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

Abstract

A method for diagnosing bacterial vaginosis in a subject is disclosed, comprising the step of performing a assay for detecting any one or more of Lactobacillus species, Atopobium virginiana, and Gardnerella virginalis in a subject sample. Additionally, a composition and a method for detecting Lactobacillus species, Atopobium virginiana, and / or Gardnerella virginalis nucleic acids in a sample are disclosed.

Description

Technology Field

[0001] As a method for diagnosing bacterial vaginosis in subjects, in subject samples Lactobacillus bell, Atopobium virginiana and Gardnerella Virginalis A method is disclosed comprising the step of performing a assay for detecting any one or more of the following: in a sample. Lactobacillus bell, Atopobium virginiana and / or Gardnerella Virginalis A composition and a method for detecting nucleic acids are disclosed. Background Technology

[0002] According to the National Health and Nutrition Examination Survey, nearly one-third of women aged 14 to 49 have bacterial vaginosis (BV). (Allsworth and Peipert, Obstetrics and Gynecology (See 109: 114-120, 2007). BV is the most common cause of vaginal discharge and is the reason many women seek treatment. It is also associated with premature birth, low birth weight, pelvic inflammatory disease, an increased incidence of STDs including HIV, and a greater risk of transmitting HIV to sexual partners. Srinivasan and Fredricks, Interdisciplinary Perspectives on Infectious Diseases See , Vol. 2008, Article ID 750479, 22 pages, 2008. Women with bacterial vaginosis may experience symptoms including foul-smelling vaginal discharge or itching, but about half of women with diagnosable BV have no obvious symptoms (see Srivinvasan and Fredricks, cited above).

[0003] There is no single causative agent of BV. Most researchers and the CDC consider bacterial vaginosis to be the result of a disruption of the normal vaginal flora. Unlike common infections, this dysbiosis is not the result of individual bacterial species. See CDC Factsheet, 2014 (BV-Fact-Sheet-March-2014.pdf from the CDC website). Dysbiosis is the disruption of the normal microbiota within a bodily environment, such as the vagina. Nibali et al. , Journal of Oral Microbiology See 6: 22962, 2014.

[0004] BV is diagnosed in hospitals using the Amsel screening criteria and in laboratories using the Nugent scoring system. The latter relies on counting bacterial morphotypes with the aid of Gram staining. In this way, the Nugent score is a visual assessment of incompatibility and assigns a score to bad bacteria relative to good bacteria. Nugent et al. , Journal of Clinical Microbiology See 29: 297-301, 1991. The Amsel criteria evaluate the sample for the presence of clue cells, pH, color, and odor, which are key symptoms associated with BV. et al. , Am. J. Med. See 74: 14-22, 1983. Wet mounts of the sample were examined under a microscope and predominantly G. Virginalis as It detects clue cells, which are human epithelial cells coated with bacteria thought to be composed.

[0005] Molecular tests generally target multiple organisms that are strongly correlated with bacterial vaginosis. The target organisms vary from test to test. In almost all cases Atopobium ( Atopobium ), Gardnera ( Gardnerella ) and Megaspaera ( Megasphaera Highly concentrated anaerobic bacteria, such as ) species, are the target.

[0006] The FDA-approved test for BV is only BD Affirm TM VPⅢ microbial identification test (2010) and BD MAX TM It is only the Quality Panel (2016). Both Affirm and MAX products are the only indicators of BV. G. Virginalis It detects. The BD Affirm VPⅢ test was found to have a sensitivity of 67.6% and a specificity of 76.4% in a study by Cartwright et al. et al., Journal of Clinical Microbiology 51: 3694-3699, 2013). The BD Affirm VIII package insert indicates that the Affirm product is 95.1% sensitive and 83.3% specific compared to the scored Gram staining method, whereas the BD MAX package insert indicates that the MAX product is 90.5% sensitive and 85.8% specific.

[0007] The above Cartwright, etc., for the diagnosis of BV Atopobium virginiana , BVAB-2 and Megaspaera A multiplex test was used for the detection of -1. They measured the performance of this test on the combination of Nugent and Amsel results in a population of 323 women (93% African American, 7% non-Hispanic White). They reported that this test was 96.9% sensitive and 92.6% specific when compared to the combination of Nugent and Amsel scores. They did not report the results of this test when compared to Nugent scores alone. The problem to be solved

[0008] Cross-reference regarding related applications

[0009] This application claims priority to U.S. provisional application 62 / 722,627 filed on August 24, 2018, the full text of which is incorporated herein by reference for all purposes.

[0010] In one aspect, the present invention provides a method for determining the presence or absence of bacterial vaginosis (BV) in a subject. In some embodiments, the method generally comprises the steps of: (a) providing a sample from a subject suspected of having BV; and (b) from the sample Lactobacillus bell ( Lactobacillus sp.), A father ( A. vaginal ) and G. Virginalis ( G. vaginalis Step of performing a test method for the detection of ); (c) Lactobacillus bell, A father and G. Virginalis A step of assigning a quantitative value based on a detection test method to each; (d) A . Virgin The quantitative value of and G . Virginalis From the larger of the quantitative values ​​of Lactobacillus (e) a step of subtracting a quantitative value of the species; (e) a step of assigning a single BV score based on step (d); and (f) a step of determining the presence or absence of BV in the subject based on a comparison of the BV score and a cutoff value: comprising. In some embodiments, in step (c) Lactobacillus species and / or A father and G. Virginalis A minimum quantitative value is assigned to . Typically, Lactobacillus bell, A father and G. Virginalis The quantitative values ​​for each are standardized or / or weighted. In some variations that include standardized quantitative values, Lactobacillus bell, A father and G. VirginalisThe quantitative value for each is in units of log copies, and standardization involves subtracting the median population value from the value determined from the detection test. Step (d) may further include the addition of an adjustment constant. In a specific embodiment, step (d) further includes the addition of an internal control (IC) adjustment factor that compensates for sample suppression in the detection test, wherein the IC adjustment factor is based on the ratio of (i) the observed IC value generated from the detection test to (ii) the expected IC value for the detection test. In a detailed variation, the BV score is assigned using a formula:

[0011] BV Score = C0 + W L Maximum value (L S , F LS ) + W GA Maximum value (A S , G S , F GAS ) + W IC log2(IC ratio),

[0012] Here, C0 is the adjustment constant; W L silver Lactobacillus It is a weighting constant for the species; and the maximum value (L S , F LS ) is L S and F LS Larger than, L S Is Lactobacillus It is the observed standardized quantitative value of the species, and F LS Is Lactobacillus It is the minimum standardized quantitative value assigned to the species; W GA Is A father and G. Virginalis It is a weighting constant for; and the maximum value (A S , G S , F GAS ) is A S , G S and F GAS Greater than, and A S Is A father is the observed standardized quantitative value of, and G S Is G. Virginalisis the observed standardized quantitative value of, and F GAS Is A father and G. Virginalis It is the assigned minimum standardized quantitative value of (e.g., 0); W IC is an internal control (IC) weighting constant; the IC ratio is the ratio of (i) the observed internal control (IC) value generated from the detection test to (ii) the expected IC value for the detection test.

[0013] In some embodiments of the method for diagnosing BV as described above, Lactobacillus bell, A father and G. Virginalis The assay for the detection of is a nucleic acid-based detection assay. Specifically, a suitable nucleic acid-based detection assay includes an amplification-based assay, for example, an assay including an isothermal amplification reaction (e.g., a transcription-mediated amplification (TMA) reaction), which can be performed in real time. In a specific embodiment, the nucleic acid-based detection assay Lactobacillus bell, A father and G. Virginalis It targets the 16S rRNA of. In a specific modification, a nucleic acid-based detection assay (i) corresponding to the region of SEQ ID NO. 1 from approximately nucleotide position 40 to approximately nucleotide position 265. L. Crispatus 16S rRNA region; (ii) corresponding to the region of SEQ ID NO. 2 at approximately nucleotide position 43 to approximately nucleotide position 247 L. Genseni 16S rRNA region; (iii) corresponding to the region of SEQ ID NO. 3 at approximately nucleotide position 93 to approximately nucleotide position 298 L. Gasseri 16S rRNA region; (iv) corresponding to the region of SEQ ID NO. 4 at approximately nucleotide position 540 to approximately nucleotide position 625 A. Virgin 16S rRNA region; and / or (v) corresponding to the region of SEQ ID NO. 5 at about nucleotide position 172 to about nucleotide position 227 G. Virginalis Targets the 16S rRNA region.

[0014] In a specific embodiment of the method for diagnosing BV as described above, comprising a nucleic acid-based detection assay, the assay is an amplification-based assay comprising the following steps:

[0015] (1) Sample

[0016] Lactobacillus The first, second, third, and fourth amplifying the target region of the species target nucleic acid Lactobacillus As a specific amplification oligomer, (i) a first corresponding to the nucleotide sequences of residues 28 to 45 of SEQ ID NO. 10 substantially thereof, of a method for diagnosing BV comprising an amplification-based detection assay as described above. Lactobacillus (ii) comprising a specific target-hybridization sequence; and (ii) the second Lactobacillus The specific amplification oligomer is a second one substantially corresponding to the nucleotide sequence of SEQ ID NO. 7. Lactobacillus It includes a specific target-hybridization sequence; (iii) the third Lactobacillus The specific amplification oligomer is a third one substantially corresponding to the nucleotide sequence of SEQ ID NO. 8. Lactobacillus It includes a specific target-hybridization sequence; (iv) the 4 Lactobacillus The specific amplification oligomer is the 4th one, which substantially corresponds to the nucleotide sequence of SEQ ID NO. 9. Lactobacillus including a specific target-hybridization sequence, Lactobacillus Specific amplification oligomer;

[0017] A . Virgin First and second amplifying the target region of the target nucleic acid A . Virgin As a specific amplification oligomer, (i) the 1 A . Virgin The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18. A . Virgin (ii) comprising a specific target-hybridization sequence; and (ii) the second A . VirginThe specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 17. A . Virgin including a specific target-hybridization sequence, A . Virgin Specific amplification oligomers; and

[0018] G . Virginalis First and second amplifying the target region of the target nucleic acid G . Virginalis As a specific amplification oligomer, (i) the 1 G . Virginalis The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15. G . Virginalis (ii) comprising a specific target-hybridization sequence; and (ii) the second G . Virginalis The specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 14. G . Virginalis including a specific target-hybridization sequence, G . Virginalis Step of contacting with a specific amplification oligomer;

[0019] (2) In vitro As a step of performing a nucleic acid amplification reaction, any Lactobacillus bell, A . Virgin and G . Virginalis If the target nucleic acid is present in the sample, Lactobacillus bell, A . Virgin and G . Virginalis A step used as a template for generating one or more amplification products corresponding to a target region; and

[0020] (3) A step of detecting the presence or absence of one or more amplification products.

[0021] In a partial variation of a method for diagnosing BV including an amplification-based detection verification method as described above, the first Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10; and the 2 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 7; and the 3 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 8; and the 4 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 9; and the 1 A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18; and the 2 A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 17; and the 1 G . Virginalis The specific target-hybridization sequence comprises the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15; and the 2 G . Virginalis The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 14. In some of these embodiments, the first Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10; and the 2 Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 7; and the 3 Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 8; and the 4 Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 9; and the 1 A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18; and the 2 A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 17; and the 1 G . VirginalisThe specific target-hybridization sequence consists of the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15; and the 2 G . Virginalis The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 14.

[0022] In a partial embodiment of a method for diagnosing BV including an amplification-based detection verification method as described above, the first Lactobacillus Specific amplification oligomer, 1 A father Specific amplification oligomer and the first G. Virginalis At least one of the specific amplification oligomers is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence. Specifically suitable promoter sequences are T7 promoter sequences, such as the promoter sequence having the nucleotide sequence of residues 1 through 27 of SEQ ID NO. 10 (also indicated, for example, residues 1 through 27 of SEQ ID NO. 18 or residues 9 through 35 of SEQ ID NO. 15). In a specific variation, the first Lactobacillus The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 10; and the 1 A father The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 18, and the first G. Virginalis The specific amplification oligomer comprises the nucleotide sequence of residues 9 through 52 of SEQ No. 15. In some embodiments of SEQ, the 1 G. Virginalis The specific amplification oligomer contains the nucleotide sequence of SEQ ID NO. 15.

