequence consists of the nucleotide sequence of Sequence ID No. 8. The fourth Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of Sequence ID No. 9. The first Atopobium vaginae-specific target hybridize sequence consists of the nucleotide sequence from residues 28-45 of SEQ ID NO: 18. The second Atopobium vaginae-specific target hybridization sequence consists of the nucleotide sequence of Sequence ID No. 17. The first Gardnerella vaginalis-specific target hybridize sequence consists of the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or The second Gardnerella vaginalis-specific target hybridize sequence consists of the nucleotide sequence of Sequence ID No. 14.

[0100] Embodiment 38 is a composition or kit according to any one of Embodiments 35 to 37. At least one of the first Lactobacillus-specific amplification oligomer, the first Atopobium 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 at 5' relative to its respective target hybridize sequence.

[0101] Embodiment 39 is the composition or kit of Embodiment 38, wherein the promoter sequence is the 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 amplified oligomer contains the nucleotide sequence of SEQ ID NO: 10. The first Atopobium vaginae-specific amplified oligomer contains the nucleotide sequence of SEQ ID NO: 18, and / or The first Gardnerella vaginalis-specific amplified oligomer contains the nucleotide sequence of residues 9-52 of SEQ ID NO: 15.

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

[0105] Embodiment 43 is a composition or kit of any one of Embodiments 35 to 42, further comprising at least one capture probe oligomer containing a target hybridized sequence covalently bound to a sequence or portion bound to an immobilized probe.

[0106] Embodiment 44 is the composition or kit of Embodiment 43, the composition or kit comprising first and second capture probe oligomers,

[0107] The first capture probe oligomer contains 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 contains a target hybridizing sequence that specifically hybridizes to a target sequence within the target nucleic acid of Atopovium vaginae and Gardnerella vaginalis, respectively. Each of the first and second capture probe target hybridized sequences is covalently bound to a sequence or portion 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 the target sequence within the target nucleic acids of Lactobacillus crispatus (L. crispatus), Lactobacillus jensenii (L. jensenii), and Lactobacillus gasseri (L. gasseriri).

[0109] Embodiment 46 is the composition or kit of Embodiment 45, The first capture probe target hybridize 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 hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO: 13.

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

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

[0113] Embodiment 50 is the composition or kit of Embodiment 49, The first Atopobium vaginae-specific detection probe target hybridized sequence substantially corresponds to the nucleotide sequence of residues 6-21 of SEQ ID NO: 19, and / or The first Gardnerella vaginalis-specific detection probe target hybridize 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 Atopobium vaginae-specific detection probe target hybridized sequence includes the nucleotide sequence of residues 6-21 of SEQ ID NO: 19, and / or The first Gardnerella vaginalis-specific detection probe target hybridize sequence contains the nucleotide sequence of residues 1-18 of SEQ ID NO: 16.

[0115] Embodiment 52 is a composition or kit according to any one of Embodiments 49 to 51, wherein the first Lactobacillus-specific detection probe target hybridize sequence to the respective target regions of the target nucleic acids of Lactobacillus crispatus and Lactobacillus jensenii.

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

[0117] Embodiment 53 is the composition or kit of Embodiment 52, The first Lactobacillus-specific detection probe target hybridized 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 hybridized sequence substantially corresponds 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 hybridized sequence includes the nucleotide sequence of residues 1-17 of SEQ ID NO: 11, and / or The second Lactobacillus-specific detection probe target hybridized sequence contains the nucleotide sequence from residues 7 to 23 of sequence number 12.

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

[0120] Embodiment 56 is a composition or kit according to any one of Embodiments 52 to 54, each of which includes a label: a first Lactobacillus-specific detection probe, a second Lactobacillus-specific detection probe, a first Atopovium vaginae-specific detection probe, and a first Gardnerella vaginalis-specific detection probe.

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

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

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

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

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

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

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

[0128] Embodiment 64 is a method for determining the presence or absence of bacterial vaginosis (BV) in a subject, and the method includes the following: (a) Provide samples from subjects suspected of having BV, (b) Perform assays to detect Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in the sample. (c) Assign quantitative values ​​to each of the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis based on the detection assay. (d) Subtract the quantitative value of Lactobacillus species from the larger of the quantitative values ​​of Atopobium vaginae and Gardnerella vaginalis. (e) Assigning a single BV score based on step (d), and (f) Determine the presence or absence of the target BV based on a comparison of the BV score and the cutoff value.

[0129] Embodiment 65 is the method of Embodiment 64, in which a minimum quantitative value is imposed on the Lactobacillus species in step (c).

[0130] Embodiment 66 is the method of Embodiment 64 or 65, wherein a minimum quantitative value is imposed on Atopobium vaginae and Gardnerella vaginalis in step (c).

[0131] Embodiment 67 is the method according to any one of Embodiments 64 to 66, in which the quantitative values ​​of each of the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are standardized.

[0132] Embodiment 68 is the method of Embodiment 67, in which the quantitative values ​​for each of the Lactobacillus species, Atopovium vaginae and Gardnerella vaginalis, are in log copy units, and standardization includes subtracting the median of the population values ​​from the values ​​determined from the detection assay.

[0133] Embodiment 69 is a method according to any one of Embodiments 64 to 68, in which the respective quantitative values ​​for Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are weighted.

[0134] Embodiment 70 is the method according to any one of Embodiments 64 to 69, wherein step (d) further includes adding an adjustment constant.

[0135] Embodiment 71 is a method according to any one of Embodiments 64 to 69, wherein step (d) further includes 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.

[0136] Embodiment 72 is the method of Embodiment 64, and 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 an adjustment constant, W L This is a weighting constant for Lactobacillus species, Max(L S ,F LS ) is L S and F LS It is the larger of the two, and in the formula, L S This is the observed standardized quantitative value for the Lactobacillus species, F LS This is the minimum standardized quantitative value imposed for Lactobacillus species. W GAThis is a weighting constant for Atopobium vaginae and Gardnerella vaginalis. Max(A S ,G S ,F GAS ) is A S , G S , and F GAS It is the larger of the two, and in the formula, A S This is the observed standardized quantitative value for Atopobium vaginae, G S This is the observed standardized quantitative value for Gardnerella vaginalis (G. vaginalis), and F GAS These are the smallest standardized quantitative values ​​for Atopobium vaginae and Gardnerella vaginalis. W IC This is the weighting constant of 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, F GAS It is 0.

[0138] Embodiment 74 is a method according to any one of Embodiments 64 to 73, wherein the assay for detecting Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis is a nucleic acid-based detection assay.

[0139] Embodiment 75 is the method of Embodiment 74, in which a nucleic acid-based detection assay targets 16S rRNA of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis.

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

[0141] Embodiment 77 is a method according to 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, and the amplification-based assay includes an isothermal amplification reaction.

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

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

[0145] Embodiment 81 is the method of Embodiment 79, and the nucleic acid-based detection assay is an amplification-based assay comprising the following: (1) Bring the sample into contact with the following: First, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 7 (iii) The third Lactobacillus-specific amplified oligomer comprises a substantially corresponding second Lactobacillus-specific target hybridize sequence, (iv) The fourth Lactobacillus-specific amplified oligomer comprises a fourth Lactobacillus-specific target hybridize sequence, which substantially corresponds to the nucleotide sequence of SEQ ID NO: 8. First and second Atopobium vaginae (A. vaginae) specific amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae (A. vaginae), wherein (i) the first Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a first Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and (ii) the second Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a second Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. First and second Gardnerella vaginalis-specific amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first Gardnerella vaginalis-specific amplification oligomer comprises a first Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and (ii) the second Gardnerella vaginalis-specific amplification oligomer comprises a second Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14. (2) If target nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are present in the sample, perform an in vitro nucleic acid amplification reaction using them as templates to produce one or more amplification products corresponding to the target regions of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis. (3) Detect the presence or absence of one or more amplification products.

[0146] Embodiment 82 is the method of Embodiment 81, The first Lactobacillus-specific target hybridize sequence includes the nucleotide sequence from residues 28-45 of SEQ ID NO: 10. The second Lactobacillus-specific target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 7. The third Lactobacillus-specific target hybridize sequence contains the nucleotide sequence of Sequence ID No. 8. The fourth Lactobacillus-specific target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 9. The first Atopobium vaginae-specific target hybridize sequence includes the nucleotide sequence of residues 28-45 of SEQ ID NO: 18. The second Atopobium vaginae-specific target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 17. The first Gardnerella vaginalis-specific target hybridization sequence includes the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or The second Gardnerella vaginalis-specific target hybridize sequence contains the nucleotide sequence of Sequence ID No. 14.

[0147] Embodiment 83 is the method of Embodiment 81 or 82, The first Lactobacillus-specific target hybridize sequence consists of the nucleotide sequence from residues 28-45 of SEQ ID NO: 10. The second Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of Sequence ID No. 7. The third Lactobacillus-specific target hybridize sequence consists of the nucleotide sequence of Sequence ID No. 8. The fourth Lactobacillus-specific target hybridization sequence consists of the nucleotide sequence of Sequence ID No. 9. The first Atopobium vaginae-specific target hybridize sequence consists of the nucleotide sequence from residues 28-45 of SEQ ID NO: 18. The second Atopobium vaginae-specific target hybridization sequence consists of the nucleotide sequence of Sequence ID No. 17. The first Gardnerella vaginalis-specific target hybridize sequence consists of the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or The second Gardnerella vaginalis-specific target hybridize sequence consists of the nucleotide sequence of Sequence ID No. 14.

[0148] Embodiment 84 is the method according to any one of Embodiments 81 to 83, At least one of the first Lactobacillus-specific amplification oligomer, the first Atopobium 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 at 5' relative to its respective target hybridize sequence.

[0149] Embodiment 85 is the method of Embodiment 84, and the promoter sequence is the T7 promoter sequence.

[0150] Embodiment 86 is the method of Embodiment 85, and 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 amplified oligomer contains the nucleotide sequence of SEQ ID NO: 10. The first Atopobium vaginae-specific amplified oligomer contains the nucleotide sequence of SEQ ID NO: 18, and / or The first Gardnerella vaginalis-specific amplified oligomer contains the nucleotide sequence of residues 9-52 of SEQ ID NO: 15.

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

[0153] Embodiment 89 is a method according to any one of Embodiments 81 to 88, further comprising, prior to step (2), purifying, if present, the target nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis from other components in the sample.

[0154] Embodiment 90 is the method of Embodiment 89, wherein the purification step includes contacting the sample with at least one capture probe oligomer containing a target hybridized sequence covalently bound to a sequence or portion bound to an immobilized probe.

[0155] Embodiment 91 is the method of Embodiment 90, in which the sample is contacted with first and second capture probe oligomers, The first capture probe oligomer contains 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 contains a target hybridizing sequence that specifically hybridizes to a target sequence within the target nucleic acid of Atopovium vaginae and Gardnerella vaginalis, respectively.

[0156] Each of the first and second capture probe target hybridized sequences is covalently bound to a sequence or portion 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 the target sequence within the target nucleic acids of Lactobacillus crispatus (L. crispatus), Lactobacillus jensenii (L. jensenii), and Lactobacillus gasseri (L. gasseriri).

[0158] Embodiment 93 is the method of Embodiment 92, The first capture probe target hybridize 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 hybridize sequence substantially corresponds to the nucleotide sequence of residues 1-20 of SEQ ID NO: 13.

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

[0161] Embodiment 96 is a method according to any one of Embodiments 81 to 95, wherein detection step (3) is to contact one or more amplification products with a first Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of a Lactobacillus species, a first Atopovium vaginae-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of Atopovium vaginae, and a first Gardnerella vaginalis-specific detection probe containing a target hybridize 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 Atopovium vaginae-specific detection probe, and / or a Gardnerella vaginalis-specific detection probe.

[0162] Embodiment 97 is the method of Embodiment 96, The first Atopobium vaginae-specific detection probe target hybridized sequence substantially corresponds to the nucleotide sequence of residues 6-21 of SEQ ID NO: 19, and / or The first Gardnerella vaginalis-specific detection probe target hybridize 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 Atopobium vaginae-specific detection probe target hybridized sequence includes the nucleotide sequence of residues 6-21 of SEQ ID NO: 19, and / or The first Gardnerella vaginalis-specific detection probe target hybridize sequence contains the nucleotide sequence of residues 1-18 of SEQ ID NO: 16.

[0164] Embodiment 99 is a method according to any one of Embodiments 96 to 98, wherein the first Lactobacillus-specific detection probe target hybridize sequence to the respective target regions of the target nucleic acids of Lactobacillus crispatus and Lactobacillus jensenii. The method further comprises contacting one or more amplification products with a second Lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of a target nucleic acid of Lactobacillus gasseri.

[0165] Embodiment 100 is the method of Embodiment 99, The first Lactobacillus-specific detection probe target hybridized 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 hybridized sequence substantially corresponds 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 hybridized sequence includes the nucleotide sequence of residues 1-17 of SEQ ID NO: 11, and / or The second Lactobacillus-specific detection probe target hybridized sequence contains the nucleotide sequence from residues 7 to 23 of sequence number 12.

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

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

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

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

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

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

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

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

[0175] Embodiment 110 is a method according to 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 Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridized 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 Atopovium vaginae-specific detection probe, and the first Gardnerella vaginalis-specific detection probe is a molecular torch or molecular beacon.

[0177] Embodiment 112 is the method according to 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, and the amplification reaction is a transcription-mediated amplification (TMA) reaction.

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

[0180] Embodiment 115 is a method according to any one of Embodiments 64 to 114, the method comprising the detection of 10 or fewer bacterial genera associated with BV.

[0181] Embodiment 116 is a method according to any one of Embodiments 64 to 114, the method comprising the detection of five or fewer bacterial genera associated with BV.