[0023] In a specific embodiment of a method for diagnosing BV including an amplification-based detection assay as described above, the method, prior to step (2), if present in the sample Lactobacillus bell, A father and G. VirginalisThe method further includes a step of purifying the target nucleic acid from other components. In some of these embodiments, the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence that binds to an immobilized probe or a target-hybridization sequence covalently attached to a moiety. For example, the sample is Lactobacillus A first capture probe oligomer comprising a target-hybridization sequence that specifically hybridizes to a target sequence within a species target nucleic acid, and A father and G. Virginalis A second capture probe target-hybridization sequence may be in contact with a second capture probe target-hybridization sequence comprising a target-hybridization sequence that specifically hybridizes to a target sequence within each of the target nucleic acids, wherein each of the first and second capture probe target-hybridization sequences is covalently attached to a sequence or moiety that binds to an immobilized probe; in some such embodiments, the first capture probe target-hybridization sequence is L. Crispatus , L. Genseni and L. Gasseri It specifically hybridizes to the target sequence within each target nucleic acid. In a detailed variation comprising the first and second target capture probes as described above, the first capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 19 of SEQ ID NO. 6, and / or the second capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 20 of SEQ ID NO. 13. In some embodiments, the first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 6, and / or the second capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 13.

[0024] In a partial embodiment of a method for diagnosing BV including an amplification-based detection test method as described above, the detection step (3) is (i) Lactobacillus A first target-hybridization sequence comprising a target-hybridization sequence that specifically hybridizes to a species target region Lactobacillus Specific detection probe, A. VirginA first target-hybridization sequence comprising a target region that specifically hybridizes to the target region A. Virgin Specific detection probe, and G. Virginalis A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region G. Virginalis A step of contacting a specific detection probe with one or more amplification products; and (ii) any target-hybridized Lactobacillus Specific, A father Specific and G. Virginalis It includes the step of detecting the presence or absence of a specific detection probe. In some embodiments, the first A. Virgin The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19, and / or the first G. Virginalis The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 18 of SEQ ID NO. 16; in a more detailed variation, the 1 A. Virgin The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 6 to 21 of SEQ ID NO. 19 and / or the first G. Virginalis The specific detection probe target-hybridization sequence substantially comprises the nucleotide sequence of residues 1 through 18 of SEQ ID NO. 16. In some embodiments, the first Lactobacillus The specific detection probe target-hybridization sequence is L. Crispatus and L. Genseni The method specifically hybridizes to the target region of each target nucleic acid, and one or more amplification products L. Gasseri specifically hybridizing to the target nucleic acid A second target-hybridization sequence comprising Lactobacillus The method further includes the step of contacting a specific detection probe; in some such embodiments, the first Lactobacillus The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 17 of SEQ ID NO. 11, and / or the second LactobacillusThe specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 7 through 23 of SEQ ID NO. 12; in a more detailed variation, the 1 Lactobacillus The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 to 17 of SEQ ID NO. 11, and the 2 Lactobacillus The specific detection probe target-hybridization sequence includes the nucleotide sequence of residues 7 to 23 of SEQ ID NO. 12.

[0025] The 1st Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis In a specific embodiment of a method for diagnosing BV comprising the use of specific detection probes, each probe comprises a label. The second Lactobacillus In some embodiments further comprising the use of a specific detection probe, the second Lactobacillus Specific detection probes include labels. Specifically suitable labels include chemiluminescent and fluorescent labels.

[0026] In some embodiments of a method for diagnosing Bv, including the use of a detection probe labeled as described above, the detection step (3) occurs during the amplification step (2). In some variations of this, each detection probe includes a fluorescent label and a quencher. Specifically, suitable detection probes including a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0027] In a specific embodiment of a method for diagnosing BV including the use of a detection probe as described above, the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis At least one of the specific detection probes further comprises a non-target-hybridization sequence. The second Lactobacillus In some embodiments further comprising the use of a specific detection probe, the second LactobacillusThe specific detection probe further includes a non-target-hybridization sequence. In some of these variations, the first Lactobacillus Specific, first A father Specific and first G. Virginalis Each specific detection probe (or the first Lactobacillus Specific, first Lactobacillus Specific, first A father Specific and first G. Virginalis Each specific detection probe is a molecular torch or molecular beacon.

[0028] In some embodiments of the method for diagnosing BV as described above, the method comprises the detection of 10 or fewer bacterial genera associated with BV. For example, in a specific variant, the method comprises the detection of 5 or fewer bacterial genera associated with BV. In a detailed variant, the method Lactobacillus , Atopobium , Gardnera It does not include the detection of other BV-related bacterial genera.

[0029] In some embodiments of the method for diagnosing BV as described above, when the presence of BV appears in a subject, the method further includes administering a therapeutic diet for BV to the subject.

[0030] In some embodiments of the method for diagnosing BV as described above, the method is a method for monitoring BV in a subject, and the subject is receiving therapeutic care for BV prior to step (a). In some variations of this, when the presence of BV is detected in the subject, the method further comprises (i) administering therapeutic care for BV to the subject, or (ii) administering a different therapeutic care for BV to the subject.

[0031] In one embodiment, the present invention in a sample Lactobacillus bell, A . Virgin and G . Virginalis A multi-composite method for determining the presence or absence of each is provided. This method generally includes the following steps:

[0032] (1) Lactobacillus bell, A . Virgin and G . Virginalis A sample suspected of containing at least one of the following,

[0033] (a) Lactobacillus The first, second, third, and fourth amplifying the target region of the species target nucleic acid Lactobacillus As a specific amplification oligomer, (i) the 1 Lactobacillus The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10. Lactobacillus (ii) comprising a specific target-hybridization sequence; and (ii) the second Lactobacillus The specific amplification oligomer is a second one substantially corresponding to the nucleotide sequence of SEQ ID NO. 7. Lactobacillus It includes a specific target-hybridization sequence; (iii) the third Lactobacillus The specific amplification oligomer is a third one substantially corresponding to the nucleotide sequence of SEQ ID NO. 8. Lactobacillus It includes a specific target-hybridization sequence; (iv) the 4 Lactobacillus The specific amplification oligomer is the 4th one, which substantially corresponds to the nucleotide sequence of SEQ ID NO. 9. Lactobacillus including a specific target-hybridization sequence, Lactobacillus Specific amplification oligomer;

[0034] (b) A . Virgin First and second amplifying the target region of the target nucleic acid A . Virgin As a specific amplification oligomer, (i) the 1 A . Virgin The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18. A . Virgin (ii) comprising a specific target-hybridization sequence; and (ii) the second A . VirginThe specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 17. A . Virgin including a specific target-hybridization sequence, A . Virgin Specific amplification oligomers; and

[0035] (c) G . Virginalis First and second amplifying the target region of the target nucleic acid G . Virginalis As a specific amplification oligomer, (i) the 1 G . Virginalis The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15. G . Virginalis (ii) comprising a specific target-hybridization sequence; and (ii) the second G . Virginalis The specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 14. G . Virginalis including a specific target-hybridization sequence, G . Virginalis Step of contacting with a specific amplification oligomer;

[0036] (2) In vitro As a step of performing a nucleic acid amplification reaction, any Lactobacillus bell, A . Virgin and G . Virginalis If the target nucleic acid is present in the sample, Lactobacillus bell, A . Virgin and G . Virginalis A step used as a template for generating one or more amplification products corresponding to a target region; and

[0037] (3) Detect the presence or absence of one or more amplification products, thereby from the sample Lactobacillus bell, A . Virgin and G . VirginalisA step to determine the presence or absence of.

[0038] In a partial variation of the multi-composite method as described above, the first Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10; and the 2 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 7; and the 3 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 8; and the 4 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 9; and the 1 A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18; and the 2 A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 17; and the 1 G . Virginalis The specific target-hybridization sequence comprises the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15; and the 2 G . Virginalis The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 14. In some of these embodiments, the first Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10; and the 2 Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 7; and the 3 Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 8; and the 4 Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 9; and the 1 A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18; and the 2 A . VirginThe specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 17; and the 1 G . Virginalis The specific target-hybridization sequence consists of the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15; and the 2 G . Virginalis The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 14.

[0039] In some embodiments of the multi-composite method as described above, the first Lactobacillus Specific amplification oligomer, 1 A father Specific amplification oligomer and the first G. Virginalis At least one of the specific amplification oligomers is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence. Specifically suitable promoter sequences are T7 promoter sequences, such as the promoter sequence having the nucleotide sequence of residues 1 through 27 of SEQ ID NO. 10 (also indicated, for example, residues 1 through 27 of SEQ ID NO. 18 or residues 9 through 35 of SEQ ID NO. 15). In a specific variation, the first Lactobacillus The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 10; and the 1 A father The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 18, and the first G. Virginalis The specific amplification oligomer comprises the nucleotide sequence of residues 9 through 52 of SEQ ID NO. 15. In some embodiments, the first G. Virginalis The specific amplification oligomer contains the nucleotide sequence of SEQ ID NO. 15.

[0040] In a specific embodiment of the multi-composite method as described above, the method is used when, prior to step (2), the sample is present Lactobacillus bell, A father and G. VirginalisThe method further includes a step of purifying the target nucleic acid from other components. In some of these embodiments, the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence that binds to an immobilized probe or a target-hybridization sequence covalently attached to a moiety. For example, the sample is Lactobacillus A first capture probe oligomer comprising a target-hybridization sequence that specifically hybridizes to a target sequence within a species target nucleic acid, and A father and G. Virginalis A second capture probe target-hybridization sequence may be in contact with a second capture probe target-hybridization sequence comprising a target-hybridization sequence that specifically hybridizes to a target sequence within each of the target nucleic acids, wherein each of the first and second capture probe target-hybridization sequences is covalently attached to a sequence or moiety that binds to an immobilized probe; in some such embodiments, the first capture probe target-hybridization sequence is L. Crispatus , L. Genseni and L. Gasseri Target nucleic acid Each specifically hybridizes to a target sequence within. In a detailed variation comprising the first and second target capture probes as described above, the first capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 19 of SEQ ID NO. 6, and / or the second capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 20 of SEQ ID NO. 13. In some embodiments, the first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 6, and / or the second capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 13.

[0041] In some embodiments of the multi-composite method as described above, the detection step (3) is (i) Lactobacillus A first target-hybridization sequence comprising a target-hybridization sequence that specifically hybridizes to a species target region Lactobacillus Specific detection probe, A. VirginA first target-hybridization sequence comprising a target region that specifically hybridizes to the target region A. Virgin Specific detection probe, and G. Virginalis A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region G. Virginalis A step of contacting a specific detection probe with one or more amplification products; and (ii) any target-hybridized Lactobacillus Specific, A father Specific and G. Virginalis It includes the step of detecting the presence or absence of a specific detection probe. In some embodiments, the first A. Virgin The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19, and / or the first G. Virginalis The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 18 of SEQ ID NO. 16; in a more detailed variation, the 1 A. Virgin The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 6 to 21 of SEQ ID NO. 19 and / or the first G. Virginalis The specific detection probe target-hybridization sequence substantially comprises the nucleotide sequence of residues 1 through 18 of SEQ ID NO. 16. In some embodiments, the first Lactobacillus The specific detection probe target-hybridization sequence is L. Crispatus and L. Genseni The method specifically hybridizes to the target region of each target nucleic acid, and one or more amplification products L. Gasseri specifically hybridizing to the target nucleic acid A second target-hybridization sequence comprising Lactobacillus The method further includes the step of contacting a specific detection probe; in some such embodiments, the first Lactobacillus The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 17 of SEQ ID NO. 11, and / or the second LactobacillusThe specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 7 through 23 of SEQ ID NO. 12; in a more detailed variation, the 1 Lactobacillus The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 to 17 of SEQ ID NO. 11, and the 2 Lactobacillus The specific detection probe target-hybridization sequence includes the nucleotide sequence of residues 7 to 23 of SEQ ID NO. 12.

[0042] The 1st Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis In a specific embodiment of a multiple composite method including the use of specific detection probes, each probe includes a label. The second Lactobacillus In some embodiments further comprising the use of a specific detection probe, the second Lactobacillus The specific detection probe includes a label. Specifically, suitable labels include chemiluminescent and fluorescent labels.