[0182] Embodiment 117 is a method according to any one of Embodiments 64 to 114, the method not including the detection of bacterial genera associated with BV other than Lactobacillus, Atopobium, and Gardnerella.

[0183] Embodiment 118 is a method according to any one of Embodiments 64 to 117, and if the presence of BV is indicated in the subject, the method further includes administering a treatment plan for BV to the subject.

[0184] Embodiment 119 is a method according to any one of Embodiments 64 to 117, the method being a method for monitoring a subject's BV, the subject having received a treatment plan for the BV prior to step (a).

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

[0186] Embodiment 121 is a method for determining the presence or absence of Lactobacillus species in a sample, and the method includes the following: (1) Contact a sample suspected of containing the Lactobacillus species with first, second, third, and fourth amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first amplification oligomer comprises a first target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, (ii) the amplification oligomer comprises a second target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 7, (iii) the third amplification oligomer comprises a third target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 8, and (iv) the fourth amplification oligomer comprises a fourth target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 9. (2) If the target nucleic acid of a Lactobacillus species is present in the sample, an in vitro nucleic acid amplification reaction is performed, which is used as a template to produce one or more amplification products corresponding to the target region of the Lactobacillus species. (3) Detect 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 hybridize sequence includes the nucleotide sequence from residues 28-45 of SEQ ID NO: 10. The second target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 7, The third target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 8, and / or The fourth target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 9.

[0188] Embodiment 123 is the method of Embodiment 122, The first target hybridization sequence consists of the nucleotide sequence from residues 28-45 of SEQ ID NO: 10. The second target hybridization sequence consists of the nucleotide sequence of Sequence ID No. 7. The third target hybridize sequence consists of the nucleotide sequence of SEQ ID NO: 8, and / or The fourth target hybridize sequence consists of the nucleotide sequence of sequence number 9.

[0189] Embodiment 124 is a method according to any one of Embodiments 121 to 123, wherein the first amplified oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' relative to the respective target hybridize sequence.

[0190] Embodiment 125 is the method of Embodiment 124, and the promoter sequence is the T7 promoter sequence.

[0191] Embodiment 126 is the method of Embodiment 125, and 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 amplified oligomer contains the nucleotide sequence of SEQ ID NO: 10.

[0194] Embodiment 128 is the method of any one of Embodiments 121 to 127, and further includes purifying a target nucleic acid of the Lactobacillus species from other components in the sample, if present, before step (2).

[0195] Embodiment 129 is the method of Embodiment 128, and the purification step includes contacting the sample with at least one capture probe oligomer including a target hybridizing sequence covalently bonded to a sequence or portion that binds to an immobilized probe, and the capture probe target hybridizing sequence specifically hybridizes to a target sequence within the target nucleic acid of the Lactobacillus species.

[0196] Embodiment 130 is the method of Embodiment 129, and the capture probe target hybridizing sequence specifically hybridizes to a target sequence within each of the target nucleic acids of Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus 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 to 19 of SEQ ID NO: 6.

[0198] Embodiment 132 is the method of Embodiment 131, and the capture probe oligomer includes the nucleotide sequence of SEQ ID NO: 6.

[0199] Embodiment 133 is the method of any one of Embodiments 121 to 132, and the detection step (3) includes contacting a first detection probe including a target hybridizing sequence that specifically hybridizes to a target region of the Lactobacillus species with one or more amplification products, and detecting the presence or absence of the target hybridizing detection probe.

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

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

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

[0203] Embodiment 137 is the method of Embodiment 133, and the first detection probe includes a label.

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

[0205] Embodiment 139 is the method of Embodiment 137, and the detection step (3) occurs during the amplification step (2).

[0206] Embodiment 140 is the method of Embodiment 139, and the detection probe includes a fluorescent label and a quencher.

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

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

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

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

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

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

[0213] Embodiment 147 is the method of Embodiment 133, wherein the first detection probe further comprises a non-target hybridized sequence.

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

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

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

[0217] Embodiment 151 is a method for determining the presence or absence of Atopobium vaginae (A. vaginae) in a sample, and the method includes the following. (1) Contacting a sample suspected of containing Atopobium vaginae (A. vaginae) with first and second amplification oligomers for amplifying a target region of the target nucleic acid of Atopobium vaginae (A. vaginae), wherein (i) the first amplification oligomer includes a first target hybridizing sequence substantially corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18, and (ii) the second amplification oligomer includes a second target hybridizing sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. (2) Performing an in vitro nucleic acid amplification reaction using, as a template for producing one or more amplification products corresponding to the target region of Atopobium vaginae (A. vaginae), the target nucleic acid of Atopobium vaginae (A. vaginae) if it is present in the sample. (3) Detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Atopobium vaginae (A. vaginae) in the sample. Embodiment 152 is the method of Embodiment 151, the first target hybridizing sequence includes the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18, and / or the second target hybridizing sequence includes 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 a method according to any one of Embodiments 151 to 153, wherein the first amplified oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' relative to the respective target hybridize sequence.

[0220] Embodiment 155 is the method of Embodiment 154, and the promoter sequence is the T7 promoter sequence.

[0221] Embodiment 156 is the method of Embodiment 155, and 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, wherein the first amplified oligomer contains the nucleotide sequence of SEQ ID NO: 18.

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

[0224] Embodiment 159 is the method of Embodiment 158, wherein the purification step includes contacting the sample with at least one capture probe oligomer containing a target hybridize sequence covalently bound to a sequence or portion bound to an immobilized probe, the capture probe target hybridize sequence specifically to a target sequence within the target nucleic acid of Atopovium vaginae.

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

[0226] Embodiment 161 is the method of Embodiment 160, wherein the capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 13.

[0227] Embodiment 162 is a method according to any one of Embodiments 151 to 161, wherein detection step (3) involves contacting a detection probe containing a target hybridize sequence that specifically hybridizes to a target region of Atopovium vaginae with one or more amplification products, This includes detecting the presence or absence of a target hybridize detection probe.

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

[0229] Embodiment 164 is the method of Embodiment 163, wherein the detection probe target hybridize sequence includes the nucleotide sequence of residues 6-21 of SEQ ID NO: 19.

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

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

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

[0233] Embodiment 168 is the method of Embodiment 167, wherein the detection probe includes a fluorescent label and a quencher.

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

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

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

[0237] Embodiment 172 is a method for determining the presence or absence of Gardnerella vaginalis in a sample, the method comprising the following: (1) Contact a sample suspected of containing Gardnerella vaginalis with first and second amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first amplification oligomer comprises a first target hybridize 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 hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14. (2) If the target nucleic acid of Gardnerella vaginalis is present in the sample, an in vitro nucleic acid amplification reaction is performed using it as a template to produce one or more amplification products corresponding to the target region of Gardnerella vaginalis. (3) To detect the presence or absence of one or more amplification products, thereby determining the presence or absence of Gardnerella vaginalis in the sample. Embodiment 173 is the method of Embodiment 172, The first target hybridize sequence includes the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and / or The second target hybridize sequence contains the nucleotide sequence of SEQ ID NO: 14.

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

[0239] Embodiment 175 is a method according to any one of Embodiments 172 to 174, wherein the first amplified oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' relative to the respective target hybridize sequence.

[0240] Embodiment 176 is the method of Embodiment 175, and the promoter sequence is the T7 promoter sequence.

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

[0242] Embodiment 178 is the method of Embodiment 175, wherein the first amplified oligomer contains the nucleotide sequence of residues 9-52 of SEQ ID NO: 15.

[0243] Embodiment 179 is the method of Embodiment 178, wherein the first amplified oligomer contains the nucleotide sequence of SEQ ID NO: 15.

[0244] Embodiment 180 is a method according to any one of Embodiments 172 to 179, further comprising, prior to step (2), purifying the target nucleic acid of Gardnerella vaginalis (G. vaginalis), 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 containing a target hybridize sequence covalently bound to a sequence or portion bound to an immobilized probe, the capture probe target hybridize sequence to a target sequence within the target nucleic acid of Gardnerella vaginalis (G. vaginalis).

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

[0247] Embodiment 183 is the method of Embodiment 182, wherein the capture probe oligomer contains the nucleotide sequence of SEQ ID NO: 13.

[0248] Embodiment 184 is a method according to any one of Embodiments 172 to 183, wherein detection step (3) involves contacting a detection probe containing a target hybridize sequence that specifically hybridizes to a target region of Gardnerella vaginalis with one or more amplification products, This includes detecting the presence or absence of a target hybridize detection probe.

[0249] Embodiment 185 is the method of Embodiment 184, in which the detection probe target hybridize 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, wherein the detection probe target hybridize sequence includes the nucleotide sequence of residues 1-18 of SEQ ID NO: 16.

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

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

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

[0254] Embodiment 190 is the method of Embodiment 189, in which each detection probe includes a fluorescent label and a quencher.

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

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

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

[0258] Embodiment 194 is the method according to 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, and the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0260] Embodiment 196 is the method of Embodiment 194 or 195, and 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 those generally understood by those skilled in the art relating to the methods and compositions described herein. Where used herein, the following terms and phrases have the same meanings unless otherwise specified.

[0262] The terms "a," "an," and "the" refer to multiple objects unless explicitly indicated otherwise in the context.

[0263] The conjunction "or" should be interpreted in a comprehensive sense, i.e., equivalent to "and / or," unless such a comprehensive meaning is inappropriate in the context.

[0264] The term "approximately" indicates a very small variation in the amount of a component of the composition that does not have any significant effect on the activity or stability of the composition. In some embodiments, "approximately" includes variations of 10%, 5%, 2%, 1%, or 0.5% or less of the stated value.

[0265] All ranges should be interpreted as including the endpoints unless there is an explicit exclusion such as "excluding the endpoints." Therefore, "within the range of 10 to 15" includes the values ​​10 and 15, as well as all integer and (where possible) non-integer values ​​between the endpoints, such as 11, 11.5, 12 and 1 / 3, 4π, etc.

[0266] Furthermore, the terms "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" are non-exclusive and not intended to be restrictive.

[0267] "Sample" includes any specimen that may contain Lactobacillus species, Atopobium vaginae, or Gardnerella vaginalis, or components thereof such as nucleic acids or nucleic acid fragments. The “biological sample” includes any tissue or material derived from a living or deceased human being, which may contain the vaginal cavity (or Gardnerella vaginalis) or components thereof (e.g., target nucleic acids derived therefrom), such as vaginal swab samples, cervical brush samples, respiratory tissue or exudates such as bronchoscopy, bronchoalveolar lavage (BAL) or lung biopsy, sputum, saliva, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, feces, urine, semen or other body fluids or materials. The biological sample may be processed to physically or mechanically destroy the tissue or cellular structure, thereby releasing intracellular components into a solution which may further contain enzymes, buffers, salts, detergents, etc., used to prepare the biological sample for analysis using standard methods. The sample may also include processed samples, such as those obtained by passing the sample over a filter, passing it through a filter, or following centrifugation, or by attaching it to a medium, matrix, or support.

[0268] "Nucleic acid" refers to a polymeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs having a nitrogen heterocyclic base or base analogue, where nucleosides are linked to one another by phosphodiester bonds or other bonds to form polynucleotides. Nucleic acids include RNA, DNA, or chimeric DNA-RNA polymers or oligonucleotides, and their analogues. The nucleic acid "backbone" may consist of various bonds, including one or more of sugar-phosphodiester bonds, peptide-nucleic acid bonds ("peptide nucleic acid" or PNA, see PCT No. WO95 / 32305), phosphorothioate bonds, methylphosphonate bonds, or combinations thereof. The sugar portion of a nucleic acid may be either ribose or deoxyribose, or similar compounds having known substitutions, e.g., 2'-methoxy substitutions and 2'-halide substitutions (e.g., 2'-F). Nitrogen bases include conventional bases (A, G, C, T, U) and their analogues (e.g., inosine, 5-methylisocytosine, isoguanine, The Biochemistry of the Nucleic Acid) Acids) 5-36, edited by Adams et al. 第 (11th edition, 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-purine, deaza- or aza-pyrimidine, pyrimidine bases having substituents at position 5 or 6, purine bases having modified or substituted substituents at position 2, 6, and / or 8, e.g., 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thiopyrimidine, 4-aminopyrimidine, 4-dimethylhydrazinepyrimidine, and O 4This includes alkylpyrimidines and pyrazolo compounds, such as unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines (U.S. Patent No. 5,378,825, U.S. Patent No. 6,949,367, and PCT Patent No. WO93 / 13121). Nucleic acids may contain "debased" residues in which the backbone does not contain a nitrogen base for one or more residues (U.S. Patent No. 5,585,481). Nucleic acids may contain only conventional sugars, bases, and bonds as found in RNA and DNA, or they may contain conventional components and substitutions (e.g., nucleic acids containing conventional bases bonded by a 2'-methoxy backbone, or a mixture of conventional bases and one or more base analogs). Nucleic acids may include "locked nucleic acids" (LNAs), where one or more nucleotide monomers have a bicyclic furanose unit locked to an RNA-mimicking glycospheric 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 include modified bases to alter the function or behavior of the nucleic acid, such as the addition of a 3'-terminal dideoxynucleotide to block the addition of further nucleotides to the nucleic acid. While synthetic methods for producing nucleic acids in vitro are well known in the art, nucleic acids can be purified from natural sources using conventional techniques.

[0269] As used herein, the term “polynucleotide” means a nucleic acid chain. Throughout this application, nucleic acids are indicated from the 5' end to the 3' end. Typical nucleic acids, such as DNA and RNA, are typically synthesized “from 5' to 3',” i.e., by adding nucleotides to the 3' end of an elongated nucleic acid.

[0270] As used herein, “nucleotide” refers to a nucleic acid subunit consisting of a phosphate group, a pentagonal sugar, and a nitrogen base. The pentagonal sugar found in RNA is ribose. In DNA, the pentagonal sugar is 2'-deoxyribose. The term also includes analogues of such subunits, such as the methoxy group (2'-O-Me) at the 2' position of ribose.