[0043] In some embodiments of a multi-composite method including the use of labeled detection probes as described above, the detection step (3) occurs during the amplification step (2). In some variations of this method, each detection probe includes a fluorescent label and a quencher. Specifically, suitable detection probes including a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0044] In a specific embodiment of a multi-composite method including the use of a detection probe as described above, the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis At least one of the specific detection probes further comprises a non-target-hybridization sequence. The second Lactobacillus In some embodiments further comprising the use of a specific detection probe, the second LactobacillusThe specific detection probe further includes a non-target-hybridization sequence. In some of these variations, the first Lactobacillus Specific, first A father Specific and first G. Virginalis Each specific detection probe (or the first Lactobacillus Specific, first Lactobacillus Specific, first A father Specific and first G. Virginalis Each specific detection probe is a molecular torch or molecular beacon.

[0045] In some embodiments of the multi-composite method as described above, the amplification reaction in step (2) is an isothermal amplification reaction. In specific variations, the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction. In certain embodiments, the isothermal amplification reaction is a real-time amplification reaction.

[0046] In another aspect, the present invention in a sample Lactobacillus bell, A . Virgin and G . Virginalis A composition or kit for determining the presence or absence of each is provided. The composition or kit generally comprises the following oligomers:

[0047] (a) Lactobacillus The first, second, third, and fourth amplifying the target region of the species target nucleic acid Lactobacillus As a specific amplification oligomer, (i) the 1 Lactobacillus The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10. Lactobacillus (ii) comprising a specific target-hybridization sequence; and (ii) the second Lactobacillus The specific amplification oligomer is a second one substantially corresponding to the nucleotide sequence of SEQ ID NO. 7. Lactobacillus It includes a specific target-hybridization sequence; (iii) the third Lactobacillus The specific amplification oligomer is a third one substantially corresponding to the nucleotide sequence of SEQ ID NO. 8. LactobacillusIt includes a specific target-hybridization sequence; (iv) the 4 Lactobacillus The specific amplification oligomer is the 4th one, which substantially corresponds to the nucleotide sequence of SEQ ID NO. 9. Lactobacillus including a specific target-hybridization sequence, Lactobacillus Specific amplification oligomer;

[0048] (b) A . Virgin First and second amplifying the target region of the target nucleic acid A . Virgin As a specific amplification oligomer, (i) the 1 A . Virgin The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18. A . Virgin (ii) comprising a specific target-hybridization sequence; and (ii) the second A . Virgin The specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 17. A . Virgin including a specific target-hybridization sequence, A . Virgin Specific amplification oligomers; and

[0049] (c) G . Virginalis First and second amplifying the target region of the target nucleic acid G . Virginalis As a specific amplification oligomer, (i) the 1 G . Virginalis The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15. G . Virginalis (ii) comprising a specific target-hybridization sequence; and (ii) the second G . Virginalis The specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 14. G . Virginalis including a specific target-hybridization sequence, G . Virginalis Specific amplification oligomer.

[0050] In some variations of the composition or kit as described above, the first Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10; and the 2 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 7; and the 3 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 8; and the 4 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 9; and the 1 A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18; and the 2 A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 17; and the 1 G . Virginalis The specific target-hybridization sequence comprises the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15; and the 2 G . Virginalis The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 14. In some of these embodiments, the first Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10; and the 2 Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 7; and the 3 Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 8; and the 4 Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 9; and the 1 A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18; and the 2 A . VirginThe specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 17; and the 1 G . Virginalis The specific target-hybridization sequence consists of the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15; and the 2 G . Virginalis The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 14.

[0051] In some embodiments of the composition or kit as described above, the first Lactobacillus Specific amplification oligomer, 1 A father Specific amplification oligomer and the first G. Virginalis At least one of the specific amplification oligomers is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence. Specifically suitable promoter sequences are T7 promoter sequences, such as the promoter sequence having the nucleotide sequence of residues 1 through 27 of SEQ ID NO. 10 (also indicated, for example, residues 1 through 27 of SEQ ID NO. 18 or residues 9 through 35 of SEQ ID NO. 15). In a specific variation, the first Lactobacillus The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 10; and the 1 A father The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 18, and the first G. Virginalis The specific amplification oligomer comprises the nucleotide sequence of residues 9 through 52 of SEQ ID NO. 15. In some embodiments, the first G. Virginalis The specific amplification oligomer contains the nucleotide sequence of SEQ ID NO. 15.

[0052] In a specific embodiment of the composition or kit as described above, the composition or kit further comprises at least one capture probe oligomer comprising a target-hybridization sequence covalently attached to a sequence or moiety that binds to an immobilized probe. For example, the composition or kit LactobacillusA first capture probe oligomer comprising a target-hybridization sequence that specifically hybridizes to a target sequence within a species target nucleic acid, and A father and G. Virginalis It may include a second capture probe target-hybridization sequence comprising a target-hybridization sequence that specifically hybridizes to a target sequence within each of the target nucleic acids, wherein each of the first and second capture probe target-hybridization sequences is covalently attached to a sequence or moiety that binds to an immobilized probe; in some such embodiments, the first capture probe target-hybridization sequence is L. Crispatus , L. Genseni and L. Gasseri It specifically hybridizes to the target sequence within each target nucleic acid. In a detailed variation comprising the first and second target capture probes as described above, the first capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 19 of SEQ ID NO. 6, and / or the second capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 20 of SEQ ID NO. 13. In some embodiments, the first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 6, and / or the second capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 13.

[0053] In some embodiments of the composition or kit as described above, the composition or kit is Lactobacillus A first target-hybridization sequence comprising a target-hybridization sequence that specifically hybridizes to a species target region Lactobacillus Specific detection probe, A. Virgin A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region A. Virgin Specific detection probe, and G. Virginalis A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region G. Virginalis It further includes a specific detection probe. In some embodiments, the first A. VirginThe specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19, and / or the first G. Virginalis The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 18 of SEQ ID NO. 16; in a more detailed variation, the 1 A. Virgin The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 6 to 21 of SEQ ID NO. 19 and / or the first G. Virginalis The specific detection probe target-hybridization sequence substantially comprises the nucleotide sequence of residues 1 through 18 of SEQ ID NO. 16. In some embodiments, the first Lactobacillus The specific detection probe target-hybridization sequence is L. Crispatus and L. Genseni The composition or kit specifically hybridizes to the target region of each target nucleic acid, and produces one or more amplification products L. Gasseri specifically hybridizing to the target nucleic acid A second target-hybridization sequence comprising Lactobacillus It further comprises a specific detection probe; in some such embodiments, the first Lactobacillus The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 17 of SEQ ID NO. 11, and / or the second Lactobacillus The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 7 through 23 of SEQ ID NO. 12; in a more detailed variation, the 1 Lactobacillus The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 to 17 of SEQ ID NO. 11, and the 2 Lactobacillus The specific detection probe target-hybridization sequence includes the nucleotide sequence of residues 7 to 23 of SEQ ID NO. 12.

[0054] The 1st Lactobacillus Specific detection probe, 1 A fatherSpecific detection probe and first G. Virginalis In a specific embodiment of a composition or kit comprising a specific detection probe, each probe comprises a label. The second Lactobacillus In some embodiments further comprising a specific detection probe, the second Lactobacillus Specific detection probes include labels. Specifically, suitable labels include chemiluminescent and fluorescent labels. In some variations, each detection probe includes a fluorescent label and a quencher; specifically, suitable detection probes including a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0055] In a specific embodiment of a composition or kit comprising a detection probe as described above, the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis At least one of the specific detection probes further comprises a non-target-hybridization sequence. The second Lactobacillus In some embodiments further comprising a specific detection probe, the second Lactobacillus The specific detection probe further includes a non-target-hybridization sequence. In some of these variations, the first Lactobacillus Specific, first A father Specific and first G. Virginalis Each specific detection probe (or the first Lactobacillus Specific, first Lactobacillus Specific, first A father Specific and first G. Virginalis Each specific detection probe is a molecular torch or molecular beacon.

[0056] In another aspect, the present invention in a sample Lactobacillus Provides a method for determining the presence or absence of a species. This method generally includes the following steps:

[0057] (1) Lactobacillus Samples suspected of containing species LactobacillusA step of contacting a first, second, third, and fourth amplification oligomers for amplifying a target region of a target nucleic acid of a species, wherein (i) the first amplification oligomer comprises a first target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10; (ii) the second amplification oligomer comprises a second target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 7; (iii) the third amplification oligomer comprises a third target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 8; and (iv) the fourth amplification oligomer comprises a fourth target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 9;

[0058] (2) In vitro As a step of performing a nucleic acid amplification reaction, any Lactobacillus If species target nucleic acids are present in the sample, Lactobacillus A step used as a template for generating one or more amplification products corresponding to a species target region; and

[0059] (3) Detect the presence or absence of one or more amplification products, thereby from the sample Lactobacillus A step to determine the presence or absence of a species.

[0060] As mentioned above Lactobacillus In some variations of the method for detecting species, the first Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10 and / or; the 2 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 7 and / or; the 3 Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 8 and / or; the 4 LactobacillusThe specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 9. In some such embodiments, the first Lactobacillus-specific target-hybridization sequence is composed of the nucleotide sequence of residues 28 through 45 of SEQ ID NO. 10 and / or; the second Lactobacillus-specific target-hybridization sequence is composed of the nucleotide sequence of SEQ ID NO. 7 and / or; the third Lactobacillus-specific target-hybridization sequence is composed of the nucleotide sequence of SEQ ID NO. 8 and / or; and the fourth Lactobacillus-specific target-hybridization sequence is composed of the nucleotide sequence of SEQ ID NO. 9.

[0061] As mentioned above Lactobacillus In some embodiments of the method for detecting a species, the first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence. Specifically suitable promoter sequences are, for example, T7 promoter sequences such as the promoter sequence having the nucleotide sequence of residues 1 through 27 of SEQ ID NO. 10. In a detailed variation, the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 10.

[0062] As mentioned above Lactobacillus In a specific embodiment of the method for detecting a species, the method is used if, prior to step (2), the species is present in the sample Lactobacillus The method further includes a step of purifying the species target nucleic acid from other components. In some of these embodiments, the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence binding to an immobilized probe or a target-hybridization sequence covalently attached to a moiety, wherein the capture probe target-hybridization sequence Lactobacillus It specifically hybridizes to a target sequence within the species target nucleic acid. In some embodiments, the capture probe target-hybridization sequence is L. Crispatus , L. Genseni and L. GasseriIt specifically hybridizes to a target sequence within each target nucleic acid; in some such variations, the first capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 19 of SEQ ID NO. 6; and in a more detailed embodiment, the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 6.

[0063] As mentioned above Lactobacillus In some embodiments of the method for detecting species, the detection step (3) is (i) Lactobacillus The method comprises: (ii) contacting a first detection probe comprising a target-hybridization sequence that specifically hybridizes to a species target region with one or more amplification products; and (ii) detecting the presence or absence of any target-hybridized detection probe. In some embodiments, the first specific detection probe target-hybridization sequence is L. Crispatus and L. Genseni The method specifically hybridizes to the target region of each target nucleic acid, and one or more amplification products L. Gasseri specifically hybridizing to the target nucleic acid A second target-hybridization sequence comprising The method further comprises the step of contacting a specific detection probe; in some such embodiments, the first specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 17 of SEQ ID NO. 11, and / or the second specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 7 through 23 of SEQ ID NO. 12; in a more detailed variation, the first specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 through 17 of SEQ ID NO. 11, and the second specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 7 through 23 of SEQ ID NO. 12.

[0064] As mentioned above LactobacillusIn a specific embodiment of the method for detecting a species, where the method includes the use of a first detection probe, the first detection probe includes a label. In some embodiments further comprising contacting one or more amplified oligomers with a second detection probe, the second detection probe includes a label. Specifically suitable labels include chemiluminescent and fluorescent labels.

[0065] Includes the use of a detection probe labeled as above. Lactobacillus In some embodiments of the method for detecting species, the detection step (3) occurs during the amplification step (2). In some variations of this, each detection probe includes a fluorescent label and a quencher. Specifically, suitable detection probes including a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0066] including the use of detection probe(s) as described above Lactobacillus In a specific embodiment of the method for detecting a species, the first detection probe further comprises a non-target-hybridization sequence. In some embodiments further comprising the use of a second detection probe, the second detection probe further comprises a non-target-hybridization sequence. In some such variations, the first detection probe (or each of the first and second detection probes) is a molecular torch or a molecular beacon.