[0271] As used herein, “nucleic acid-based detection assay” is an assay for detecting a target sequence within a target nucleic acid, utilizing another oligonucleotide that specifically hybridizes with the target sequence.

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

[0273] In other embodiments, nucleic acid-based detection assays are “non-amplification-based assays,” i.e., assays that do not depend on any step for amplifying the nucleic acid target sequence. For clarity, nucleic acid-based detection assays that involve primer extension reactions in the absence of corresponding downstream amplified oligomers (e.g., primer extension by reverse transcriptase producing RNA:DNA double strands, followed by RNase digestion of RNA to obtain a single-stranded cDNA complementary to the RNA target, but without producing a copy of the cDNA) are understood to be non-amplification-based assays.

[0274] An exemplary non-amplification-based assay is a “cleavage-based assay,” which relies on the specific cleavage by a flap endonuclease of linear double-strand cleavage structures formed by the specific hybridization of duplicate oligonucleotides to a target nucleic acid. In these assays, a probe oligonucleotide containing a non-target hybridizing flap region is cleaved by a flap endonuclease in a duplicate-dependent manner, subsequently releasing cleavage products that are then detected. The principles of cleavage-based assays are well known in the art, and exemplary assays are described, for example, by Lyamichev et al. (Nature Biotechnology 17:292-296, 1999), Ryan et al. (Molecular Diagnostics 4:135-144, 1999), Allawi et al. (Journal of Clinical Microbiology 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, Wisconsin).

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

[0276] "Isolated" means that the sample containing the target nucleic acid is taken from its natural environment, but this 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. The “target sequence” includes complex sequences into which oligonucleotides (e.g., probe oligonucleotides, priming oligonucleotides, and / or promoter oligonucleotides) complex during a detection process (e.g., amplification-based detection assays such as TMA or PCR, or non-amplification-based detection assays such as cleavage-based assays). If the target nucleic acid is originally single-stranded, the term “target sequence” also refers to sequences 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 unrelated target nucleic acids from closely related ones.

[0278] In this specification, “target hybridizing sequence” is used to refer to an oligomeric portion configured to hybridize with a target nucleic acid sequence. Preferably, the target hybridizing sequence is configured to specifically hybridize with the target nucleic acid sequence. Target hybridizing sequences may, but may not, be 100% complementary to the portion of the target sequence to which they are configured to hybridize. Target hybridizing sequences may also include nucleotide residues that are inserted, deleted, and / or substituted relative to the target sequence. Less than 100% complementarity of the target hybridizing sequence to the target sequence may occur when the target nucleic acid is multiple strains within a species, for example, in the case of an oligomer configured to hybridize with various strains of Lactobacillus. It is understood that there may be other reasons for configuring the target hybridizing sequence to have less than 100% complementarity to the target nucleic acid.

[0279] As used herein with respect to the nucleic acid region of a Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis, the term “sequence targeting” refers to the process by which an oligonucleotide hybridizes to a target sequence in a manner that enables the detection described herein. In one embodiment, the oligonucleotide is complementary to the nucleic acid sequence of the targeted Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis and contains no mismatches. In another embodiment, the oligonucleotide is complementary but contains 1, 2, 3, 4, or 5 mismatches with the nucleic acid sequence of the targeted Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis. Preferably, the oligonucleotide that hybridizes to the target nucleic acid sequence contains at least 10 to a maximum of 50 nucleotides complementary to the target sequence. Since at least 10 and a maximum of 50 is an inclusive range, it is understood that it includes integers of 10, 50, and each in between. Preferably, the oligomer hybridizes specifically to the target sequence.

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

[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 the target region sequence of a referenced Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis. Such oligonucleotides are not limited to targeting only that sequence, but are rather useful as a composition, in a kit, or in a method for targeting the target nucleic acid of a Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis. The oligonucleotides are designed to function as components of assays for the detection of Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis from a sample, and are therefore designed to target Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis in the presence of other nucleic acids commonly found in the test sample. “Specifically hybridizes” does not mean exclusively hybridizing, as understood in the art, since some small level of hybridization to non-target nucleic acids may occur. Rather, “specifically hybridizes” means that the oligonucleotides are configured to function in assays that primarily hybridize the target, allowing for the precise detection of the target nucleic acid in the sample. The term “configured to ~” indicates the actual arrangement of the polynucleotide sequence configuration of the oligonucleotide target hybridizing sequence.

[0282] As used herein with respect to targeted nucleic acids of the Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis, the term “fragment” refers to one adjacent nucleic acid. In certain embodiments, the fragment comprises adjacent nucleotides from the 16S ribosomal RNA of the Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis, where the number of 16S adjacent nucleotides in the fragment is less than the total number of 16S nucleotides.

[0283] As used herein, the term “region” refers to a portion of a nucleic acid, which is smaller than the entire nucleic acid. For example, if the referenced nucleic acid is an oligonucleotide promoter primer, the term “region” may be used to refer to a smaller promoter portion of the entire oligonucleotide. Similarly, and only illustratively, if the nucleic acid is 16S ribosomal RNA, the term “region” may be used to refer to a smaller region of the nucleic acid, which is targeted by one or more oligonucleotides of the present invention. In another non-limiting example, if the referenced nucleic acid is an amplicon, the term “region” may be used to refer to a smaller nucleotide sequence identified for hybridization by the target hybridize sequence of the 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 and an upper limit of about 500–900 nt. In some embodiments, oligonucleotides are in a size range having a lower limit of about 12–15 nt and an upper limit of about 50–600 nt, while in other embodiments, they are in 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 the various well-known enzymatic or chemical methods. The term oligonucleotide does not indicate any particular function of a reagent, but rather is used generally to cover all such reagents described herein. Oligonucleotides can perform a variety of different functions. For example, an oligonucleotide can function as a primer if it is specific to and can hybridize to a complementary strand and can be further extended in the presence of nucleic acid polymerase; an oligonucleotide can function as a primer and provide a promoter if it contains a sequence recognized by RNA polymerase and enables transcription (e.g., a T7 primer); and an oligonucleotide can function to detect a target nucleic acid if it is capable of hybridizing to a target nucleic acid or its amplicon and further provides a detectable portion (e.g., an acridinium ester compound).

[0285] As used herein, an oligonucleotide that “substantially corresponds” to a particular reference nucleic acid sequence means that the oligonucleotide is sufficiently similar to the reference nucleic acid sequence that it has similar hybridization properties to the reference nucleic acid sequence in that it 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, yet still be able to 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, and its complements, unless otherwise explicitly indicated in the context. This variation from nucleic acids may be described by the proportion of identical bases in the sequence or the proportion of bases that are perfectly complementary between a probe or primer and its target sequence. Thus, in certain embodiments, an oligonucleotide “substantially corresponds” to a reference nucleic acid sequence if these proportions of base identity or complementarity are between 100% and about 80%. In preferred embodiments, the proportion is between 100% and about 85%. In a more preferred embodiment, the ratio is 100% to about 90%, and in another preferred embodiment, the ratio is 100% to about 95%. Similarly, a region of 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 the hybridization conditions that may be required with varying proportions of complementarity to enable hybridization to a specific target sequence without causing an unacceptable level of nonspecific hybridization.

[0286] An "amplifying oligomer" is an oligomer, or its complement, that is complementary to the target nucleic acid at least at its 3' end, hybridizes to the target nucleic acid or its complement, and participates in the nucleic acid amplification reaction. An example of an amplifying oligomer is a "primer" that contains a 3'OH end that hybridizes to the target nucleic acid and is extended by polymerase in the amplification process. Another example of an amplifying oligomer is an oligomer that is not extended by polymerase (e.g., because it has a 3' blocked end) but participates in or facilitates amplification. For example, the 5' region of an amplifying oligonucleotide may contain a promoter sequence that is non-complementary to the target nucleic acid (this may be called a "promoter primer" or "promoter provider"). Those skilled in the art will understand that an amplifying oligomer that functions as a primer may be modified to include a 5' promoter sequence and thus function as a promoter primer. Incorporating a 3' blocked end further modifies the promoter primer, which provides an upstream promoter sequence that can hybridize to the target nucleic acid and help initiate transcription, but does not provide a primer for oligo extension. Such modified oligonucleotides are referred to herein as “promoter provider” oligomers. The size range of the amplification oligonucleotides is approximately 10 nt to approximately 70 nt in length (excluding any promoter sequence or poly(A) tail) and includes at least approximately 10 consecutive bases, or at least 12 consecutive bases complementary to a region of the target nucleic acid sequence (or its complementary strand). The consecutive bases are at least 80%, at least 90%, or fully complementary to the target sequence to which the amplification oligomer binds. The amplification oligomer may contain modified nucleotides or analogues, or further nucleotides that are involved in the amplification reaction but are neither complementary to nor contained in the target nucleic acid or template sequence. For example, the amplification oligomer may contain further nucleotides at its 5' end that are complementary to its 3' end for the purpose of forming a hairpin to modulate priming efficiency (e.g., promoter primers may contain further nucleotides upstream of the promoter sequence for this purpose).When referring to a range of lengths for oligonucleotides, amplicons, or other nucleic acids, it is understood that the range includes all integers (for example, the length of 19 to 25 consecutive nucleotides includes 19, 20, 21, 22, 23, 24, and 25).

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

[0288] As used herein, “promoter provider” or “provider” refers to an oligonucleotide comprising first and second regions and modified to prevent the initiation of DNA synthesis from its 3' end. The “first region” of the promoter provider oligonucleotide contains a nucleotide sequence that hybridizes to a DNA template, the hybridizing sequence being located at 3' of the promoter region but not necessarily adjacent to it. The hybridizing portion of the promoter oligonucleotide is typically at least 10 nucleotides long and may be extended to a length of 50 nucleotides or more. The “second region” contains a promoter sequence for RNA polymerase. The promoter oligonucleotide is preferably engineered to prevent it from being extended by RNA or DNA-dependent DNA polymerases, such as reverse transcriptase, and preferably contains a blocking portion at its 3' end as described above. As referred herein, “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 fragment. These 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-exclusive examples of nucleic acid amplification methods. Replicase-mediated amplification uses a self-replicating RNA molecule and a replicase such as QB replicase (e.g., U.S. Patent No. 4,786,600). PCR amplification uses DNA polymerase, primer pairs, and a thermal cycle to synthesize multiple copies of two complementary strands of dsDNA or multiple copies from cDNA (e.g., U.S. Patents No. 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 by using multiple cycles of hybridization, ligation, and denaturation (e.g., U.S. Patent No. 5,427,930 and U.S. Patent No. 5,516,663). SDA uses primers containing a recognition site for restriction endonucleases and an endonuclease that cleaves one strand of a semi-modified DNA double helix containing the target sequence, thereby resulting in amplification in a series of primer extension and strand substitution steps (e.g., U.S. Patent No. 5,422,252, U.S. Patent No. 5,547,861, and U.S. Patent No. 5,648,211). Preferred embodiments use amplification methods suitable for amplifying RNA-targeted nucleic acids such as transcription-mediated amplification (TMA) or NASBA, but it will be apparent to those skilled in the art that the oligomers disclosed herein can be readily used as primers in other amplification methods.

[0290] Transcription-mediated amplification, also referred to herein as "transcription-mediated amplification" (TMA), refers to nucleic acid amplification that uses RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods generally use RNA polymerase, DNA polymerase, deoxyribonucleoside triphosphate, ribonucleoside triphosphate, and template complementary oligonucleotides containing a promoter sequence, and may optionally include one or more other oligonucleotides. The TMA method is an embodiment of amplification methods used to amplify and detect target sequences as described herein. Modifications of amplification related to transcription are well known in the art, as previously disclosed in detail (e.g., U.S. Patents 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 Patents 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 polymerase.

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

[0292] The term "amplicon," used interchangeably with "amplification product," refers to a nucleic acid molecule produced during the amplification procedure that is complementary or homologous to the sequence contained within the 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] The terms "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 or within amplified nucleic acid under conditions that promote hybridization, in order to enable the detection of a target sequence or amplified nucleic acid. Detection may be direct (e.g., a probe that directly hybridizes to its target sequence) or indirect (e.g., a probe ligated to its target via an intermediate molecular structure). The probe may be DNA, RNA, their analogues, or a combination thereof, and may 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 by standard base pairing. The probe may include a target-specific sequence and other sequences that contribute to the probe's three-dimensional conformation (e.g., U.S. Patents 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 20060068417). In a preferred embodiment, the detection probe includes a 2'-methoxy skeleton, 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 its 5' base and a non-fluorescent quenching dye (quencher) at its 3' base. Upon irradiation, the excited fluorescent dye transfers energy to a nearby quenching dye molecule rather than to a fluorescent substrate. During amplification, the exonuclease activity of polymerase cleaves the TaqMan probe, separating the fluorophore from the quencher, thereby releasing a non-quenching signal from the fluorophore as an indicator of amplification.

[0295] As used herein, “labeling” refers to a moiety or compound directly or indirectly bound to a probe that is detected or gives rise to a detectable signal. Direct labeling may occur through binding or interactions that link the label to the probe, including covalent or non-covalent interactions, e.g., hydrogen bonding, hydrophobic and ionic interactions, or the formation of chelates or coordination complexes. Indirect labeling may occur through the use of a cross-linking moiety or “linker,” such as a binding pair member, antibody, or further oligomer, which is labeled directly or indirectly and can amplify a detectable signal. Examples of 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), luminescent compounds (e.g., bioluminescence, phosphorescence, or chemiluminescence labeling), or fluorophores. Labeling may be detectable in homogeneous assays where a bound labeled probe in a mixture exhibits a detectable change different from an unbound labeled probe, e.g., instability or different degradation characteristics. A “homogeneous, detectable label” can be detected without physically removing the bound label from an unbound label or label probe (e.g., U.S. Patents 5,283,174, 5,656,207, and 5,658,737). Examples of labels include chemiluminescent compounds, such as standard acridinium esters ("AE") and AE compounds including derivatives (e.g., U.S. Patents 5,656,207, 5,658,737, and 5,639,604). Methods for synthesis, as well as methods for binding labels to nucleic acids and detecting labels, are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition, (Cold Spring Harbor Laboratory Publication, Cold Spring Harbor, New York, 1989), Chapter 10, and U.S. Patents No. 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, each labeled with a compound that produces a detectable signal (see, for example, U.S. Patent Nos. 6,180,340 and 6,350,579).

[0296] As used herein, a structure referred to as a “molecular torch” is designed to contain distinct, self-complementary regions (“closed domains”) that are linked by a binding region (“target-binding domain”) and hybridize with one another under given hybridization assay conditions. All or part of a nucleotide sequence containing a target closed domain may also function as a target-binding domain. Therefore, a target closed sequence may include a target-binding sequence, a non-target-binding sequence, and combinations thereof.

[0297] "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 to capture the target nucleic acid to a support. An example of a capture oligomer is an oligonucleotide comprising two binding regions: a target hybridize sequence and an immobilized probe binding region. In a variation of this example, the two regions may be located on two different oligomers linked to each other by one or more linkers. In another embodiment of the capture oligomer, the target hybridize sequence is a sequence comprising random or non-random polyGU, polyGT, or polyU sequences for non-specific binding to the target nucleic acid and for binding it to an immobilized probe on a support (see, for example, PCT WO2008 / 016988). The immobilized probe binding region may be a nucleic acid sequence called a tail. The tail consists of about 10 to 40 nucleotides (e.g., A) that bind to a complementary immobilized sequence attached to a support particle or support matrix. 10 ~A 40 ) or about 14-33 nts (e.g., T3A14 ~T3A 30 ) includes a tail that is substantially homopolymerized. Therefore, non-limiting examples of preferred nucleic acid tails include, in some embodiments, T 0~4 A 10~40 Sequences may be included. Another example of a capture oligomer includes two regions, a target hybridization 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 captured oligomer to a support. An immobilized probe bound to a support facilitates the separation of the captured probe-bound target from unbound material in a sample. One embodiment of an immobilized probe is an oligomer bound to a support that facilitates the separation of a binding target sequence from unbound material in a sample. Examples of supports include known materials such as nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane, polypropylene, metal, or matrices and particles freed in solution made from other compositions, one embodiment of which is magnetically attracted particles. The support may be a monodisperse magnetic sphere (e.g., uniform size ±5%) to which the immobilized probe is directly (via covalent bonding, chelation, or ionic interactions) or indirectly (via one or more linkers), and the binding or interaction between the probe and the support is stable under hybridization conditions. [Modes for carrying out 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-exclusive embodiments, the present invention provides methods for detecting one or more of the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in a sample, the methods comprising performing amplification-based detection of 16S rRNA-targeted nucleic acids from one or more of the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis. The present invention further provides compositions (including reaction mixtures) and kits comprising combinations of oligomers for detecting one or more of the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis. The combination of oligomers generally comprises at least two amplification oligomers for detecting one or more of the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in a sample, and may further comprise one or more of the oligomers described herein, such as capture probes and / or detection probes, for amplification-based detection of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis.

[0300] Methods for diagnosing BV generally involve detecting the presence or absence of one or more of the following species in a sample from a subject suspected of having BV: Lactobacillus, Atopovium vaginae, and Gardnerella vaginalis. Specifically, assays are performed for the specific detection of each of these species in the sample. Based on the results of the detection assay, quantitative values ​​are assigned to each of the Lactobacillus, Atopovium vaginae, and Gardnerella 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 with a cutoff value, which is usually predetermined. Generally, when a BV score exceeding a cutoff value indicates a BV-positive state, and the quantitative value is in log copies, determining the BV score involves subtracting the quantitative value of the Lactobacillus species from the larger of the values ​​for Atopovium vaginae and Gardnerella vaginalis. Determining 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 a percentage of clinical sensitivity or specificity for the infection status of BV patients established by clinical reference methods (e.g., Nugent score including intermediate, resolved by Amsel Criteria). In some embodiments, a minimum quantitative value is imposed on Lactobacillus species and / or Atopovium vaginae and Gardnerella vaginalis, and if the qualitative value measured by the detection assay is smaller than the imposed minimum value for each species, the minimum value is used as the quantitative value for determining the BV score.

[0301] The quantitative values ​​for Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis may also be standardized and / or weighted. For example, the BV score could be determined using only the maximum values ​​of these two species, and since observed Gardnerella vaginalis concentrations tend to be higher than Atopovium vaginae concentrations in the clinical sample population, standardization could be used to balance the potential influence of each quantitative value of Atopovium vaginae and Gardnerella vaginalis on the BV score calculation. In some variations, standardization involves adjusting for quantitative values ​​measured by detection assays using population statistics, such as the population median (e.g., dividing the concentration in units of copies by the population median, or subtracting the median of log copies if the quantitative data is in units of log copies). Weighting can be used to influence the effect of changes in term values ​​(e.g., quantitative values ​​for Lactobacillus species, Atopovium vaginae or Gardnerella vaginalis, or quantitative values ​​for other terms in the prediction formula) on the score value. Higher weights may result in a greater effect on the score from changes in term values, while lower weights may result in a smaller effect.

[0302] The formula for calculating the BV score may include additional terms. In some variations, the calculation of the BV score further includes adding or subtracting adjustment constants to adjust for a desired cutoff value (e.g., a cutoff value of zero). In some variations, the calculation of the BV score further includes adding an internal standard (IC) adjustment factor that may be used to compensate for sample inhibition in the detection assay. A particularly appropriate 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 a specific variation 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), The elements of the formula are as explained in Table 1. [Table 1]

[0304] In some specific embodiments of the method for diagnosing BV, in which the BV score is determined by the above formula, the term for Atopovium vaginalis (A. vaginalis) / Gardenerella vaginalis (G. vaginalis) (F GA The minimum value imposed on ) is set to zero (0) before standardization.

[0305] Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis can be detected using any suitable method, but currently, these bacteria are preferably detected using nucleic acid-based detection assays. The nucleic acid-based detection assays according to the present invention generally utilize oligonucleotides that specifically hybridize to target nucleic acids of Lactobacillus species, Atopovium vaginae, or Gardnerella vaginalis, while minimizing cross-reactivity with other nucleic acids suspected to be present in the sample.Therefore, oligonucleotides for nucleic acid-based detection of selected species of Lactobacillus, Atopobium vaginae, or Gardnerella vaginalis are used, for example, for Trichomonas, Trichomonas vaginalis, Candida, bacteria from the Clostridiales order, Clostridium-like species, Eggerthella, Enterobacteriaceae, Peptostreptococcus micros, Aerococcus christensenii, and Leptotrichia amnionyi. The cross-reactivity to species within the bacterial genera, including *Peptoniphilus*, *Dialister*, *Mycoplasma hominis*, *Sneathia sanguinegens*, *Anaerococcus tetradius*, *Mobiluncus*, *Mobiluncus hominis*, *Megasphaera*, *Prevotella*, *Leptotrichia sanguinegens*, and *Finegoldia magna*, is minimal. In one embodiment, the nucleic acid-based detection assay according to the present invention further comprises components for detecting one or more of these organisms, or other bacterial genera associated with BV.

[0306] In certain embodiments, nucleic acid-based detection assays target 16S rRNA or genes encoding 16S rRNA in Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis. Particularly suitable target regions of 16S rRNA or genes encoding it are: (i) the 16S rRNA region of Lactobacillus crispatus, corresponding to the region of SEQ ID NO: 1, from nucleotide position approximately 40 to nucleotide position approximately 265; (ii) the 16S rRNA region of Lactobacillus jensenii, corresponding to the region of SEQ ID NO: 2, from nucleotide position approximately 43 to nucleotide position approximately 247; (iii) the 16S rRNA region of Lactobacillus gasseri, corresponding to the region of SEQ ID NO: 3, from nucleotide position approximately 93 to nucleotide position approximately 298; (iv) the 16S rRNA region of Atopobium vaginae, corresponding to the region of SEQ ID NO: 4, from nucleotide position approximately 540 to nucleotide position approximately 625; and (v) the 16S region of Gardnerella vaginalis, corresponding to the region of SEQ ID NO: 5, from nucleotide position approximately 172 to nucleotide position approximately 227. This is the rRNA region.In a specific variant of the nucleic acid-based detection assay targeting the 16S rRNA region as described above, (a) the Lactobacillus-specific oligonucleotide comprises a target hybridized region including 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 residues 28-45 of SEQ ID NO: 10, a sequence substantially corresponding to residues 1-17 of SEQ ID NO: 11, or a sequence substantially corresponding to residues 7-23 of SEQ ID NO: 12, and (b) Atopobium vaginae e) The specific oligonucleotide comprises a target hybridize region including a sequence substantially corresponding to the sequence of SEQ ID NO: 17, a sequence substantially corresponding to residues 28-45 of SEQ ID NO: 18, or a sequence substantially corresponding to residues 6-21 of SEQ ID NO: 19, and / or (c) the Gardnerella vaginalis (G. vaginalis) specific oligonucleotide comprises a target hybridize region including a sequence substantially corresponding to the sequence of SEQ ID NO: 14, a sequence substantially corresponding to residues 36-52 of SEQ ID NO: 15, or a sequence substantially corresponding to residues 1-18 of SEQ ID NO: 16. In some such embodiments, (a) a Lactobacillus-specific oligonucleotide comprises a target hybridize region including or consisting of the sequence of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, the sequence of residues 28-45 of SEQ ID NO: 10, the sequence of residues 1-17 of SEQ ID NO: 11, or the sequence of residues 7-23 of SEQ ID NO: 12; (b) an Atopobium vaginae-specific oligonucleotide comprises a target hybridize region including or consisting of the sequence of SEQ ID NO: 17, the sequence of residues 28-45 of SEQ ID NO: 18, or the sequence of residues 6-21 of SEQ ID NO: 19; and / or (c) a Gardnerella vaginalis-specific oligonucleotide comprises a target hybridize region including or consisting of the sequence of SEQ ID NO: 14, the sequence of residues 36-52 of SEQ ID NO: 15, or the sequence of 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 Atopobium 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 the method, which involves the use of nucleic acid-based detection assays, amplification-based assays are used to detect Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis. Such variations generally involve amplifying a target sequence in a bacterial target nucleic acid utilizing an in vitro nucleic acid amplification reaction, and detecting the amplification product by specifically hybridizing the amplification product with a nucleic acid detection probe that provides a signal indicating the presence of the bacterial target in the sample. The amplification step involves contacting the sample with two or more amplification oligomers specific to a target sequence in the target nucleic acid (e.g., a target sequence in 16S rRNA) to produce an amplification product, provided the target nucleic acid is present in the sample. Amplification synthesizes additional copies of the target sequence or its complement by, for example, using at least one nucleic acid polymerase and a template strand to extend the sequence from the amplification oligomers (primers). One embodiment for detecting amplification products involves a hybridization step that includes contacting the amplification product with at least one probe specific to a sequence amplified by a selected amplification oligomer, for example, a sequence contained in a target sequence adjacent to a pair of selected amplification oligomers. 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-related amplification (TMA). Such amplification methods are well known in the art (see, for example, the description of amplification methods in the definition section above) and are readily used by the method of the present invention.

[0308] For example, several amplification methods using TMA amplification involve the following steps. Briefly, the target nucleic acid containing the 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 thawing 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 double strand. RNase digests the RNA strand of the RNA:DNA double strand, and a second primer specifically binds to its target sequence located on the cDNA strand downstream from the promoter primer end. RT synthesizes a new DNA strand by extending the 3' end of the second primer using the first cDNA template to produce a dsDNA containing the functional promoter sequence. Next, RNA polymerase specific to the promoter sequence initiates transcription, producing an RNA transcript that is approximately 100–1000 amplified copies ("amplicons") of the initial target strand during the reaction. Amplification continues as a second primer specifically binds to its target sequence in each amplicon, and the RT generates DNA copies from the amplicon RNA template to produce an RNA:DNA double strand. RNase in the reaction mixture digests the amplicon RNA from the RNA:DNA double strand, and the promoter primer specifically binds to its complementary sequence in the newly synthesized DNA. The RT extends the 3' end of the promoter primer, generating dsDNA containing a functional promoter to which RNA polymerase binds and transcribes further amplicons complementary to the target strand. This autocatalytic cycle, which generates more amplicon copies, is repeated during the reaction, resulting in an amplification of approximately 1 billion times the target nucleic acid present in the sample. The amplification product 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 sequences contained in the amplification product. Detection of the signal emanating from the binding 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 variant forms, the initiation 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. A second or “reverse” amplification oligomer is a promoter primer or promoter provider having a target hybridize 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 produce a second cDNA copy of the target sequence strand, thereby producing a dsDNA containing the functional promoter sequence. Amplification then essentially continues as described above for transcription initiation from the promoter sequence utilizing RNA polymerase. Alternatively, if the second amplification oligomer is a promoter provider, a termination oligonucleotide that hybridizes to a target sequence near the 5' end of the target region is typically used to terminate the elongation of the priming oligomer at its 3' end, thereby providing a defined 3' end for the initial cDNA strand synthesized by elongation from the priming oligomer. The target hybridization 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 elongated to append 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, which does not contain the promoter portion, using an RNA polymerase that recognizes the double-stranded promoter and then initiates transcription. Each of these RNA transcripts is then available to serve as a template for further amplification from the first priming amplification oligomer.