[0067] In another aspect, the present invention in a sample A. Virgin Provides a method for determining the presence or absence of. This method generally includes the following steps:

[0068] (1) A . Virgin Samples suspected of containing A . VirginA step of contacting a first and second amplification oligomers for amplifying a target region of a target nucleic acid, wherein (i) the first amplification oligomer comprises a first target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18; and (ii) the second amplification oligomer comprises a second target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 17;

[0069] (2) In vitro As a step of performing a nucleic acid amplification reaction, any A . Virgin If the target nucleic acid is present in the sample, A . Virgin A step used as a template for generating one or more amplification products corresponding to a target region; and

[0070] (3) Detect the presence or absence of one or more amplification products, thereby from the sample A . Virgin A step to determine the presence or absence of.

[0071] As mentioned above A . Virgin In some variations of the method for detecting, the first target-hybridization sequence comprises the nucleotide sequence of residues 28 through 45 of SEQ ID NO. 18 and / or; and the second target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 17. In some such embodiments, the first target-hybridization sequence is composed of the nucleotide sequence of residues 28 through 45 of SEQ ID NO. 18 and / or; and the second target-hybridization sequence is composed of the nucleotide sequence of SEQ ID NO. 17.

[0072] As mentioned above A. VirginIn some embodiments of the method for detecting, the first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence. Specifically suitable promoter sequences are, for example, T7 promoter sequences such as the promoter sequence having the nucleotide sequence of residues 1 through 27 of SEQ ID NO. 18. In a detailed variation, the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 18.

[0073] As mentioned above A. Virgin In a specific embodiment of the method for detecting, the method is used if, prior to step (2), it is present in the sample A. Virgin The method further includes a step of purifying the target nucleic acid from other components. In some of these embodiments, the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence binding to an immobilized probe or a target-hybridization sequence covalently attached to a moiety, wherein the capture probe target-hybridization sequence A. Virgin It specifically hybridizes to a target sequence within the target nucleic acid. In some embodiments, the capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 20 of SEQ ID NO. 13. In a more detailed variation, the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 13.

[0074] As mentioned above A. Virgin In some embodiments of the method for detecting, the detection step (3) is A. VirginThe method comprises the steps of: contacting one or more amplification products with a detection probe comprising a target-hybridization sequence that specifically hybridizes to a target region; and detecting the presence or absence of any target-hybridized detection probe. In some embodiments, the detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19. In a more detailed variation, the detection probe target-hybridization sequence comprises the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19.

[0075] As mentioned above A. Virgin In a specific embodiment of the method for detecting, where the method includes the use of a detection probe, the detection probe includes a label. Specifically suitable labels include fluorescent and chemiluminescent labels.

[0076] Includes the use of a detection probe labeled as above. A. Virgin In some embodiments of the method for detecting, the detection step (3) occurs during the amplification step (2). In some such variations, the detection probe includes a fluorescent label and a quencher. Specifically, suitable detection probes including a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0077] including the use of a detection probe as described above A. Virgin In a specific embodiment of the method for detecting, the detection probe further comprises a non-target-hybridization sequence. In some of these variations, the detection probe is a molecular torch or a molecular beacon.

[0078] In another aspect, the present invention in a sample G. Virginalis Provides a method for determining the presence or absence of. This method generally includes the following steps:

[0079] (1) G . Virginalis Samples suspected of containing G . VirginalisA step of contacting a first and second amplification oligomers for amplifying a target region of a target nucleic acid, wherein (i) the first amplification oligomer comprises a first target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15; and (ii) the second amplification oligomer comprises a second target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 14;

[0080] (2) In vitro As a step of performing a nucleic acid amplification reaction, any G . Virginalis If the target nucleic acid is present in the sample, G . Virginalis A step used as a template for generating one or more amplification products corresponding to a target region; and

[0081] (3) Detect the presence or absence of one or more amplification products, thereby from the sample G . Virginalis A step to determine the presence or absence of.

[0082] As mentioned above G . Virginalis In some variations of the method for detecting, the first target-hybridization sequence comprises the nucleotide sequence of residues 36 through 52 of SEQ ID NO. 15 and / or; and the second target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 14. In some such embodiments, the first target-hybridization sequence is composed of the nucleotide sequence of residues 36 through 52 of SEQ ID NO. 15 and / or; and the second target-hybridization sequence is composed of the nucleotide sequence of SEQ ID NO. 14.

[0083] As mentioned above G . VirginalisIn some embodiments of the method for detecting, the first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence. Specifically suitable promoter sequences are, for example, T7 promoter sequences such as the promoter sequence having the nucleotide sequence of residues 9 through 36 of SEQ ID NO. 15. In a detailed variation, the first amplification oligomer comprises the nucleotide sequence of residues 9 through 52 of SEQ ID NO. 15; and in some such embodiments, the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 15.

[0084] As mentioned above G . Virginalis In a specific embodiment of the method for detecting, the method is used if, prior to step (2), it is present in the sample G . Virginalis The method further includes a step of purifying the target nucleic acid from other components. In some of these embodiments, the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence binding to an immobilized probe or a target-hybridization sequence covalently attached to a moiety, wherein the capture probe target-hybridization sequence G . Virginalis It specifically hybridizes to a target sequence within the target nucleic acid. In some embodiments, the capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 20 of SEQ ID NO. 13. In a more detailed variation, the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 13.

[0085] As mentioned above G . Virginalis In some embodiments of the method for detecting, the detection step (3) is G . VirginalisThe method comprises the steps of: contacting one or more amplification products with a detection probe comprising a target-hybridization sequence that specifically hybridizes to a target region; and detecting the presence or absence of any target-hybridized detection probe. In some embodiments, the detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 18 of SEQ ID NO. 16. In a more detailed variation, the detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 through 18 of SEQ ID NO. 16.

[0086] As mentioned above G . Virginalis In a specific embodiment of the method for detecting, where the method includes the use of a detection probe, the detection probe includes a label. Specifically suitable labels include fluorescent and chemiluminescent labels.

[0087] Includes the use of a detection probe labeled as above. G . Virginalis In some embodiments of the method for detecting, the detection step (3) occurs during the amplification step (2). In some such variations, the detection probe includes a fluorescent label and a quencher. Specifically, suitable detection probes including a fluorescent label and a quencher include molecular torches, molecular beacons, and TaqMan detection probes.

[0088] including the use of a detection probe as described above G . Virginalis In a specific embodiment of the method for detecting, the detection probe further comprises a non-target-hybridization sequence. In some of these variations, the detection probe is a molecular torch or a molecular beacon.

[0089] As mentioned above Lactobacillus bell, A father and G. VirginalisIn some embodiments of the method for detecting, the amplification reaction in step (2) is an isothermal amplification reaction. In specific variations, the isothermal amplification reaction is a transcription-mediated amplification (TMA) reaction. In certain embodiments, the isothermal amplification reaction is a real-time amplification reaction. means of solving the problem

[0090] These and other aspects of the present invention will become apparent from the following detailed description of the invention. Additionally disclosed embodiments are as follows.

[0091] Embodiment 1 is in a sample Lactobacillus ( Lactobacillus ) bell, A . Virgin ( A. vaginal ) and G . Virginalis ( G. vaginalis A multi-composite method for determining the presence or absence of each, comprising the following steps:

[0092] (1) Lactobacillus bell, A . Inside the pants and G . Jeannalis A sample suspected of containing at least one of the following,

[0093] (a) Lactobacillus The first, second, third, and fourth amplifying the target region of the species target nucleic acid Lactobacillus As a specific amplification oligomer, (i) the 1 Lactobacillus The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10. Lactobacillus (ii) comprising a specific target-hybridization sequence; and (ii) the second Lactobacillus The specific amplification oligomer is a second one substantially corresponding to the nucleotide sequence of SEQ ID NO. 7. Lactobacillus It includes a specific target-hybridization sequence; (iii) the third Lactobacillus The specific amplification oligomer is a third one substantially corresponding to the nucleotide sequence of SEQ ID NO. 8. LactobacillusIt includes a specific target-hybridization sequence; (iv) the 4 Lactobacillus The specific amplification oligomer is the 4th one, which substantially corresponds to the nucleotide sequence of SEQ ID NO. 9. Lactobacillus including a specific target-hybridization sequence, Lactobacillus Specific amplification oligomer;

[0094] (b) A . Inside the pants First and second amplifying the target region of the target nucleic acid A . Inside the pants As a specific amplification oligomer, (i) the 1 A . Inside the pants The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18. A . Inside the pants (ii) comprising a specific target-hybridization sequence; and (ii) the second A . Inside the pants The specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 17. A . Inside the pants including a specific target-hybridization sequence, A . Inside the pants Specific amplification oligomers; and

[0095] (c) G . Jeannalis First and second amplifying the target region of the target nucleic acid G . Jeannalis As a specific amplification oligomer, (i) the 1 G . Jeannalis The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15. G . Jeannalis (ii) comprising a specific target-hybridization sequence; and (ii) the second G . Jeannalis The specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 14. G . Jeannalis including a specific target-hybridization sequence, G . Jeannalis Step of contacting with a specific amplification oligomer;

[0096] (2) In vitro As a step of performing a nucleic acid amplification reaction, any Lactobacillus bell, A . Virgin and G . Virginalis If the target nucleic acid is present in the sample, Lactobacillus bell, A . Virgin and G . Virginalis A step used as a template for generating one or more amplification products corresponding to a target region; and

[0097] (3) Detect the presence or absence of one or more amplification products, thereby from the sample Lactobacillus bell, A . Virgin and G . Virginalis A step to determine the presence or absence of.

[0098] Embodiment 2 is the method of Embodiment 1,

[0099] (1) The 1st Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10 and / or;

[0100] (2) The 2nd Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 7 and / or;

[0101] (3) The 3rd Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 8 and / or;

[0102] (4) The 4th Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 9 and / or;

[0103] (5) The 1st A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18 and / or;

[0104] (6) The 2nd A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 17 and / or;

[0105] (7) The 1st G . Virginalis The specific target-hybridization sequence comprises the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15 and / or;

[0106] (8) The 2nd G . Virginalis The specific target-hybridization sequence is a method comprising the nucleotide sequence of SEQ ID NO. 14.

[0107] Embodiment 3 is a method of Embodiment 1 or 2,

[0108] (1) The 1st Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10 and / or;

[0109] (2) The 2nd Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 7 and / or;

[0110] (3) The 3rd Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 8 and / or;

[0111] (4) The 4th Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 9 and / or;

[0112] (5) The 1st A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18 and / or;

[0113] (6) The 2nd A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 17 and / or;

[0114] (7) The 1st G . Virginalis The specific target-hybridization sequence consists of the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15 and / or;

[0115] (8) The 2nd G . Virginalis The specific target-hybridization sequence is a method consisting of the nucleotide sequence of SEQ ID NO. 14.

[0116] Embodiment 4 is a method of any one of Embodiments 1 to 3, wherein the first Lactobacillus Specific amplification oligomer, 1 A father Specific amplification oligomer and the first G. Virginalis A method in which at least one of the specific amplification oligomers is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence.

[0117] Embodiment 5 is a method of Embodiment 4, wherein the promoter sequence is a T7 promoter sequence.

[0118] Embodiment 6 is a method of Embodiment 5, wherein the promoter sequence has a nucleotide sequence of residues 1 to 27 of SEQ ID NO. 10.

[0119] Embodiment 7 is the method of Embodiment 4,

[0120] The 1st Lactobacillus The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 10 and / or;

[0121] The 1st A. Virgin The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 18 and / or;

[0122] The 1st G. Virginalis The specific amplification oligomer is a method comprising the nucleotide sequence of residues 9 to 52 of SEQ ID NO. 15.

[0123] Embodiment 8 is the method of Embodiment 7, wherein the first G. VirginalisThe specific amplification oligomer is a method comprising the nucleotide sequence of SEQ ID NO. 15.

[0124] Embodiment 9 is a method of any one of Embodiments 1 to 8, wherein, prior to step (2), in the sample, if Lactobacillus bell, A father and G. Virginalis The method further comprises the step of purifying the target nucleic acid from other components.

[0125] Embodiment 10 is a method of Embodiment 9, wherein the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence that binds to an immobilized probe or a target-hybridization sequence covalently attached to a moiety.

[0126] Embodiment 11 is the method of Embodiment 10, wherein a sample is contacted with the first and second capture probe oligomers, and

[0127] The first capture probe oligomer is Lactobacillus It includes a target-hybridization sequence that specifically hybridizes to a target sequence within the species, and the second capture probe target-hybridization sequence is each A. Virgin and G. Virginalis It includes a target-hybridization sequence that specifically hybridizes to a target sequence within a target nucleic acid, and

[0128] The first and second capture probe target-hybridization sequences are each covalently attached to a sequence or moiety that binds to an immobilized probe, in a method.

[0129] Embodiment 12 is the method of Embodiment 11, wherein the first capture probe target-hybridization sequence is L. Crispatus , L. Genseni , and L. Gasseri It is a method that specifically hybridizes to the target sequence within each target nucleic acid.

[0130] Embodiment 13 is the method of Embodiment 12,

[0131] The first capture probe target-hybridization sequence is a method that substantially corresponds to the nucleotide sequence of residues 1 to 19 of SEQ ID NO. 6.

[0132] Embodiment 14 is a method of Embodiment 12 or 13, wherein the second capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO. 13.

[0133] Embodiment 15 is the method of Embodiment 14,

[0134] The first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 6 and / or,

[0135] The second capture probe oligomer is a method comprising the nucleotide sequence of SEQ ID NO. 13.

[0136] Embodiment 16 is a method of any one of Embodiments 1 to 15, wherein the detection step (3) Lactobacillus A first target-hybridization sequence comprising a target-hybridization sequence that specifically hybridizes to a species target region Lactobacillus Specific detection probe, A. Virgin A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region A. Virgin Specific detection probe, and G. Virginalis A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region G. Virginalis A step of contacting a specific detection probe with one or more amplification products; and

[0137] Arbitrary target-hybridized Lactobacillus Specific, A father Specific and G. Virginalis A method comprising the step of detecting the presence or absence of a specific detection probe.

[0138] Embodiment 17 is the method of Embodiment 16,

[0139] The 1st A. VirginThe specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19;

[0140] The 1st G . Virginalis The specific detection probe target-hybridization sequence is a method corresponding to the nucleotide sequences of residues 1 to 18 of SEQ ID NO. 16.

[0141] Embodiment 18 is the method of Embodiment 17,

[0142] The 1st A. Virgin The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19;

[0143] The 1st G . Virginalis The specific detection probe target-hybridization sequence is a method corresponding to the nucleotide sequences of residues 1 to 18 of SEQ ID NO. 16.

[0144] Embodiment 19 is a method of any one of Embodiments 16 to 18, wherein the first Lactobacillus The specific detection probe target-hybridization sequence is L. Crispatus and L. Genseni It specifically hybridizes to the target region of each target nucleic acid, and

[0145] The method involves one or more amplification products L. Gasseri A second target-hybridization sequence comprising a target region of the target nucleic acid that specifically hybridizes to the target region of the target nucleic acid. Lactobacillus It is a method that further includes contacting a specific detection probe.

[0146] Embodiment 20 is the method of Embodiment 19,

[0147] The 1st Lactobacillus The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 17 of SEQ ID NO. 11;

[0148] The 2nd LactobacillusThe specific detection probe target-hybridization sequence is a method that substantially corresponds to the nucleotide sequence of residues 7 through 23 of SEQ ID NO. 12.

[0149] Embodiment 21 is the method of Embodiment 20,

[0150] The 1st Lactobacillus The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 to 17 of SEQ ID NO. 11 and / or;

[0151] The 2nd Lactobacillus The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 7 to 23 of SEQ ID NO. 12.

[0152] Embodiment 22 is a method of any one of Embodiments 16 to 18, wherein the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis Each specific detection probe is a method comprising a label.

[0153] Embodiment 23 is a method of any one of Embodiments 19 to 21, wherein the first Lactobacillus Specific detection probe, 2 Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis Each specific detection probe is a method comprising a label.

[0154] Embodiment 24 is a method of Embodiment 22 or 23, wherein the label is a chemiluminescent label or a fluorescent label.

[0155] Embodiment 25 is a method of Embodiment 22 or 23, wherein the detection step (3) occurs during the amplification step (2).

[0156] Embodiment 26 is a method of Embodiment 25, wherein each detection probe comprises a fluorescent label and a quenching agent.

[0157] Embodiment 27 is a method of Embodiment 26, wherein each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0158] Embodiment 28 is a method of any one of Embodiments 16 to 18, wherein the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis A method in which at least one of the specific detection probes further comprises a non-target-hybridization sequence.

[0159] Embodiment 29 is the method of Embodiment 28, wherein the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis It is a method in which each specific detection probe is a molecular torch or a molecular beacon.

[0160] Embodiment 30 is a method of any one of Embodiments 19 to 21, wherein the first Lactobacillus Specific detection probe, 2 Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis A method in which at least one of the specific detection probes further comprises a non-target-hybridization sequence.

[0161] Embodiment 31 is the method of Embodiment 30, wherein the first Lactobacillus Specific detection probe, 2 Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis It is a method in which each specific detection probe is a molecular torch or a molecular beacon.

[0162] Embodiment 32 is a method of any one of Embodiments 1 to 31, wherein the amplification reaction in step (2) is an isothermal amplification reaction.

[0163] Embodiment 33 is a method of Embodiment 32, wherein the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0164] Embodiment 34 is a method of Embodiment 32 or 33, wherein the amplification reaction is a real-time amplification reaction.

[0165] Embodiment 35 is in a sample Lactobacillus bell, A . Virgin and G . Virginalis A composition or kit for determining the presence or absence of each, comprising the following:

[0166] (a) Lactobacillus The first, second, third, and fourth amplifying the target region of the species target nucleic acid Lactobacillus As a specific amplification oligomer, (i) the 1 Lactobacillus The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10. Lactobacillus (ii) comprising a specific target-hybridization sequence; and (ii) the second Lactobacillus The specific amplification oligomer is a second one substantially corresponding to the nucleotide sequence of SEQ ID NO. 7. Lactobacillus It includes a specific target-hybridization sequence; (iii) the third Lactobacillus The specific amplification oligomer is a third one substantially corresponding to the nucleotide sequence of SEQ ID NO. 8. Lactobacillus It includes a specific target-hybridization sequence; (iv) the 4 Lactobacillus The specific amplification oligomer is the 4th one, which substantially corresponds to the nucleotide sequence of SEQ ID NO. 9. Lactobacillus including a specific target-hybridization sequence, Lactobacillus Specific amplification oligomer;

[0167] (b) A . Inside the pants First and second amplifying the target region of the target nucleic acid A . Inside the pants As a specific amplification oligomer, (i) the 1 A . Inside the pantsThe specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18. A . Inside the pants (ii) comprising a specific target-hybridization sequence; and (ii) the second A . Inside the pants The specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 17. A . Inside the pants including a specific target-hybridization sequence, A . Inside the pants Specific amplification oligomers; and

[0168] (c) G . Virginalis First and second amplifying the target region of the target nucleic acid G . Virginalis As a specific amplification oligomer, (i) the 1 G . Virginalis The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15. G . Virginalis (ii) comprising a specific target-hybridization sequence; and (ii) the second G . Virginalis The specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 14. G . Virginalis including a specific target-hybridization sequence, G . Virginalis Specific amplification oligomer.

[0169] Embodiment 36 is a composition or kit of Embodiment 35,

[0170] The 1st Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10 and / or;

[0171] The 2nd Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 7 and / or;

[0172] The 3rd LactobacillusThe specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 8 and / or;

[0173] The 4th Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 9 and / or;

[0174] The 1st A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18 and / or;

[0175] The 2nd A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 17 and / or;

[0176] The 1st G . Virginalis The specific target-hybridization sequence comprises the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15 and / or;

[0177] The 2nd G . Virginalis The specific target-hybridization sequence is a composition or kit comprising the nucleotide sequence of SEQ ID NO. 14.

[0178] Embodiment 37 is a composition or kit of Embodiment 35 or 36,

[0179] The 1st Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10 and / or;

[0180] The 2nd Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 7 and / or;

[0181] The 3rd Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 8 and / or;

[0182] The 4th Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 9 and / or;

[0183] The 1st A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18 and / or;

[0184] The 2nd A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 17 and / or;

[0185] The 1st G . Virginalis The specific target-hybridization sequence consists of the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15 and / or;

[0186] The 2nd G . Virginalis The specific target-hybridization sequence is a composition or kit consisting of the nucleotide sequence of SEQ ID NO. 14.

[0187] Embodiment 38 is a composition or kit of any one of Embodiments 35 to 37,

[0188] The 1st Lactobacillus Specific amplification oligomer, 1 A father Specific amplification oligomer and the first G. Virginalis At least one of the specific amplification oligomers is a composition or kit that is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence.

[0189] Embodiment 39 is a composition or kit of Embodiment 38 in which the promoter sequence is a T7 promoter sequence.

[0190] Embodiment 40 is a composition or kit of Embodiment 39, wherein the promoter sequence has a nucleotide sequence of residues 1 to 27 of SEQ ID NO. 10.

[0191] Embodiment 41 is a composition or kit of Embodiment 38,

[0192] The 1st LactobacillusThe specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 10 and / or;

[0193] The 1st A. Virgin The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 18 and / or;

[0194] The 1st G. Virginalis The specific amplification oligomer is a composition or kit comprising the nucleotide sequence of residues 9 to 52 of SEQ ID NO. 15.

[0195] Embodiment 42 is a composition or kit of Embodiment 41, wherein the first G. Virginalis It is a composition or kit in which a specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 15.

[0196] Embodiment 43 is a composition or kit of any one of Embodiments 35 to 42, further comprising at least one capture probe oligomer comprising a target-hybridization sequence covalently attached to a sequence or moiety that binds to an immobilized probe.

[0197] Embodiment 44 comprises first and second capture probe oligomers as the composition or kit of Embodiment 43, and

[0198] The first capture probe oligomer is Lactobacillus It includes a target-hybridization sequence that specifically hybridizes to a target sequence within the species, and the second capture probe target-hybridization sequence is each A. Virgin and G. Virginalis It includes a target-hybridization sequence that specifically hybridizes to a target sequence within a target nucleic acid, and

[0199] Each of the first and second capture probe target-hybridization sequences is a composition or kit that is covalently attached to a sequence or moiety that binds to an immobilized probe.

[0200] Embodiment 45 is a composition or kit of Embodiment 44, wherein the first capture probe target-hybridization sequence is L. Crispatus , L. Genseni , and L. Gasseri It is a composition or kit that specifically hybridizes to the target sequence within each target nucleic acid.

[0201] Embodiment 46 is a composition or kit of Embodiment 45,

[0202] The first capture probe target-hybridization sequence is a composition or kit substantially corresponding to the nucleotide sequence of residues 1 to 19 of SEQ ID NO. 6.

[0203] Embodiment 47 is a composition or kit of Embodiment 45 or 46, wherein the second capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO. 13.

[0204] Embodiment 48 is a composition or kit of Embodiment 47,

[0205] The first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 6 and / or,

[0206] The second capture probe oligomer is a composition or kit comprising the nucleotide sequence of SEQ ID NO. 13.

[0207] Embodiment 49 is a composition or kit of any one of Embodiments 35 to 48, Lactobacillus A first target-hybridization sequence comprising a target-hybridization sequence that specifically hybridizes to a species target region Lactobacillus Specific detection probe, A. Virgin A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region A. Virgin Specific detection probe, and G. Virginalis A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region G. Virginalis It is a composition or kit that further comprises a specific detection probe.

[0208] Embodiment 50 is a composition or kit of Embodiment 49,

[0209] The 1st A. VirginThe specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19;

[0210] The 1st G . Virginalis The specific detection probe target-hybridization sequence is a composition or kit substantially corresponding to the nucleotide sequence of residues 1 to 18 of SEQ ID NO. 16.

[0211] Embodiment 51 is a composition or kit of Embodiment 50,

[0212] The 1st A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19 and / or;

[0213] The 1st G . Virginalis The specific target-hybridization sequence is a composition or kit comprising the nucleotide sequence of residues 1 to 18 of SEQ ID NO. 16.

[0214] Embodiment 52 is a composition or kit of any one of Embodiments 49 to 51, wherein the first Lactobacillus The specific detection probe target-hybridization sequence is L. Crispatus and L. Genseni It specifically hybridizes to the target region of each target nucleic acid, and

[0215] The composition or kit L. Gasseri A second target-hybridization sequence comprising a target region of the target nucleic acid that specifically hybridizes to the target region of the target nucleic acid. Lactobacillus It is a composition or kit that further comprises a specific detection probe.