[0310] Therefore, in certain embodiments including amplification-based detection assays, a combination of at least two amplification oligomers is used to detect 16S rRNA of Lactobacillus species or genes encoding 16S rRNA of Lactobacillus species. The oligomer combination may include first and second amplification oligomers for amplifying the 16S rRNA region of Lactobacillus crispatus (L. crispatus) corresponding to the region of SEQ ID NO: 1 from nucleotide position approximately 40 to nucleotide position approximately 265, the 16S rRNA region of Lactobacillus jensenii (L. jensenii) corresponding to the region of SEQ ID NO: 2 from nucleotide position approximately 43 to nucleotide position approximately 247, and / or the 16S rRNA region of Lactobacillus gasseri (L. gasserii) corresponding to the region of SEQ ID NO: 3 from nucleotide position approximately 93 to nucleotide position approximately 298. For example, in some embodiments, the first amplification oligomer comprises a target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and the second amplification oligomer comprises a target hybridize sequence substantially corresponding to the nucleotides of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9. In some such variations, the first amplification oligomer comprises a target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and the second amplification oligomer comprises a target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 7. The oligomer combination further comprises third and fourth amplification oligomers, the third amplification oligomer comprising a target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 8, and the fourth amplification oligomer comprising a target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 9.In more specific modifications, the first amplified oligomer includes the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, or a target hybridize sequence consisting thereof; the second amplified oligomer includes the nucleotide sequence of SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or a target hybridize sequence consisting thereof; in some such embodiments, the first amplified oligomer includes a target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10; the second amplified oligomer includes a target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 7; and the oligomer combination further includes third and fourth amplified oligomers, the third amplified oligomer includes the nucleotide sequence of SEQ ID NO: 8, or a target hybridize sequence consisting thereof; and the fourth amplified oligomer includes the nucleotide sequence of SEQ ID NO: 9, or a target hybridize sequence consisting thereof. In some embodiments as described above, at least one amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' of each target hybridize sequence (e.g., a T7 promoter sequence, such as the nucleotide sequence of residues 1-27 of SEQ ID NO: 10), and in some such embodiments, the first amplification oligomer is a promoter primer or promoter provider. In a more specific modification, 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, SEQ ID NO: 8, or SEQ ID NO: 9, and 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 third and fourth amplification oligomers, the third amplification oligomer consisting of the nucleotide sequence of SEQ ID NO: 8, and the fourth amplification oligomer consisting of the nucleotide sequence of SEQ ID NO: 9.

[0311] In some embodiments, including amplification-based detection assays, a combination of at least two amplification oligomers is used to detect Atopobium vaginae 16S rRNA or the gene encoding Atopobium vaginae 16S rRNA. The oligomer combination may include first and second amplification oligomers for amplifying the nucleic acid target region of Atopobium vaginae corresponding to the region of SEQ ID NO: 4 from nucleotide position approximately 540 to nucleotide position approximately 625. For example, in some embodiments, the first amplification oligomer includes a target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17, and / or the second amplification oligomer includes a target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18. In a more specific modification, the first amplification oligomer comprises a target hybridize sequence containing or consisting of the nucleotide sequence of SEQ ID NO: 17, and / or the second amplification oligomer comprises a target hybridize sequence containing 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 at 5' of each target hybridize 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 a more specific modification, 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, including amplification-based detection assays, a combination of at least two amplification oligomers is used to detect 16S rRNA of Gardnerella vaginalis (G. vaginalis) or the gene encoding 16S rRNA of Gardnerella vaginalis (G. vaginalis). The oligomer combination may include first and second amplification oligomers for amplifying the nucleic acid target region of Gardnerella vaginalis (G. vaginalis) corresponding to the region of SEQ ID NO: 5, from nucleotide position approximately 172 to nucleotide position approximately 227. For example, in some embodiments, the first amplification oligomer includes a target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14, and / or the second amplification oligomer includes a target hybridize sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO: 15. In a more specific modification, the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 14 or a target hybridize sequence consisting thereof, and / or the second amplification oligomer comprises the nucleotide sequence of residues 36-52 of SEQ ID NO: 15 or a target hybridize sequence consisting thereof. In some embodiments as described above, at least one amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' of each target hybridize 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 a more specific modification, 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 amplification target sequence. Nucleic acids may associate with surfaces that produce physical changes, such as detectable electrical changes. Amplified nucleic acids can be detected by concentrating them in or on a matrix and detecting the nucleic acid or a nucleic acid-associated dye (e.g., an insert agent such as ethidium bromide or Cybergreen), or by detecting an increase in the nucleic acid-associated dye in the solution phase. Other detection methods may use nucleic acid detection probes configured to specifically hybridize with sequences in the amplification product and detect the presence of a probe:product complex, or use probe complexes capable of amplifying a detectable signal that associates with the amplification product (e.g., U.S. Patents 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 the 16S rRNA of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis, the amplification product will contain the target sequence within the 16S rRNA or a sequence complementary to the sequence within the 16S rRNA, and the probe will bind directly or indirectly to the sequence contained in the amplification product to indicate the presence of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis 16S rRNA in the test sample.

[0314] The detection probe that hybridizes to a 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, for example, an oligomer containing one or more 2'-methoxysubstituted ribonucleotides. The probe used to detect the amplified sequence may be detected indirectly and unlabeled (e.g., by the binding of another binding partner to a portion on the probe), or it may be labeled with various detectable labels. In some embodiments of the method 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 may also provide further information about the amplified sequence, such as all or part of its nucleic acid base sequence. Detection may be performed after the amplification reaction is complete or simultaneously with the amplification of the target region, for example, in real time. In one embodiment, the detection step enables homogeneous detection, such as detection of hybridized probes without removing non-hybridized probes from the mixture (see, for example, U.S. Patents 5,639,604 and 5,283,174).

[0316] In embodiments where the amplification product is detected near or at the end of the amplification step, a linear detection probe can be used to provide a signal and indicate hybridization of the probe to the amplification product. One example of such detection uses a luminescently labeled probe that hybridizes to a target nucleic acid. The luminescent label is then hydrolyzed from the non-hybridizing probe. Detection is performed by chemiluminescence using a luminometer (see, for example, International Patent Application Publication WO89 / 002476, incorporated herein by reference). In other embodiments using real-time detection, the detection probe may be a hairpin probe such as a molecular beacon, molecular torch, or hybridization switch probe, which is labeled with a reporter portion that is detected when bound to the amplified product. Such a probe may include a target hybridizing sequence and a non-target hybridizing sequence. Various forms of such probes have been previously described (see, for example, U.S. Patents No. 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 No. 20060068417A1, and U.S. Patent Application Publication No. 20060194240A1, respectively, which are incorporated herein by reference).

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

[0318] In some embodiments of the method, which involves the use of nucleic acid-based detection assays, non-amplified assays are used to detect Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis. In some such embodiments, the non-amplified assay is a hybridization assay that involves hybridization of a specific detection probe to a target nucleic acid. Methods for carrying out polynucleotide hybridization assays have been well developed in the art. The procedures and conditions for hybridization assays vary depending on the application and are selected according to known common binding methods, including, for example, Maniatis et al., Molecular Cloning: A Laboratory Manual (3rd edition, Cold Spring Harbor, New York, 2002), and Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, San Diego, California, 1987). Generally, the probe and sample are mixed under conditions that allow for specific nucleic acid hybridization, and then the specific hybridization of the probe to each target is detected. Nucleic acid hybridization is adaptable to various assay formats. One suitable format is the sandwich assay format, which is particularly suitable for hybridization under non-denaturing conditions. The main component of a sandwich assay is a solid support adsorbed or covalently bound to an immobilized nucleic acid probe that is unlabeled and complementary to a portion of the DNA sequence. 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 that the immobilized unlabeled nucleic acid probe hybridizes to the [target nucleic acid]:[immobilized probe] double strand to detect the target nucleic acid.Another exemplary form utilizes the electrochemical detection of a target nucleic acid hybridized to an unlabeled detection probe immobilized on a suitable electrode surface, acting as a signal transducer. See, for example, Drummond et al., Nature Biotechnology 21:1192, 2003; Gooding, Electroanalysis 14:1149, 2002; Wang, Analytica 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, including hybridization assays, the detection probe is used to detect 16S rRNA of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis, or genes encoding 16S rRNA of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis. In some such embodiments, (A) a probe for detecting the 16S rRNA of a Lactobacillus species, or a gene encoding the 16S rRNA of a Lactobacillus species, specifically hybridizes to (1) a nucleic acid target region corresponding to the region of SEQ ID NO: 1 from nucleotide position approximately 40 to nucleotide position approximately 265, (2) a nucleic acid target region corresponding to the region of SEQ ID NO: 2 from nucleotide position approximately 43 to nucleotide position approximately 247, and / or (3) a nucleic acid target region corresponding to the region of SEQ ID NO: 3 from nucleotide position approximately 93 to nucleotide position approximately 298, and (B) the 16S rRNA of Atopobium vaginae, or the 16S of Atopobium vaginae A probe for detecting the gene encoding rRNA specifically hybridizes to the nucleic acid target region corresponding to the region of SEQ ID NO: 4, from nucleotide position approximately 540 to nucleotide position approximately 625, and / or (C) Gardnerella vaginalis 16S rRNA, or a probe for detecting the gene encoding Gardnerella vaginalis 16S rRNA specifically hybridizes to the nucleic acid target region corresponding to the region of SEQ ID NO: 5, from nucleotide position approximately 172 to nucleotide position approximately 227.For example, in some variations, the first probe for detecting the 16S rRNA of Lactobacillus species, or the gene encoding the 16S rRNA of Lactobacillus species, contains a target hybridize sequence that specifically hybridizes to the target region of Lactobacillus crispatus and Lactobacillus jensenii, respectively, and contains the 16S rRNA of Lactobacillus species, or the 16S A second probe for detecting the gene encoding rRNA comprises a target hybridize sequence that specifically hybridizes to a target region of the target nucleic acid of Lactobacillus gasseri, and in some such embodiments, the first Lactobacillus-specific detection probe comprises a target hybridize 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 hybridize sequence substantially corresponding to the sequence of residues 7-23 of SEQ ID NO: 12 (e.g., the first probe comprising the target hybridize sequence of residues 1-17 of SEQ ID NO: 11, and / or the second probe comprising the target hybridize sequence of residues 7-23 of SEQ ID NO: 12). In some variations, a probe for detecting the 16S rRNA of Atopobium vaginae, or the gene encoding the 16S rRNA of Atopobium vaginae, contains a target hybridize sequence substantially corresponding to residues 6-21 of SEQ ID NO: 19 (e.g., a probe containing the target hybridize sequence of residues 6-21 of SEQ ID NO: 19). In some variations, a probe for detecting the 16S rRNA of Gardnerella vaginalis, or the gene encoding the 16S rRNA of Gardnerella vaginalis, contains a target hybridize sequence substantially corresponding to residues 1-18 of SEQ ID NO: 16 (e.g., a probe containing the target hybridize sequence of residues 1-18 of SEQ ID NO: 16).In certain embodiments, a first probe for detecting 16S rRNA of a Lactobacillus species, or a gene encoding 16S rRNA of a Lactobacillus species, includes or comprises the sequence of SEQ ID NO: 11; a second probe for detecting 16S rRNA of a Lactobacillus species, or a gene encoding 16S rRNA of a Lactobacillus species, includes or comprises the sequence of SEQ ID NO: 12; a probe for detecting 16S rRNA of Atopovium vaginae, or a gene encoding 16S rRNA of Atopovium vaginae, includes or comprises the sequence of SEQ ID NO: 19; and / or 16S rRNA of Gardnerella vaginalis. A probe for detecting rRNA, or the gene encoding the 16S rRNA of Gardnerella vaginalis (G. vaginalis), contains or consists of the sequence of Sequence ID No. 16.

[0320] In some embodiments, non-amplification-based assays for detecting Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis are cleavage-based assays in which a probe oligonucleotide containing a non-target hybridizing flap region is cleaved in a duplication-dependent manner by a flap endonuclease, subsequently releasing the cleavage product to be detected. Exemplary cleavage-based assay reagents are described, for example, by Lyamichev et al. (Nature Biotechnology 17:292-296, 1999), Ryan et al. (Molecular Diagnostics 4:135-144, 1999), and Allawi et al. (Journal of Clinical Microbiology 44:3443-3447, 2006). Suitable conditions for flap endonuclease reactions are known or can be readily determined using methods well known in the art (see, for example, Kaiser et al., Journal of Biological Chemistry 274:2138-721394, 1999). Examples of flap-end nucleases that can be used in this 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, and Thermus thermophilus. Examples include thermophilus)FEN-1, CLEAVASE® (Hologic, Madison, Wisconsin), Saccharomyces cerevisiae RTH1, Saccharomyces cerevisiae RAD27, Schizosaccharomyces pombe rad2, bacteriophage T5 5'-3' exonuclease, Pyrococcus horikoshii FEN-1, human endonuclease 1, calf thymus 5'-3' exonuclease, their congeners in bacteria, eukaryotes, and archaea, such as members of the class II family of structure-specific enzymes, as well as their enzymatically active variants or modifiers. For explanations of flap endonucleases, see, for example, Lyamichev et al., Science 260:778-783, 1993; Eis et al., Nature Biotechnology 19:673-676, 2001; Shen et al., Trends in Bio.Sci. 23:171-173, 1998; Kaiser et al., Journal of Biological Chemistry 274:21387-21394, 1999; Ma et al., Journal of Biological Chemistry 275:24693-24700, 2000; Allawi et al., Journal of Molecular Biology. This can be found in J.Mol.Biol.)328:537-554, 2003, Sharma et al., Journal of Biological Chemistry (J.Biol.Chem.)278:23487-23496, 2003, and Feng et al., Nature Structural and Molecular Biology (Nat.Struct.Mol.Biol.)11:450-456, 2004.