[0216] Embodiment 53 is a composition or kit of Embodiment 52,

[0217] The 1st Lactobacillus The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 17 of SEQ ID NO. 11;

[0218] The 2nd Lactobacillus The specific detection probe target-hybridization sequence is a composition or kit that substantially corresponds to the nucleotide sequence of residues 7 to 23 of SEQ ID NO. 12.

[0219] Embodiment 54 is a composition or kit of Embodiment 53,

[0220] The 1st Lactobacillus The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 to 17 of SEQ ID NO. 11 and / or;

[0221] The 2nd Lactobacillus The specific detection probe target-hybridization sequence is a composition or kit comprising the nucleotide sequence of residues 7 to 23 of SEQ ID NO. 12.

[0222] Embodiment 55 is a composition or kit of any one of Embodiments 49 to 51, wherein the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis Each specific detection probe is a composition or kit containing a label.

[0223] Embodiment 56 is a composition or kit of any one of Embodiments 52 to 54, wherein the first Lactobacillus Specific detection probe, 2 Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis Each specific detection probe is a composition or kit containing a label.

[0224] Embodiment 57 is a composition or kit of Embodiment 55 or 56, wherein the label is a chemiluminescent label or a fluorescent label.

[0225] Embodiment 58 is a composition or kit of Embodiment 55 or 56, wherein each detection probe comprises a fluorescent label and a quenching agent.

[0226] Embodiment 59 is a composition or kit of Embodiment 58, wherein each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0227] Embodiment 60 is a composition or kit of any one of Embodiments 49 to 51, wherein the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis At least one of the specific detection probes is a composition or kit that further comprises a non-target hybridization sequence.

[0228] Embodiment 61 is a composition or kit of Embodiment 60, wherein the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis Each specific detection probe is a composition or kit that is a molecular torch or molecular beacon.

[0229] Embodiment 62 is a composition or kit of any one of Embodiments 52 to 54, wherein the first Lactobacillus Specific detection probe, 2 Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis At least one of the specific detection probes is a composition or kit that further comprises a non-target hybridization sequence.

[0230] Embodiment 63 is a composition or kit of Embodiment 62, wherein the first Lactobacillus Specific detection probe, 2 Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis Each specific detection probe is a composition or kit that is a molecular torch or molecular beacon.

[0231] Embodiment 64 is a method for determining the presence or absence of bacterial vaginosis (BV) in a subject, comprising the following steps:

[0232] (a) A step of providing a sample from a subject suspected of having BV;

[0233] (b) In the sample Lactobacillus bell, A . Virgin and G . Virginalis A step of performing a test method for the detection of;

[0234] (c) Lactobacillus bell, A . Virgin and G . Virginalis A step of assigning a quantitative value based on a detection test method to each;

[0235] (d) A . Virgin The quantitative value of and G . Virginalis From the larger of the quantitative values ​​of Lactobacillus A step of subtracting the quantitative value of the species;

[0236] (e) a step of assigning a single BV score based on step (d); and

[0237] (f) A step of determining the presence or absence of BV in the subject based on a comparison of the BV score and the cutoff value.

[0238] Embodiment 65 is the method of Embodiment 64, in step (c) Lactobacillus It is a method in which a minimum quantitative value is assigned to the species.

[0239] Embodiment 66 is the method of Embodiment 64 or 65, in step (c) A . Virgin and G . Virginalis It is a method in which a minimum quantitative value is assigned to it.

[0240] Embodiment 67 is a method of any one of Embodiments 64 to 66, Lactobacillus bell, A father and G. Virginalis The quantitative value for each is a method of standardization.

[0241] Embodiment 68 is the method of Embodiment 67, Lactobacillus bell, A father and G. Virginalis The quantitative value for each is in units of log copies, and standardization is a method involving subtracting the median population value from the value determined by the detection test.

[0242] Embodiment 69 is a method of any one of Embodiments 64 to 68, Lactobacillus bell, A father and G. Virginalis The quantitative value for each is a method in which weights are assigned.

[0243] Embodiment 70 is a method of any one of Embodiments 64 to 69, wherein step (d) is a method of additionally adding an adjustment constant.

[0244] Embodiment 71 is a method of any one of Embodiments 64 to 69, wherein step (d) further comprises adding an internal control (IC) adjustment factor that compensates for sample inhibition of the detection test, wherein the IC adjustment factor is based on the ratio of (ii) the expected IC value for the detection test to (ii) the observed IC value generated from the detection test.

[0245] Embodiment 72 is the method of Embodiment 64, wherein the BV score is assigned using the following formula, and

[0246] BV Score = C0 + W L Maximum value (L S , F LS ) + W GA Maximum value (A S , G S , F GAS ) + W IC log2(IC ratio),

[0247] Here, C0 is the adjustment constant;

[0248] W L silver Lactobacillus It is a weighting constant for the species;

[0249] Maximum value (L S , FLS ) is L S and F LS Larger than, L S Is Lactobacillus It is the observed standardized quantitative value of the species, and F LS Is Lactobacillus It is the minimum standardized quantitative value assigned to the species;

[0250] W GA Is A. Virgin and G. Virginalis It is a weight constant for;

[0251] Maximum value (A S , G S , F GAS ) is A S , G S and F GAS Greater than, and A S Is A father is the observed standardized quantitative value of, and G S Is G. Virginalis is the observed standardized quantitative value of, and F GAS Is A father and G. Virginalis It is the assigned minimum standardized quantitative value;

[0252] W IC is an internal control group (IC) weighting constant;

[0253] The IC ratio is the ratio of (i) the observed internal control (IC) value generated from the detection test to (ii) the expected IC value for the detection test.

[0254] Example 73 is F GAS This is the method of Implementation Example 72, where ga is 0.

[0255] Embodiment 74 is a method of any one of Embodiments 64 to 73, Lactobacillus bell, A father and G. Virginalis The assay for the detection of is a nucleic acid-based detection assay.

[0256] Embodiment 75 is the method of Embodiment 74, wherein the nucleic acid-based detection assay method Lactobacillus bell, A father and G. Virginalis It is a method that targets the 16S rRNA.

[0257] Embodiment 76 is the method of Embodiment 75, wherein the nucleic acid-based detection assay targets the following:

[0258] (a) Corresponding to the region of approximately nucleotide positions 40 to 265 of SEQ ID NO. 1 L. Crispatus 16S rRNA region;

[0259] (b) Corresponding to the region of approximately nucleotide positions 43 to 247 of SEQ ID NO. 2 L. Crispatus 16S rRNA region;

[0260] (c) Corresponding to the region of approximately nucleotide positions 93 to 298 of SEQ ID NO. 3 L. Crispatus 16S rRNA region;

[0261] (d) Corresponding to the region of approximately nucleotide positions 540 to 625 of SEQ ID NO. 4 L. Crispatus 16S rRNA region; and / or

[0262] (e) corresponding to the region of approximately nucleotide positions 172 to 227 of SEQ ID NO. 5 L. Crispatus 16S rRNA region.

[0263] Embodiment 77 is a method of any one of Embodiments 74 to 76, wherein the nucleic acid-based detection assay is an amplification-based assay.

[0264] Embodiment 78 is the method of Embodiment 77, in which the amplification-based test method includes an isothermal amplification reaction.

[0265] Embodiment 79 is a method of Embodiment 78, wherein the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0266] Embodiment 80 is a method of Embodiment 78 or 79, wherein the amplification reaction is a real-time amplification reaction.

[0267] Embodiment 81 is a method of Embodiment 79, wherein the nucleic acid-based detection assay is an amplification-based assay comprising the following steps:

[0268] (1) Sample

[0269] Lactobacillus The first, second, third, and fourth amplifying the target region of the species target nucleic acid Lactobacillus As a specific amplification oligomer, (i) a first corresponding to the nucleotide sequences of residues 28 to 45 of SEQ ID NO. 10 substantially thereof, of a method for diagnosing BV comprising an amplification-based detection assay as described above. Lactobacillus (ii) comprising a specific target-hybridization sequence; and (ii) the second Lactobacillus The specific amplification oligomer is a second one substantially corresponding to the nucleotide sequence of SEQ ID NO. 7. Lactobacillus It includes a specific target-hybridization sequence; (iii) the third Lactobacillus The specific amplification oligomer is a third one substantially corresponding to the nucleotide sequence of SEQ ID NO. 8. Lactobacillus It includes a specific target-hybridization sequence; (iv) the 4 Lactobacillus The specific amplification oligomer is the 4th one, which substantially corresponds to the nucleotide sequence of SEQ ID NO. 9. Lactobacillus including a specific target-hybridization sequence, Lactobacillus Specific amplification oligomer;

[0270] A . Virgin First and second amplifying the target region of the target nucleic acid A . Virgin As a specific amplification oligomer, (i) the 1 A . Virgin The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18. A . Virgin (ii) comprising a specific target-hybridization sequence; and (ii) the second A . VirginThe specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 17. A . Virgin including a specific target-hybridization sequence, A . Virgin Specific amplification oligomers; and

[0271] G . Virginalis First and second amplifying the target region of the target nucleic acid G . Virginalis As a specific amplification oligomer, (i) the 1 G . Virginalis The specific amplification oligomer is substantially the first corresponding to the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15. G . Virginalis (ii) comprising a specific target-hybridization sequence; and (ii) the second G . Virginalis The specific amplification oligomer is substantially the second corresponding to the nucleotide sequence of SEQ ID NO. 14. G . Virginalis including a specific target-hybridization sequence, G . Virginalis Step of contacting with a specific amplification oligomer;

[0272] (2) In vitro As a step of performing a nucleic acid amplification reaction, any Lactobacillus bell, A . Virgin and G . Virginalis If the target nucleic acid is present in the sample, Lactobacillus bell, A . Virgin and G . Virginalis A step used as a template for generating one or more amplification products corresponding to a target region; and

[0273] (3) A step of detecting the presence or absence of one or more amplification products.

[0274] Embodiment 82 is the method of Embodiment 81,

[0275] The 1st Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10 and / or;

[0276] The 2nd Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 7 and / or;

[0277] The 3rd Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 8 and / or;

[0278] The 4th Lactobacillus The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 9 and / or;

[0279] The 1st A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18 and / or;

[0280] The 2nd A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 17 and / or;

[0281] The 1st G . Virginalis The specific target-hybridization sequence comprises the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15 and / or;

[0282] The 2nd G . Virginalis The specific target-hybridization sequence is a method comprising the nucleotide sequence of SEQ ID NO. 14.

[0283] Embodiment 83 is a method of Embodiment 81 or 82,

[0284] The 1st Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10 and / or;

[0285] The 2nd Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 7 and / or;

[0286] The 3rd Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 8 and / or;

[0287] The 4th Lactobacillus The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 9 and / or;

[0288] The 1st A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18 and / or;

[0289] The 2nd A . Virgin The specific target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 17 and / or;

[0290] The 1st G . Virginalis The specific target-hybridization sequence consists of the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15 and / or;

[0291] The 2nd G . Virginalis The specific target-hybridization sequence is a method consisting of the nucleotide sequence of SEQ ID NO. 14.

[0292] Embodiment 84 is a method of any one of Embodiments 81 to 83,

[0293] The 1st Lactobacillus Specific amplification oligomer, 1 A father Specific amplification oligomer and the first G. Virginalis At least one of the specific amplification oligomers is a composition or kit that is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence.

[0294] Embodiment 85 is a method of Embodiment 84, wherein the promoter sequence is a T7 promoter sequence.

[0295] Embodiment 86 is a method of Embodiment 85, wherein the promoter sequence has a nucleotide sequence of residues 1 to 27 of SEQ ID NO. 10.

[0296] Embodiment 87 is the method of Embodiment 84,

[0297] The 1st Lactobacillus The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 10 and / or;

[0298] The 1st A. Virgin The specific amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 18 and / or;

[0299] The 1st G. Virginalis The specific amplification oligomer is a method comprising the nucleotide sequence of residues 9 to 52 of SEQ ID NO. 15.

[0300] Embodiment 88 is the method of Embodiment 87, wherein the first G. Virginalis The specific amplification oligomer is a method comprising the nucleotide sequence of SEQ ID NO. 15.

[0301] Embodiment 89 is a method of any one of Embodiments 81 to 88, wherein, prior to step (2), in the sample, if Lactobacillus bell, A father and G. Virginalis The method further comprises the step of purifying the target nucleic acid from other components.

[0302] Embodiment 90 is a method of Embodiment 89, wherein the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence that binds to an immobilized probe or a target-hybridization sequence covalently attached to a moiety.