[0321] In certain modifications, the cleavage-based assay detects RNA target nucleic acids of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis, and the cleavage-based assay utilizes flap endonucleases that can cleave RNA:DNA linear double-stranded structures. In some alternative embodiments, the cleavage-based assay detects DNA target nucleic acids of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis, and the cleavage-based assay utilizes flap endonucleases that can cleave DNA:DNA linear double-stranded structures. Exemplary flap-end nucleases capable of cleaving RNA:DNA double strands include Thermus polymerase-deficient 5' nucleases, as well as specific CLEAVASE® enzymes (Hologic, Madison, Wisconsin), such as CLEAVASE® BN (BstX-NotI deletion of Taq polymerase, see U.S. Patent No. 5,614,402), CLEAVASE® II ("AG" mutant of full-length Taq polymerase, see U.S. Patent No. 5,614,402), CLEAVASE® VII (synthesis-deficient mutant of full-length Thermus thermophilus polymerase), CLEAVASE® IX (polymerase-deficient mutant of TthDNA polymerase), and CLEAVASE® XII (polymerase-deficient chimeric polymerase constructed from fragments of taqDNA polymerase and TthDNA polymerase).DNA: Exemplary flap endonucleases capable of cleaving DNA double strands include the flap endonucleases listed above, as well as CLEAVASE® 2.0 (Archaeoglobus fulgidus FEN-1), CLEAVASE® 2.1 (Archaeoglobus fulgidus FEN-1 with six histidines at the C-terminus), CLEAVASE® 3.0 (Archaeoglobus veneficus FEN-1), and CLEAVASE® 3.1 (Archaeoglobus veneficus FEN-1 with six histidines at the C-terminus).

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

[0323] In certain embodiments utilizing nucleic acid-based detection assays, the method further includes purifying target nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis from other components in the sample. Such purification may include methods for separating and / or concentrating organisms contained in the sample from other sample components. In certain embodiments, purifying target nucleic acids includes capturing the target nucleic acids to specifically or nonspecifically separate them from other sample components. Nonspecific target capture methods may involve selective precipitation of nucleic acids from a substantially aqueous mixture, adhesion of nucleic acids to a support that is washed to remove other sample components, or other means of physically separating nucleic acids from a mixture containing nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis 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, Atopovium vaginae, and Gardnerella vaginalis are isolated from other sample components by hybridizing the target nucleic acid to a capture probe oligomer. The capture probe oligomer contains a target hybridize sequence configured to hybridize specifically or nonspecificly with the target nucleic acid to form a [target nucleic acid]:[capture probe] complex isolated from other sample components. Capture probes containing target hybridize sequences suitable for nonspecific capture of target nucleic acids are described, for example, in International PCT Publication WO2008 / 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 containing a target hybridize sequence that specifically hybridizes to a target sequence in the target nucleic acid of a Lactobacillus species (e.g., a target hybridize sequence that specifically hybridizes to a target sequence in the target nucleic acids of Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri, respectively), and (ii) a second capture probe oligomer containing a target hybridize sequence that specifically hybridizes to a target sequence in the target nucleic acids of Atopovium vaginae and Gardnerella vaginalis, respectively.In some specific modifications, including the first and second capture probe oligomers of (i) and (ii) above, the first capture probe oligomer contains a target hybridize sequence substantially corresponding to the nucleotide sequence of residues 1-19 of SEQ ID NO: 6 (e.g., a capture probe containing the target hybridize sequence of residues 1-19 of SEQ ID NO: 6), and / or the second capture probe oligomer contains a target hybridize sequence substantially corresponding to the nucleotide sequence of residues 1-20 of SEQ ID NO: 13 (e.g., a capture probe containing the target hybridize sequence of residues 1-20 of SEQ ID NO: 13). In preferred modifications, the capture probe is conjugated to an immobilized probe to form a [target nucleic acid]:[capture probe]:[immobilized probe] complex, which is then separated from the sample and optionally washed to remove non-target sample components (see, for example, U.S. Patent Nos. 6,110,678, 6,280,952, and 6,534,273). In such modifications, the capture probe oligomer further includes a sequence or portion that allows the capture probe, along with its bound target sequence, to be attached to an immobilized probe bound to a solid support, thereby enabling 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 specifically hybridizes to the sequence on the immobilized probe, as previously described, for example, in U.S. Patent No. 6,110,678 incorporated herein by reference, and thereby enables the target nucleic acid to be separated from other sample components. Any sequence can be used in a tail region that 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 14nt to about 33nt (for example, A 14 ~A 30 Or T3A 14 ~T3A 30It includes a substantially homopolymerized tail, which binds to a solid support, such as a matrix or a complementary immobilized sequence (e.g., poly-T) bound to particles. In some such embodiments, the first capture probe oligomer comprises (i) a first capture probe oligomer comprising a target hybridize sequence that specifically hybridizes to a target sequence within each of the 16S rRNA target nucleic acids of Lactobacillus crispatus, Lactobacillus jensenii, and Lactobacillus gasseri, and / or (ii) a second capture probe oligomer comprising a target hybridize sequence that specifically hybridizes to a target sequence within each of the 16S rRNA target nucleic acids of Atopovium vaginae and Gardnerella vaginalis, wherein 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 with a double-stranded T[tail sequence]:[immobilized probe sequence]. mThe process occurs at a temperature higher than that in a liquid-phase mixture containing one or more capture probe oligomers that hybridize to the target nucleic acid. 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 then the entire complex on the solid support is separated from other sample components. The support having the bound [immobilized probe]:[capture probe]:[target nucleic acid] may be washed once or multiple times to further remove sample components. A preferred embodiment uses a particulate solid support such as paramagnetic beads, thereby allowing particles having the bound [target nucleic acid]:[capture probe]:[immobilized probe] complex to be suspended in a washing solution and recovered from the washing solution, preferably by using magnetic attraction. In embodiments of the method including the use of an amplification-based detection assay, the target nucleic acid can be amplified by simply mixing the target nucleic acid in the complex on the support with an amplification oligomer and proceeding with the amplification step, in order to limit the number of handling steps.

[0327] In some embodiments of a method for diagnosing BV, in which the detection of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis indicates BV in a subject, the method further includes treating the BV in the subject. Treatment plans for BV are generally known in the art and include, for example, the administration of antibiotics such as metronidazole (e.g., Flagyl, METROGEL-VAGINAL), clindamycin (e.g., Cleosin, CLINDESSE), and tinidazole (e.g., TINDAMAX). In certain modifications, the subject has not been previously diagnosed with BV. In other embodiments, the subject has been previously diagnosed with BV and is receiving treatment for BV at the time the diagnostic method of this disclosure is performed. Such modifications are particularly useful for monitoring the treatment of BV in a subject. For example, if the method indicates that babesiosis is still present in the subject, the subject may continue treatment. In some embodiments, the same treatment plan (i.e., the same treatment the subject was receiving at the time of the diagnostic procedure) is re-administered to the subject. Alternatively, the continued presence of BV in a treated subject may indicate that a change in the ongoing treatment is necessary, and a different treatment plan (e.g., a different drug therapy, or an increase in the dose and / or frequency of the drug) is administered to the subject.

[0328] According to the present invention, the detection of the presence or absence of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis can be carried out separately for each target (e.g., sequentially or in parallel in separate reaction vessels) or together as a multiplex reaction system. Accordingly, in some embodiments, the methods described herein (e.g., methods for diagnosing BV) utilize a multiplex reaction, and the reaction mixture comprises 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 comprise multiple different target-specific oligonucleotides for performing detection assays. For example, in a method utilizing an amplification-based detection assay, the multiplex reaction may comprise multiple sets (e.g., multiple pairs) of amplified oligomers (e.g., multiple pairs of PCR primers or multiple pairs of TMA amplified oligomers (e.g., multiple pairs of promoter primers and non-promoter primers, or multiple pairs of promoter providers and non-promoter primers for TMA)). In other embodiments utilizing a cleavage-based detection assay, the multiplex reaction may include multiple probe oligonucleotides having different flaps, multiple different duplicate probe oligonucleotides, and multiple different FRET cassettes for detecting different flaps once the different flaps are cleaved.

[0329] Further microbial detection assays can be performed to determine the presence and / or relative amounts of multiple microorganisms associated with BV. As just one example, such multiple microorganisms include one or more anaerobic Gram-positive cocci, Eggerthella species, bacteria from the Clostridiales order, Clostridium-like species, Enterobacteriaceae, Peptostreptococcus micros, Aerococcus christensenii, Leptotrichia amnionii, Peptoniphilus species, Dialister species, Mycoplasma hominis, Sneathia sanguinegens, and Anaerococcus tetrazius. Examples include *Tetradius*, *Mobiluncus* species, *Mobiluncus hominis*, *Eggerthella hongkongensis*, *Prevotella* species, *Megasphaera* species, *Leptotrichia sanguinegens*, and *Finegoldia magna*. The assays may be performed individually or in combination. Therefore, the diagnosis of BV may involve identifying multiple microorganisms and, if applicable, determining their relative abundance in the sample.

[0330] In certain embodiments, the method for diagnosing BV includes the detection of 10 or fewer bacterial genera associated with BV. In other embodiments, the method includes 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 include the detection of bacterial genera associated with BV other than Lactobacillus, Atopobium, and Gardnerella.

[0331] Furthermore, the present invention provides oligomers or combinations thereof for determining the presence or absence of one or more Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in a sample. In various embodiments, the oligomers or combinations thereof include oligomers as described herein for methods of determining the presence or absence of Lactobacillus species, Atopovium vaginae, and / or Gardnerella 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 Atopobium vaginae-specific oligonucleotide (e.g., at least two or three Atopobium 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 Gardnerella 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 Atopobium vaginae-specific oligonucleotides (e.g., at least three Atopobium vaginae-specific oligonucleotides, each binding to a different target sequence), and / or at least two Gardnerella vaginalis-specific oligonucleotides (e.g., at least three Gardnerella 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 the nucleic acid target region of Lactobacillus species, at least two A. vaginae-specific amplification oligonucleotides for amplifying the nucleic acid target region of Atopovium vaginae, and / or at least two G. vaginalis-specific amplification oligonucleotides for amplifying the nucleic acid target region of G. vaginalis. In some embodiments, the composition of the present invention comprises one or more detection probe oligomers for detecting the target nucleic acid of Lactobacillus species, the target nucleic acid of Atopovium vaginae, and / or the target nucleic acid of G. vaginalis. 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 contain many optional components, such as, for example, capture probe nucleic acids or arrays of capture probe nucleic acids.For amplification reaction mixtures, the reaction mixture typically includes, for example, a buffer, a salt solution, suitable 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, which may or may not contain the target nucleic acids of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis. Kits containing oligomer combinations for amplifying one or more target nucleic acid regions of Lactobacillus species, Atopovium vaginae, and / or Gardnerella vaginalis may also contain other reagents suitable for in vitro amplification, such as buffers, salt solutions, suitable 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) containing combinations of amplification oligomers targeting a common target nucleic acid together with a detection probe, the selection of amplification oligomers and detection probe oligomers is related by a common target region (i.e., the combination includes a probe that binds to a sequence that can be amplified by the combination of amplification oligomers).

[0332] The present invention can be further illustrated by the following non-limiting examples. Example 1: BV Multivariate Algorithm 1. Summary of result interpretation

[0333] To determine the BV positive or BV negative state, quantitative information on Lactobacillus species ("L.spp", negatively correlated with BV) as well as Gardnerella vaginalis and Atopobium vaginae (respectively "Gvag" and "Avag", 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 a negative call below the cutoff and a positive call 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 larger of the Gvag or Avag values. Simplified score = max(Gvag, Avag) - L.spp

[0335] As actually applied to the interpretation of APTIMA BV results, the equation becomes more complex. A negative constant is added (to set the score to zero at the clinical decision point), additional coefficients (IC terms) are 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] Equation applied: Score = C0 + W L *Max(L - M L , F L - M L ) + W AG *Max(G - M G , A - M A , F GA - M G ) + WIC * log2(IC / calIC)

[0337] 2. Details of the interpretation of the results The interpretation of a sample consists of evaluating the validity criteria, calculating the BV score, and interpreting the score. This example begins with the TTime value of the internal standard (IC) signal and the log copy / mL quantified values ​​of L.spp, Gvag, and Avag (L, G, A) for each sample being evaluated.

[0338] 2.1 Calculation of BV Score For samples that meet the validity criteria (not described here), calculate the BV score. The calculation of the BV score consists of three steps: 1) standardization of the analyte, 2) calculation of the IC ratio, and 3) application of the general equation. 2.1.1 Standardization of analytes

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

[0340] Standardization serves two main functions. 1) (As described above) the number of concentrations is normalized, 2) Improve the confidence interval of the trained constant.

[0341] This standardization format applies to all terms present in the log copy unit. This includes the three sample analytes (L, G, A) and the baseline value F. L and F GA This includes the population median (M) before using each of these in a linear prediction formula. L M G M A It is standardized by subtracting ). The standardized value is denoted by the subscript "S" 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 to compensate for sample inhibition in order to reduce the likelihood of false negative calls. The IC ratio is the observed value of the internal standard (IC) TTime divided by the expected value of IC TTime. If the reaction is inhibited (the reaction is slower than expected), the IC ratio is greater than 1. The expected IC TTime 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 a constant ICd. The default IC TTime can be calculated as the average observed IC TTime of valid KitCalibrator replicates multiplied by ICd. An IC TTime that does not fall below the default IC TTime uses ICd as the IC ratio. IC ratio = Observed IC TTime Maximum IC ratio = ICd Expected IC TTime

[0344] 2.1.3 General Equation The equation encoded in the PANTHER (registered trademark) software to interpret the results of APTIMA (registered trademark) BV is called the general equation. In this equation, the subscript "S" refers to the value standardized by subtracting the median of the relevant analyte population. 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 the algorithm was completed. Next, W A and W G We set both to zero. Therefore, the (applied) equation can be simplified as follows: 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 up four terms to create the score. [Table 3]

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

[0348] 3. Application of multivariate algorithms to the interpretation of clinical sample results As shown in Table 4, to interpret the clinical outcomes of BV, the “true outcome” was determined by the Nugent score using the intermediate level determined by the Amsel Criteria. [Table 4]

[0349] Table 5 shows the constants used in the algorithm, along with the numerical values ​​used in this analysis. [Table 5]

[0350] Two sets of data were collected from the BV RT-TMA PANTHER trial results of the APTIMA product under development. The training set consisted of 1204 trial results, one trial per subject. This dataset was used to derive the constant values ​​shown in Table 5. The validation set consisted of 1076 trial results, one trial per subject. The subject populations were identical, but there were no common trial results between the training and validation sets. (As used herein, “training set” and “validation set” refer to sets of clinical specimens tested with the same assay design to optimize the algorithm for making clinical calls (positive or negative). The training set is the set of specimens used to design a provisional algorithm, and the validation set is a separate set of specimens to test the accuracy of the algorithm’s performance.)

[0351] Using the constants in Table 5 in the general equation, we can obtain estimated values ​​for the clinical outcomes (sensitivity / specificity) of the training and validation sets shown in Table 6. [Table 6]

[0352] BV can manifest differently in 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 (Black), Non-Hispanic White (White-NH), Hispanic White (White-Hisp), Asian (Asian), and Others. The Others category consists of Islanders, Indians, Middle Easterners, Native Americans, and other, unknown, or mixed-race individuals. [Table 7] [Table 8]

[0353] Example 2: Amplification Interference The amplified oligonucleotides should ideally specifically detect the 16S rRNA of the target species while minimizing interference from other organisms that may be present at high titers in the preferred sample type. Alignment of 16S rRNA sequences between G.vag and other vaginal organisms shows similarities between G.vag and Bifidobacteria sequences, suggesting that interference from Bifidobacteria or other related organisms may affect the detection of G.vag in some samples. Further G.vag amplified oligonucleotides were designed to reduce the potential for interference.

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

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

[0356] Test results for G. vag amplification interference. Both sets in region 2 showed relatively stable detection of G. vag in the presence of high titer Bifidobacterium breve (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 increased concentration Bifidobacterium breve (B. breve), indicating G. vag amplification interference.

[0357] Initial oligo combinations for detecting Lactobacillus species (L. gasseri, L. jensenii, and L. crispatus) showed decreased detection of L. gasseri at low concentrations 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 oligosets were used alone or in manual RT-TMA format to detect Lactobacillus gasseri at 1E4 CFU / mL in the presence of 1E6 CFU / mL of Neisseria gonorrhoeae, Clostridium difficile, Acinetobacter lwoffii, Mycoplasma hominis, or Lactobacillus iners. 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 in each condition are shown in the table below. To evaluate interference, the average TTime of each sample was divided by the average TTime of Lactobacillus gasseri (L. gasseri) 1E4 CFU / mL. A TTime ratio greater than 100% indicates TTime delay (amplification interference). [Table 10]

[0358] Results of amplification interference tests for Lactobacillus species. Under initial conditions (using a single forward primer for Lactobacillus gasseri, Lactobacillus jensenii, and Lactobacillus crispatus), amplification interference by Clostridium difficile, Acinetobacter lwoffi, and Mycoplasma hominis was observed. However, under modified conditions (using separate forward primers for each target species), no amplification interference was observed.

[0359] Example 3: Lactobacillus target capture oligoscreening To improve the initial design of the target capture oligo for Lactobacillus species (SEQ ID NO: 257), further designs were developed and screened in parallel with the existing design using samples containing 1E4 CFU / mL of Lactobacillus crispatus, Lactobacillus gasseri, or Lactobacillus jensenii. The amplified and detected 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 3 replicates per condition, and mean-normalized Ttime values ​​are shown. [Table 11]

[0360] Since the conditions are altered only by the target capture oligo, slow amplification (high Ttime) may be due to decreased target capture efficiency. In condition 1, no target capture oligo was used, resulting in delayed or absent Ttime. The use of a target capture oligo is expected to improve this by increasing capture efficiency. In each of the other conditions, a single TCO was used to capture 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 other bacteria as well as Lactobacillus.

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

[0362] Example 4: Cross-reactivity in initial multiplexing Multiplex configurations of RT-TMA reagents for detecting G.vag, A.vag, and Lactobacillus species were constructed using the oligonucleotides shown in Table 12. [Table 12]

[0363] The reagent was tested in five replicates per sample using a PANTHER instrument against a pool containing 1 to 5 non-target organisms at high titers (mostly 1E6 CFU / mL). Unexpected signals in the FAM channel were observed in pool #7, which consisted of the following four organisms: Mobiluncus curtisii (5E9 rRNA copies / mL), Mycoplasma genitalium (1E6 CFU / mL), Mycoplasma hominis (5E9 rRNA copies / mL), and Neisseria gonorrhoeae (1E6 CFU / mL). Follow-up tests were performed on samples containing each of the organisms in pool #7. The reagent was tested twice for each control condition and four times for each test condition using a PANTHER instrument. The mean normalized Ttime is shown in Table 13 below. [Table 13]

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

[0365] This experiment demonstrated that SEQ ID NOs. 259, 10, and 45 are necessary for the generation of an unexpected FAM signal. To test the sufficiency of these oligos for detecting Mycoplasma genitalium (M. genitalium) on FAM, we constructed an RT-TMA reagent in which these three oligomers (SEQ ID NOs. 259, 10, and 45) represent only the torch, promoter primer, and primer. Mycoplasma genitalium (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 cross-reactivity risk of multiplexed Mycoplasma genitalium (M. genitalium), as listed in Table 12, was not flagged in the initial bioinformatics assessment. Sequence IDs 10 and 259 did not meet the initial criteria for predicted hybridization to Mycoplasma genitalium (M. genitalium). Therefore, the observed cross-reactivity was unexpected.

[0367] Example 5 To enhance the sensitivity of 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 started with the oligos shown in Table 16. [Table 16]

[0368] The A.vag biphasic system was too rapid and could not provide TTime from clinically relevant high concentrations of A.vag (≧1E11 cp / mL) comparable to IVT. Previously designed A.vag T7 oligonucleotides were re-screened. One T7 oligonucleotide with predicted secondary structure slowed TTime.

[0369] Improvements to the A.vag non-biphasic system focused on improving the slope of the A.vag curve. Repeats of multiple A.vag torches 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] The results of the analysis specificity were similar between the two A.vag systems (LAG5 and LAG6).

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

[0372] The clinical outcomes of LAG5 and LAG6 multiplex oligo combinations (with different oligos designed for A.vag, as shown in Table 17 above) were compared using two small vaginal clinical swab sample sets. 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. In the uncalibrated Ttime ratio algorithm, a sample was called positive if both of the following criteria were met (otherwise negative): 1) the ratio of internal standard Ttime to either G.vagTtime or A.vagTtime exceeds a cutoff value, and 2) the ratio of L.sppTtime to either G.vagTtime or A.vagTtime exceeds a different cutoff value. A fixed value was assigned to non-existent Ttimes. The clinical outcomes for both LAG5 and LAG6 were similar, but LAG6 performed slightly better at pre-defined log c / mL cutoff values ​​(Lacto 8.3, G.vag 10.5, and A.vag 7.4). The project proceeded using a LAG6 multiplex design, with all systems being non-biphasic and the A.vag amplicon shortened.

[0373] Example 6 Oligomers having the nucleotide sequences shown in Table 18 were evaluated for amplification and detection of Lactobacillus crispatus, Lactobacillus jensenii, Lactobacillus gasseri, Gardnerella vaginalis, and Atopovium vaginae, as well as a common internal standard (GIC), using the multiplex APTIMA® bacterial vaginosis (ABV) assay. [Table 18]

[0374] Table 34 shows the oligomers designed and screened for bacterial vaginosis assays. Based on this screening, the oligomers shown in Table 16 were selected for further evaluation in the PANTHER® system for linearity, cross-reactivity, clinical outcomes, and strain comprehensiveness.

[0375] The following bulk liquid reagents were prepared. 1) TCR: HEPES free acid dihydrate (250 mM), lithium hydroxide monohydrate (310 mM), lithium chloride (1.883 M), EDTA free acid (100 mM), polydT14 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), EDTA disodium 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 reagents: 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 prepared by adding oligonucleotides to aliquots of the amplification and promoter bulk reagents. The TCR reagents in Table 19 were prepared by adding oligonucleotides and in vitro transcripts of internal standards to the TCR bulk reagent. Oligonucleotides were not added to the enzyme reagents.

[0377] The surfactant Tween-20 was added to the amplification / promoter buffer to a final concentration of 1%, reducing aggregation and improving torch stability.

[0378] We conducted tests using the PANTHER system, analyzed the results with our in-house developed software, and interpreted the curve characteristics in real time. [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 the culture lysate to the sample transport medium, STM (sodium mononucleotide monohydrate (2.07 g / L), lithium lauryl sulfate (30 g / L), disodium EDTA dihydrate (0.372 g / L), free EGTA acid (0.38 g / L), disodium hydrogen phosphate (2.13 g / L)). The second panel was prepared by adding the in vitro transcript (IVT) to the STM. Each panel was tested in 5 replicates using the PANTHER system. Panel concentrations are shown in Table 20. [Table 20]

[0380] Table 21 summarizes the positive 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. The results presented include positive result, mean RFU range, mean Ttime, mean Tslope, and standard deviation. All reactions were valid, as indicated by the general internal standard. [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26]

[0382] Cross-reactivity with microorganisms - purity (analytical specificity) and addition (inhibition) tests To support cross-reactivity testing, 17 pools of bacterial or yeast material representing the normal genitourinary flora or organisms closely associated with APTIMA BV targets were constructed (see Table 27). The target concentration for each organism was 1.00E+06 CFU / mL or cell equivalent / mL, which is above the upper limit of the expected concentration.

[0383] To confirm the specificity of the analysis, the pool was tested in pure form (without the addition of assay targets). The pool was then tested with the addition of lysates representing each of the assay targets (A. vag, G. vag, and all three Lacto lysates) to test for inhibition of the assay by other microorganisms. Each condition was tested with 5 replicates. [Table 27-1] [Table 27-2]

[0384] The result of pure cross-reactivity 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. crispus. This cross-reactivity is acceptable because Lactobacillus acidophilus is found in the upper gastrointestinal tract and is unlikely to be present in the vaginal microbiota.

[0385] The HEX channel (G.vag) yielded 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 make up pool #13 (A. rimae, A. minimum, and A. parvulum) were isolated and retested. Component testing showed that all three Atopobium species emitted a positive signal in the ROX channel.

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

[0388] Results of cross-reactivity after addition The target assay analytes (L. crisp, L. jen, L. gas, A. vag, or G. vag) were added to each of the 17 pools in Table 27. Positive analytes were added at approximately 3x LOD concentration, and each pool and panel was tested with 5 replicates. The concentrations of the added analytes are shown in Table 28. The added STM (pool 0) was tested as a control for comparison. [Table 28]

[0389] A bulk reagent was prepared for the added cross-reactivity test. The bulk reagent was dispensed into three 100-test reagent kits for testing. Individual bottles of enzyme were reconstituted separately for each of the three kits. Plotting the Ttime results, reagent kit #2 was found to have slower Ttimes in all four channels compared to reagent kits #1 and #3. This may be due to variability in reconstitutes from the enzyme and therefore represents the variability that may occur due to inter-kit variability. The Ttime of the analytes was divided by the GIC Ttime to normalize the data to account for inter-kit variability. The Ttime results are shown in Table 29.

[0390] The FAM channel (Lacto species) showed faster Ttime results from pool #16 (L. crisp, L. jen, and L. gas in the presence of L. acidophilus) compared to the control (pool #0), while the Ttime for pool #06 (Leptotrichia bucalis, Mobilunchus curtisii, Mycoplasma genitalium, Mycoplasma hominis) was slightly slower. Pool #16 also showed cross-reactivity during the pure test, which was expected given the close association of the test organism with L. crisp in the 16S design region. Pool #06 was split into subcomponents and retested. In the retest, a 1.6-minute delay was observed for L. gas in the presence of Mycoplasma hominis.

[0391] The HEX channel (G. vag) showed decreased sensitivity in 4 out of 5 replicates when tested in the presence of pool #12 (Candida tropicalis, Candida krusei, and Candida lusitaniae). The three Candida species constituting pool #12 were isolated and retested. Component testing showed that Candida krusei caused the decreased sensitivity. Candida krusei is found in the vaginal microbiota but is uncommon, so this potential interference should be noted. (BD Max) TM In a US clinical trial of a vaginal panel, the Candida krusei (C. krusei) rate was 4 / 1647, or 0.2% (see BD PI 443710).

[0392] The ROX channel (A.vag) showed a slightly faster Ttime in pool #13 (Atopobium species) compared to the control (pool 0). This was expected, given that this is the same pool that caused cross-reactivity in the pure test, and that the properties of A.vag, the target of APTIMA BV, and the test organism are closely related.

[0393] The Cy5.5 channel (GIC) showed a slightly delayed Ttime in Pool #6 (Leptotrichia bucalis, Mobiluncus curtisii, Mycoplasma genitalium, Mycoplasma hominis). Component testing of these four microorganisms indicates that Mycoplasma hominis delays the GIC Ttime by slightly more than one minute, slightly altering the shape of the GIC appearance curve. Mycoplasma hominis is commonly found in BV. Using the BV multivariate algorithm described in Example 1, the GIC Ttime delay increases the probability of a BV-positive readout because the BV score increases via the IC ratio term. This can mitigate the effect of response inhibition caused by the presence of BV-related organisms, including Mycoplasma hominis. [Table 29]

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

[0395] Clinical samples were tested with 20 reference materials (reference material levels for five single analytes: L. crisp, A. vag, and G. vag, and reference material levels for five mixed analytes containing all three analytes). Each single analyte reference material was tested in 4 replicates, and each mixed analyte reference material was tested in 5 replicates.