[0303] Embodiment 91 is the method of Embodiment 90, wherein a sample is contacted with first and second capture probe oligomers, and

[0304] The first capture probe oligomer is LactobacillusIt includes a target-hybridization sequence that specifically hybridizes to a target sequence within the species, and the second capture probe target-hybridization sequence is each A. Virgin and G. Virginalis It includes a target-hybridization sequence that specifically hybridizes to a target sequence within a target nucleic acid, and

[0305] The first and second capture probe target-hybridization sequences are each covalently attached to a sequence or moiety that binds to an immobilized probe, in a method.

[0306] Embodiment 92 is a method of Embodiment 91, wherein the first capture probe target-hybridization sequence is L. Crispatus , L. Genseni , and L. Gasseri It is a method that specifically hybridizes to the target sequence within each target nucleic acid.

[0307] Embodiment 93 is the method of Embodiment 92,

[0308] The first capture probe target-hybridization sequence is a method that substantially corresponds to the nucleotide sequence of residues 1 to 19 of SEQ ID NO. 6.

[0309] Embodiment 94 is a method of Embodiment 92 or 93, wherein the second capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO. 13.

[0310] Embodiment 95 is the method of Embodiment 94,

[0311] The first capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 6 and / or,

[0312] The second capture probe oligomer is a method comprising the nucleotide sequence of SEQ ID NO. 13.

[0313] Embodiment 96 is a method of any one of Embodiments 81 to 95, wherein the detection step (3) Lactobacillus A first target-hybridization sequence comprising a target-hybridization sequence that specifically hybridizes to a species target region Lactobacillus Specific detection probe, A. Virgin A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region A. Virgin Specific detection probe, and G. Virginalis A first target-hybridization sequence comprising a target region that specifically hybridizes to the target region G. Virginalis A step of contacting a specific detection probe with one or more amplification products; and

[0314] Arbitrary target-hybridized Lactobacillus Specific, A father Specific and G. Virginalis A method comprising the step of detecting the presence or absence of a specific detection probe.

[0315] Embodiment 97 is the method of Embodiment 96,

[0316] The 1st A. Virgin The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19;

[0317] The 1st G . Virginalis The specific detection probe target-hybridization sequence is a composition or kit substantially corresponding to the nucleotide sequence of residues 1 to 18 of SEQ ID NO. 16.

[0318] Embodiment 98 is the method of Embodiment 97,

[0319] The 1st A . Virgin The specific target-hybridization sequence comprises the nucleotide sequence of residues 6 through 21 of SEQ ID NO. 19 and / or;

[0320] The 1st G . Virginalis The specific target-hybridization sequence is a method comprising the nucleotide sequence of residues 1 to 18 of SEQ ID NO. 16.

[0321] Embodiment 99 is a method of any one of Embodiments 96 to 98, wherein the first Lactobacillus The specific detection probe target-hybridization sequence is L. Crispatus and L. GenseniIt specifically hybridizes to the target region of each target nucleic acid, and

[0322] The method involves one or more amplification products L. Gasseri A second target-hybridization sequence comprising a target region of the target nucleic acid that specifically hybridizes to the target region of the target nucleic acid. Lactobacillus It is a method that further includes contacting a specific detection probe.

[0323] Embodiment 100 is the method of Embodiment 99,

[0324] The 1st Lactobacillus The specific detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 17 of SEQ ID NO. 11;

[0325] The 2nd Lactobacillus The specific detection probe target-hybridization sequence is a method that substantially corresponds to the nucleotide sequence of residues 7 through 23 of SEQ ID NO. 12.

[0326] Embodiment 101 is the method of Embodiment 100,

[0327] The 1st Lactobacillus The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 to 17 of SEQ ID NO. 11 and / or;

[0328] The 2nd Lactobacillus The specific detection probe target-hybridization sequence comprises the nucleotide sequence of residues 7 to 23 of SEQ ID NO. 12.

[0329] Embodiment 102 is a method of any one of Embodiments 96 to 98, wherein the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis Each specific detection probe is a method comprising a label.

[0330] Embodiment 103 is a method of any one of Embodiments 99 to 101, wherein the first Lactobacillus Specific detection probe, 2 LactobacillusSpecific detection probe, 1 A father Specific detection probe and first G. Virginalis Each specific detection probe is a method comprising a label.

[0331] Embodiment 104 is a method of Embodiment 102 or 103, wherein the label is a chemiluminescent label or a fluorescent label.

[0332] Embodiment 105 is a method of Embodiment 102 or 103, wherein the detection step (3) occurs during the amplification step (2).

[0333] Embodiment 106 is a method of Embodiment 105, wherein each detection probe comprises a fluorescent label and a quenching agent.

[0334] Embodiment 107 is a method of Embodiment 106, wherein each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0335] Embodiment 108 is a method of any one of Embodiments 96 to 98, wherein the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis A method in which at least one of the specific detection probes further comprises a non-target-hybridization sequence.

[0336] Embodiment 109 is the method of Embodiment 108, wherein the first Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis It is a method in which each specific detection probe is a molecular torch or a molecular beacon.

[0337] Embodiment 110 is a method of any one of Embodiments 99 to 101, wherein the first Lactobacillus Specific detection probe, 2 Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. VirginalisA method in which at least one of the specific detection probes further comprises a non-target-hybridization sequence.

[0338] Embodiment 111 is the method of Embodiment 110, wherein the first Lactobacillus Specific detection probe, 2 Lactobacillus Specific detection probe, 1 A father Specific detection probe and first G. Virginalis It is a method in which each specific detection probe is a molecular torch or a molecular beacon.

[0339] Embodiment 112 is a method of any one of Embodiments 81 to 111, wherein the amplification reaction in step (2) is an isothermal amplification reaction.

[0340] Embodiment 113 is a method of Embodiment 112, wherein the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0341] Embodiment 114 is a method of Embodiment 112 or 113, wherein the amplification reaction is a real-time amplification reaction.

[0342] Embodiment 115 is a method of any one of Embodiments 64 to 114, comprising the detection of 10 or fewer bacterial genera associated with BV.

[0343] Embodiment 116 is a method of any one of Embodiments 64 to 114, comprising the detection of five or fewer bacterial genera associated with BV.

[0344] Embodiment 117 is a method of any one of Embodiments 64 to 114, Lactobacillus , Atopobium , Gardnera It is a method that does not include the detection of other BV-related bacterial genera.

[0345] Embodiment 118 is a method of any one of Embodiments 64 to 117, which further comprises administering a therapeutic diet for BV to a subject when the presence of BV appears in the subject.

[0346] Embodiment 119 is a method of any one of Embodiments 64 to 117, a method for monitoring BV in a subject, and a method in which the subject undergoes treatment for BV prior to step (a).

[0347] Embodiment 120 is a method of Embodiment 119, wherein when the presence of BV appears in a subject, the method further comprises (i) administering a therapeutic diet for BV to the subject, or (ii) administering a different therapeutic diet for BV to the subject.

[0348] Embodiment 121 is in a sample Lactobacillus A method for determining the presence or absence of a species, comprising the following steps:

[0349] (1) Lactobacillus Samples suspected of containing species Lactobacillus A step of contacting a first, second, third, and fourth amplification oligomers for amplifying a target region of a target nucleic acid of a species, wherein (i) the first amplification oligomer comprises a first target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10; (ii) the second amplification oligomer comprises a second target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 7; (iii) the third amplification oligomer comprises a third target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 8; and (iv) the fourth amplification oligomer comprises a fourth target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 9;

[0350] (2) In vitro As a step of performing a nucleic acid amplification reaction, any Lactobacillus If species target nucleic acids are present in the sample, Lactobacillus A step used as a template for generating one or more amplification products corresponding to a species target region; and

[0351] (3) Detect the presence or absence of one or more amplification products, thereby from the sample Lactobacillus A step to determine the presence or absence of a species.

[0352] Embodiment 122 is the method of Embodiment 121,

[0353] The first target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10 and / or;

[0354] The second target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 7 and / or;

[0355] The third target-hybridization sequence comprises the nucleotide sequence of SEQ ID NO. 8 and / or;

[0356] The fourth target-hybridization sequence is a method comprising the nucleotide sequence of SEQ ID NO. 9.

[0357] Embodiment 123 is the method of Embodiment 122,

[0358] The first target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10 and / or;

[0359] The second target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 7 and / or;

[0360] The third target-hybridization sequence consists of the nucleotide sequence of SEQ ID NO. 8 and / or;

[0361] The fourth target-hybridization sequence is a method consisting of the nucleotide sequence of SEQ ID NO. 9.

[0362] Embodiment 124 is a method of any one of Embodiments 121 to 123, wherein the first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence.

[0363] Embodiment 125 is a method of Embodiment 124, wherein the promoter sequence is a T7 promoter sequence.

[0364] Embodiment 126 is a method of Embodiment 125, wherein the promoter sequence has a nucleotide sequence of residues 1 to 27 of SEQ ID NO. 10.

[0365] Embodiment 127 is the method of Embodiment 124,

[0366] The first amplification oligomer is a method comprising the nucleotide sequence of SEQ ID NO. 10.

[0367] Embodiment 128 is a method of any one of Embodiments 121 to 127, wherein, prior to step (2), in the sample, if Lactobacillus The method further comprises the step of purifying the species target nucleic acid from other components.

[0368] Embodiment 129 is a method of Embodiment 128, wherein the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence binding to an immobilized probe or a target-hybridization sequence covalently attached to a moiety, wherein the capture probe target-hybridization sequence Lactobacillus It is a method that specifically hybridizes to a target sequence within a species target nucleic acid.

[0369] Embodiment 130 is the method of Embodiment 129, wherein the capture probe target-hybridization sequence is L. Crispatus , L. Genseni , and L. Gasseri It is a method that specifically hybridizes to the target sequence within each target nucleic acid.

[0370] Embodiment 131 is the method of Embodiment 130,

[0371] The first capture probe target-hybridization sequence is a method that substantially corresponds to the nucleotide sequence of residues 1 to 19 of SEQ ID NO. 6.

[0372] Embodiment 132 is a method of Embodiment 131, wherein the capture probe oligomer sequence comprises the nucleotide sequence of SEQ ID NO. 6.

[0373] Embodiment 133 is a method of any one of Embodiments 121 to 132, wherein the detection step (3) Lactobacillus A step of contacting a first detection probe comprising a target-hybridization sequence that specifically hybridizes to a species target region with one or more amplification products; and

[0374] A method comprising the step of detecting the presence or absence of any target-hybridized detection probe.

[0375] Embodiment 134 is the method of Embodiment 133, wherein the first Lactobacillus The specific detection probe target-hybridization sequence is L. Crispatus and L. Genseni It specifically hybridizes to the target region of each target nucleic acid, and

[0376] One or more amplification products L. Gasseri The method further comprises contacting a second detection probe containing a target-hybridization sequence that specifically hybridizes to a target region of the target nucleic acid.

[0377] Embodiment 135 is the method of Embodiment 134,

[0378] The first detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 through 17 of SEQ ID NO. 11, and / or,

[0379] The second detection probe target-hybridization sequence is a method that substantially corresponds to the nucleotide sequence of residues 7 through 23 of SEQ ID NO. 12.

[0380] Embodiment 136 is the method of Embodiment 135,

[0381] The first detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 to 17 of SEQ ID NO. 11 and / or;

[0382] The method is one in which the second detection probe target-hybridization sequence comprises the nucleotide sequence of residues 7 to 23 of SEQ ID NO. 12.

[0383] Embodiment 137 is a method of Embodiment 133, wherein the first detection probe includes a label.

[0384] Embodiment 138 is a method of Embodiment 137, wherein the label is a chemiluminescent label or a fluorescent label.

[0385] Embodiment 139 is a method of Embodiment 137 in which the detection step (3) occurs during the amplification step (2).

[0386] Embodiment 140 is a method of Embodiment 139, wherein the detection probe comprises a fluorescent label and a quenching agent.

[0387] Embodiment 141 is a method of Embodiment 140, wherein the detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0388] Embodiment 142 is a method of any one of Embodiments 134 to 136, wherein the first and second Lactobacillus Each specific detection probe is a method comprising a label.

[0389] Embodiment 143 is a method of Embodiment 142, wherein the label is a chemiluminescent label or a fluorescent label.

[0390] Embodiment 144 is a method of Embodiment 142 in which the detection step (3) occurs during the amplification step (2).

[0391] Embodiment 145 is a method of Embodiment 144, wherein each detection probe comprises a fluorescent label and a quenching agent.