[0396] Of the 100 clinical samples tested, 93 were effective. The remaining 7 replicates were ineffective due to insufficient sample volume.

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

[0398] The diagnosis of BV status was based on a quantification and 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, and X, Y, and Z are the cutoff values ​​of L.crisp, A.vag, and G.vag, respectively) BV is negative if L > X, positive if A > Y or G > Z, and negative otherwise. For this analysis, the following cutoff values, 8.3, 10.4, and 7.4 log c / mL were used for L.crisp, G.vag, and A.vag, respectively.

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

[0400] Single analytes and mixed analytes showed the same sensitivity results (90.2%) and similar specificity results (88.5% and 86.5%, respectively), with one sample changing its calling due to quantitative differences (see Table 30). [Table 30]

[0401] Comprehensive testing The comprehensive test was performed for each of the five assay targets using one control strain and all available polymorphic IVT strains. The tests were performed at low and intermediate concentrations of each strain. The low panel results were used to determine the effect of polymorphism on the sensitivity of the analyte, and the intermediate panel results were analyzed to determine the effect of polymorphism on the Ttime of the analyte.

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

[0403] A. vag showed decreased sensitivity at low copy levels in three of the four polymorphic strains tested. At intermediate copy levels, two of the four strains showed a Ttime delay of 2–3.5 minutes. The two strains that showed Ttime delay had two mismatches for A. vag T7 oligonucleotides. The prevalence of the two strains that showed Ttime delay was less than 21.95% and 4.87%, respectively.

[0404] G.vag did not show a decrease in sensitivity of the polymorphic strain tested. At the intermediate copy level, the polymorphic strain showed a Ttime delay of slightly over 2 minutes. This polymorphism has 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. Compared to the control, one strain had a Ttime 3.5 minutes earlier, while the other had a Ttime 2 minutes later. Both of these strains had two mismatches for T7, and both had a prevalence of less than 2%.

[0406] L.gas showed 100% sensitivity for all strains tested. The intermediate copy level indicated the smallest change in Ttime in polymorphic strains compared to the control.

[0407] L. jen showed 100% sensitivity for all strains tested. Intermediate copy levels indicated the smallest change in Ttime in polymorphic strains compared to the control. [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 while specific embodiments of the present invention are described herein for illustrative purposes, various modifications can be made without departing from the spirit and scope of the invention. Therefore, the present invention is not limited to those provided for in the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. To the extent that any material incorporated by reference conflicts with the express content of this disclosure, the express content shall prevail.

[0409] This application includes a sequence listing submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The above ASCII copy, created on August 20, 2019, is named 2019-08-20_01159-0039-60PCT_SeqList_ST25.txt and is 66.8 kilobytes in size. In certain embodiments, for example, the following items are provided: (Item 1) A multiplex method for determining the presence or absence of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in a sample, wherein the method is (1) Contact a sample suspected to contain at least one of the following species: Lactobacillus, Atopovium vaginae, and Gardnerella vaginalis, (a) First, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 7 (iii) The third Lactobacillus-specific amplified oligomer comprises a second Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 8, and (iv) The fourth Lactobacillus-specific amplified oligomer comprises a fourth Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 9. (b) First and second Atopobium vaginae (A. vaginae) specific amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae (A. vaginae), wherein (i) the first Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a first Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and (ii) the second Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a second Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. (c) First and second Gardnerella vaginalis-specific amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first Gardnerella vaginalis-specific amplification oligomer comprises a first Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and (ii) the second Gardnerella vaginalis-specific amplification oligomer comprises a second Gardnerella vaginalis-specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14. (2) If target nucleic acids of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis are present in the sample, perform an in vitro nucleic acid amplification reaction using them as templates to produce one or more amplification products corresponding to the target regions of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis. (3) Detect the presence or absence of one or more amplification products, thereby determining the presence or absence of Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in the sample. Methods that include... (Item 2) A composition or kit for determining the presence or absence of each of the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in a sample, wherein the composition or kit comprises the following: (a) First, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first Lactobacillus-specific amplification oligomer comprises a first Lactobacillus-specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, and (ii) the second Lactobacillus-specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO: 7 (iii) The third Lactobacillus-specific amplified oligomer comprises a second Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 8, and (iv) The fourth Lactobacillus-specific amplified oligomer comprises a fourth Lactobacillus-specific target hybridize sequence substantially corresponding to the sequence of SEQ ID NO: 9. (b) First and second Atopobium vaginae (A. vaginae) specific amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae (A. vaginae), wherein (i) the first Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a first Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and (ii) the second Atopobium vaginae (A. vaginae) specific amplification oligomer comprises a second Atopobium vaginae (A. vaginae) specific target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. (c) A composition or kit for amplifying the target region of a target nucleic acid of Gardnerella vaginalis (G. vaginalis), wherein (i) the first Gardnerella vaginalis (G. vaginalis)-specific amplification oligomer comprises a first Gardnerella vaginalis (G. vaginalis)-specific target hybridize 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 hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14. (Item 3) A method for determining the presence or absence of bacterial vaginosis (BV) in a subject, wherein the method is (a) Provide samples from subjects suspected of having BV, (b) Perform assays to detect Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis in the sample. (c) Assign quantitative values ​​to each of the Lactobacillus species, Atopovium vaginae, and Gardnerella vaginalis based on detection assays. (d) Subtract the quantitative value of Lactobacillus species from the larger of the quantitative values ​​of Atopobium vaginae and Gardnerella vaginalis. (e) Assigning a single BV score based on step (d), and (f) Determine the presence or absence of the target BV based on a comparison of the BV score and the cutoff value. Methods that include... (Item 4) A method for determining the presence or absence of Lactobacillus species in a sample, wherein the method is (1) Contact a sample suspected of containing the Lactobacillus species with first, second, third, and fourth amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, where (i) the first amplification oligomer contains a first target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 10, (ii) the amplification oligomer contains a second target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 7, (iii) the third amplification oligomer contains a third target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 8, and (iv) the fourth amplification oligomer contains a fourth target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 9. (2) If the target nucleic acid of a Lactobacillus species is present in the sample, an in vitro nucleic acid amplification reaction is performed, which is used as a template to produce one or more amplification products corresponding to the target region of the Lactobacillus species. (3) Detect the presence or absence of one or more amplification products, thereby determining the presence or absence of Lactobacillus species in the sample. Methods that include... (Item 5) A method for determining the presence or absence of Atopobium vaginae in a sample, wherein the method is (1) Contact a sample suspected of containing Atopobium vaginae with first and second amplification oligomers for amplifying the target region of the target nucleic acid of Atopobium vaginae, where (i) the first amplification oligomer contains a first target hybridize sequence substantially corresponding to the nucleotide sequence of residues 28-45 of SEQ ID NO: 18, and (ii) the second amplification oligomer contains a second target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 17. (2) If the target nucleic acid of Atopobium vaginae is present in the sample, perform an in vitro nucleic acid amplification reaction using it as a template to produce one or more amplification products corresponding to the target region of Atopobium vaginae. (3) Detect the presence or absence of one or more amplification products, thereby determining the presence or absence of Atopobium vaginae in the sample. Methods that include... (Item 6) A method for determining the presence or absence of Gardnerella vaginalis in a sample, wherein the method is (1) Contact a sample suspected of containing Gardnerella vaginalis with first and second amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, wherein (i) the first amplification oligomer contains a first target hybridize sequence substantially corresponding to the nucleotide sequence of residues 36-52 of SEQ ID NO: 15, and (ii) the second amplification oligomer contains a second target hybridize sequence substantially corresponding to the nucleotide sequence of SEQ ID NO: 14. (2) If the target nucleic acid of Gardnerella vaginalis is present in the sample, an in vitro nucleic acid amplification reaction is performed using it as a template to produce one or more amplification products corresponding to the target region of Gardnerella vaginalis. (3) A method comprising detecting the presence or absence of one or more amplification products, thereby determining the presence or absence of Gardnerella vaginalis in the sample.

Claims

1. A composition for determining the presence or absence of the Lactobacillus species in a sample, comprising first, second, third, and fourth Lactobacillus-specific amplification oligomers for amplifying the target region of the target nucleic acid of the Lactobacillus species, (i) The first amplified oligomer comprises a first target hybridize sequence consisting of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 10, (ii) The second amplified oligomer comprises a second target hybridize sequence consisting of the nucleotide sequence of SEQ ID NO: 7, (iii) The third amplified oligomer comprises a third target hybridize sequence consisting of the nucleotide sequence of SEQ ID NO: 8, (iv) A composition wherein the fourth amplified oligomer comprises a fourth target hybridize sequence consisting of the nucleotide sequence of SEQ ID NO:

9.

2. The composition according to claim 1, wherein the first lactobacillus-specific amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' with respect to each target hybridize sequence.

3. The composition according to claim 2, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:

10.

4. The composition according to claim 1, further comprising a first Lactobacillus-specific detection probe.

5. The composition according to claim 4, wherein the target hybridize sequence of the first lactobacillus-specific detection probe specifically hybridizes to the target regions of the target nucleic acids of Lactobacillus crispatus and Lactobacillus jensenii, respectively, and the composition further comprises a second lactobacillus-specific detection probe containing a target hybridize sequence that specifically hybridizes to the target region of the target nucleic acid of Lactobacillus gasseri.

6. The composition according to claim 5, wherein the target hybridize sequence of the first lactobacillus-specific detection probe consists of the nucleotide sequence of residues 1 to 17 of SEQ ID NO: 11, and / or the target hybridize sequence of the second lactobacillus-specific detection probe consists of the nucleotide sequence of residues 7 to 23 of SEQ ID NO: 12, and / or the second lactobacillus-specific detection probe comprises the nucleotide sequence of SEQ ID NO:

12.

7. The composition according to claim 4, wherein the first Lactobacillus-specific detection probe comprises a fluorescent label and a quencher.

8. The composition according to claim 4, wherein the first lactobacillus-specific detection probe further comprises a non-target hybridized sequence.

9. The composition according to claim 7 or claim 8, wherein the first Lactobacillus-specific detection probe is a molecular torch or a molecular beacon.

10. The composition according to claim 7 or claim 8, wherein the first Lactobacillus-specific detection probe comprises the nucleotide sequence corresponding to SEQ ID NO:

11.

11. A composition for determining the presence or absence of Atopovium virginae in a sample, comprising first and second amplification oligomers for amplifying the target region of the target nucleic acid of Atopovium virginae, (i) The first amplified oligomer comprises a first target hybridize sequence consisting of the nucleotide sequence of residues 28 to 45 of SEQ ID NO: 18, and (ii) A composition wherein the second amplified oligomer comprises a second target hybridize sequence consisting of the nucleotide sequence of SEQ ID NO:

17.

12. The composition according to claim 11, wherein the first Atopobium weginae-specific amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at 5' with respect to each target hybridize sequence.

13. The composition according to claim 12, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:

10.

14. The composition according to claim 11, further comprising a first Atopovium veginae-specific detection probe containing a target hybridize sequence that specifically hybridizes to a target region of Atopovium veginae.

15. The composition according to claim 14, wherein the target hybridize sequence of the first Atopovium veginae-specific detection probe consists of the nucleotide sequence of residues 6 to 21 of SEQ ID NO:

19.

16. The composition according to claim 15, wherein the first Atopovium weginae specific detection probe comprises a fluorescent label and a quencher.

17. The composition according to claim 15, wherein the first Atopovium weginae-specific detection probe further comprises a non-target hybridized sequence.

18. The composition according to claim 17, wherein the first Atopovium weginae-specific detection probe is a molecular torch or a molecular beacon.

19. The composition according to claim 16 or claim 17, wherein the first Atopovium weginae-specific detection probe comprises the nucleotide sequence corresponding to SEQ ID NO:

19.

20. A composition for determining the presence or absence of Gardnerella vaginalis in a sample, comprising first and second amplification oligomers for amplifying the target region of the target nucleic acid of Gardnerella vaginalis, (i) The first amplified oligomer comprises a first target hybridize sequence consisting of the nucleotide sequence of residues 36 to 52 of SEQ ID NO: 15, and (ii) A composition wherein the second amplified oligomer comprises a second target hybridize sequence consisting of the nucleotide sequence of SEQ ID NO:

14.

21. The composition according to claim 20, wherein the first Gardnerella vaginalis-specific amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located 5' with respect to each target hybridize sequence.

22. The composition according to claim 21, wherein the promoter sequence has the nucleotide sequence of residues 1 to 27 of SEQ ID NO:

10.

23. The composition according to claim 22, further comprising a first Gardnerella vaginalis-specific detection probe comprising a target hybridize sequence that specifically hybridizes to a target region of Gardnerella vaginalis.

24. The composition according to claim 23, wherein the target hybridize sequence of the first Gardnerella vaginalis-specific detection probe comprises the nucleotide sequence of residues 1 to 18 of SEQ ID NO:

16.

25. The composition according to claim 23, wherein the first Gardnerella vaginalis-specific detection probe comprises a fluorescent label and a quencher.

26. The composition according to claim 23, wherein the first Gardnerella vaginalis-specific detection probe further comprises a non-target hybridized sequence.

27. ​​The composition according to claim 26, wherein the first Gardnerella vaginalis-specific detection probe is a molecular torch or a molecular beacon.

28. The composition according to claim 25 or claim 26, wherein the first Gardnerella vaginalis-specific detection probe comprises the nucleotide sequence corresponding to SEQ ID NO: 16.

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