[0392] Embodiment 146 is a method of Embodiment 145, wherein each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0393] Embodiment 147 is a method of Embodiment 133, wherein the first detection probe further comprises a non-target-hybridized sequence.

[0394] Embodiment 148 is a method of Embodiment 147, wherein the first detection probe is a molecular torch or a molecular beacon.

[0395] Embodiment 149 is a method of any one of embodiments 134 to 136, wherein at least one of the first and second detection probes further comprises a non-target-hybridization sequence.

[0396] Embodiment 150 is a method of Embodiment 149, wherein the first and second detection probes are each a molecular torch or a molecular beacon.

[0397] Embodiment 151 is in a sample A . Virgin A method for determining the presence or absence of, comprising the following steps:

[0398] (1) A . Virgin Samples suspected of containing A . Virgin A step of contacting a first and second amplification oligomers for amplifying a target region of a target nucleic acid, wherein (i) the first amplification oligomer comprises a first target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18; and (ii) the second amplification oligomer comprises a second target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 17;

[0399] (2) In vitro As a step of performing a nucleic acid amplification reaction, any A . Virgin If the target nucleic acid is present in the sample, A . Virgin A step used as a template for generating one or more amplification products corresponding to a target region; and

[0400] (3) Detect the presence or absence of one or more amplification products, thereby from the sample A . VirginA step to determine the presence or absence of.

[0401] Embodiment 152 is the method of Embodiment 151,

[0402] The first target-hybridization sequence comprises the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18 and / or;

[0403] The second target-hybridization sequence is a method comprising the nucleotide sequence of SEQ ID NO. 17.

[0404] Embodiment 153 is a method of Embodiment 151 or 152,

[0405] The first target-hybridization sequence consists of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 18 and / or;

[0406] The second target-hybridization sequence is a method consisting of the nucleotide sequence of SEQ ID NO. 17.

[0407] Embodiment 154 is a method of any one of Embodiments 151 to 153, wherein the first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence.

[0408] Embodiment 155 is a method of Embodiment 154, wherein the promoter sequence is a T7 promoter sequence.

[0409] Embodiment 156 is a method of Embodiment 155, wherein the promoter sequence has a nucleotide sequence of residues 1 to 27 of SEQ ID NO. 18.

[0410] Embodiment 157 is a method of Embodiment 154, wherein the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 18.

[0411] Embodiment 158 ​​is a method of any one of Embodiments 151 to 157, wherein, prior to step (2), in the sample, if A. VirginThe method further comprises the step of purifying the target nucleic acid from other components.

[0412] Embodiment 159 is a method of Embodiment 158, wherein the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence binding to an immobilized probe or a target-hybridization sequence covalently attached to a moiety, wherein the capture probe target-hybridization sequence A. Virgin It is a method that specifically hybridizes to a target sequence within a target nucleic acid.

[0413] Embodiment 160 is a method of Embodiment 159, wherein the capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO. 13.

[0414] Embodiment 161 is a method of Embodiment 160, wherein the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 13.

[0415] Embodiment 162 is a method of any one of Embodiments 151 to 161, wherein the detection step (3) A. Virgin A step of contacting a detection probe comprising a target-hybridization sequence that specifically hybridizes to a target region with one or more amplification products; and

[0416] A method comprising the step of detecting the presence or absence of any target-hybridized detection probe.

[0417] Embodiment 163 is a method of Embodiment 162, wherein the detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 6 to 21 of SEQ ID NO. 19.

[0418] Embodiment 164 is a method of Embodiment 163, wherein the detection probe target-hybridization sequence comprises the nucleotide sequence of residues 6 to 21 of SEQ ID NO. 19.

[0419] Embodiment 165 is a method of any one of Embodiments 162 to 164, wherein the detection probe includes a label.

[0420] Embodiment 166 is a method of Embodiment 165, wherein the label is a chemiluminescent label or a fluorescent label.

[0421] Embodiment 167 is a method of Embodiment 165, wherein the detection step (3) occurs during the amplification step (2).

[0422] Embodiment 168 is a method of Embodiment 167, wherein the detection probe comprises a fluorescent label and a quenching agent.

[0423] Embodiment 169 is a method of Embodiment 168, wherein the detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0424] Embodiment 170 is a method of any one of Embodiments 162 to 164, wherein the detection probe further comprises a non-target-hybridized sequence.

[0425] Embodiment 171 is a method of Embodiment 170, wherein the detection probe is a molecular torch or a molecular beacon.

[0426] Embodiment 172 is in a sample G . Virginalis A method for determining the presence or absence of, comprising the following steps:

[0427] (1) G . Virginalis Samples suspected of containing G . VirginalisA step of contacting a first and second amplification oligomers for amplifying a target region of a target nucleic acid, wherein (i) the first amplification oligomer comprises a first target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15; and (ii) the second amplification oligomer comprises a second target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 14;

[0428] (2) In vitro As a step of performing a nucleic acid amplification reaction, any G . Virginalis If the target nucleic acid is present in the sample, G . Virginalis A step used as a template for generating one or more amplification products corresponding to a target region; and

[0429] (3) Detect the presence or absence of one or more amplification products, thereby from the sample G . Virginalis A step to determine the presence or absence of.

[0430] Embodiment 173 is the method of Embodiment 172,

[0431] The first target-hybridization sequence comprises the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15 and / or;

[0432] The second target-hybridization sequence is a method comprising the nucleotide sequence of SEQ ID NO. 14.

[0433] Embodiment 174 is the method of Embodiment 172 or 173,

[0434] The first target-hybridization sequence consists of the nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15 and / or;

[0435] The second target-hybridization sequence is a method consisting of the nucleotide sequence of SEQ ID NO. 14.

[0436] Embodiment 175 is a method of any one of Embodiments 172 to 174, wherein the first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence.

[0437] Embodiment 176 is a method of Embodiment 175, wherein the promoter sequence is a T7 promoter sequence.

[0438] Embodiment 177 is a method of Embodiment 176, wherein the promoter sequence has a nucleotide sequence of residues 9 to 36 of SEQ ID NO. 15.

[0439] Embodiment 178 is a method of Embodiment 175, wherein the first amplification oligomer comprises the nucleotide sequence of residues 9 to 52 of SEQ ID NO. 15.

[0440] Embodiment 179 is a method of Embodiment 178, wherein the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 15.

[0441] Embodiment 180 is a method of any one of Embodiments 172 to 179, wherein, prior to step (2), in the sample, if G. Virginalis The method further comprises the step of purifying the target nucleic acid from other components.

[0442] Embodiment 181 is a method of Embodiment 180, wherein the purification step comprises contacting a sample with at least one capture probe oligomer comprising a sequence binding to an immobilized probe or a target-hybridization sequence covalently attached to a moiety, wherein the capture probe target-hybridization sequence G. Virginalis It is a method that specifically hybridizes to a target sequence within a target nucleic acid.

[0443] Embodiment 182 is a method of Embodiment 181, wherein the capture probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 to 20 of SEQ ID NO. 13.

[0444] Embodiment 183 is a method of Embodiment 182, wherein the capture probe oligomer comprises the nucleotide sequence of SEQ ID NO. 13.

[0445] Embodiment 184 is a method of any one of Embodiments 172 to 183, wherein the detection step (3) G. Virginalis A step of contacting a detection probe comprising a target-hybridization sequence that specifically hybridizes to a target region with one or more amplification products; and

[0446] A method comprising the step of detecting the presence or absence of any target-hybridized detection probe.

[0447] Embodiment 185 is a method of Embodiment 184, wherein the detection probe target-hybridization sequence substantially corresponds to the nucleotide sequence of residues 1 to 18 of SEQ ID NO. 16.

[0448] Embodiment 186 is a method of Embodiment 185, wherein the detection probe target-hybridization sequence comprises the nucleotide sequence of residues 1 to 18 of SEQ ID NO. 16.

[0449] Embodiment 187 is a method of any one of Embodiments 184 to 186, wherein the detection probe includes a label.

[0450] Embodiment 188 is a method of Embodiment 187, wherein the label is a chemiluminescent label or a fluorescent label.

[0451] Embodiment 189 is a method of Embodiment 187, wherein the detection step (3) occurs during the amplification step (2).

[0452] Embodiment 190 is a method of Embodiment 189, wherein each detection probe comprises a fluorescent label and a quenching agent.

[0453] Embodiment 191 is a method of Embodiment 190, wherein each detection probe is a molecular torch, a molecular beacon, or a TaqMan detection probe.

[0454] Embodiment 192 is a method of any one of Embodiments 184 to 186, wherein the detection probe further comprises a non-target-hybridized sequence.

[0455] Embodiment 193 is a method of Embodiment 192, wherein the detection probe is a molecular torch or a molecular beacon.

[0456] Embodiment 194 is a method of any one of Embodiments 121 to 193, wherein the amplification reaction in step (2) is an isothermal amplification reaction.

[0457] Embodiment 195 is a method of Embodiment 194, wherein the amplification reaction is a transcription-mediated amplification (TMA) reaction.

[0458] Embodiment 196 is a method of Embodiment 194 or 195, wherein the amplification reaction is a real-time amplification reaction.

[0459] definition

[0460] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in relation to the described methods and compositions. As used herein, the following terms and phrases have the meanings falling under these categories unless otherwise specified.

[0461] The terms "a," "an," and "the" include multiple referents unless otherwise specified in the context.

[0462] The conjunction "or" should be interpreted as equivalent to "and / or" in a comprehensive sense, that is, unless a different comprehensive meaning is unreasonable in the context.

[0463] The term “about” indicates an insubstantial variation in the amount of a component of the composition that has no significant effect on the activity or stability of the composition. In some embodiments, “about” includes a variation within 10%, 5%, 2%, 1%, or 0.5% of the stated value.

[0464] All ranges should be interpreted as including the endpoint unless there is an explicit exclusion such as “not including the endpoint,” and thus, for example, “within 10 to 15” includes values ​​10 and 15, as well as all integers between the endpoints and (where possible) non-integer values, e.g., 11, 11.5, 12 and 1 / 3, 4π, etc.

[0465] The terms "include," "include," "including," "include (contain)," "include," "include (include)," "include," and "including" are not intended to be restrictive.

[0466] The "sample" is Lactobacillus bell, Atopobium virginiana or Gardnerella Virginalis or any specimen that may contain components thereof, such as nucleic acids or fragments of nucleic acids. The specimen may include, for example, vaginal swab specimens, cervical brush specimens, respiratory tissues or exudates such as bronchoscopy, bronchoalveolar lavage (BAL) or lung biopsy, sputum, saliva, peripheral blood, plasma, serum, lymph nodes, gastrointestinal tissue, stool, urine, semen, or other body fluids or substances. Lactobacillus bell, Atopobium virginiana or Gardnerella VirginalisAlternatively, it includes a “biological sample” comprising any tissue or material derived from a living or deceased human that may contain components thereof (e.g., target nucleic acids derived therefrom). The biological sample may be treated to physically or mechanically destroy tissue or cell structures and thus release intracellular components into a solution that may additionally contain enzymes, buffers, salts, and detergents used to prepare biological samples for analysis using standard methods. Additionally, the sample may include a processed sample, such as a sample obtained by centrifugation following or passing the sample over a filtration device, or by attachment to a culture medium, matrix, or support.

[0467] “Nucleic acid” refers to a multimeric compound comprising two or more covalently bonded nucleosides or nucleoside analogs or base analogs having nitrogenous heterocyclic bases, wherein the nucleosides are joined together by phosphodiester bonds or other linkages to form a polynucleotide. Nucleic acid includes RNA, DNA or chimeric DNA-RNA polymers or oligonucleotides and analogs thereof. The nucleic acid “backbone” may consist of various bonds comprising one or more of sugar-phosphodiester bonds, peptide-nucleic acid bonds (in “peptide nucleic acid” or PNA, see PCT International Application WO 95 / 32305), phosphorothioate bonds, methylphosphonate bonds, or combinations thereof. The sugar moiety of the nucleic acid may be ribose or deoxyribose, or a similar compound having known substitutions, e.g., 2’ methoxy substitution and 2’ halogen substitution (e.g., 2’-F). Nitrogenous bases are common bases (A, G, C, T, U) and their analogs (e.g., inosine, 5-methyllysocytosine, isoguanine; The Biochemistry of the Nucleic Acids 5-36, Adams et al. , ed., 11th edition, 1992, Abraham et al., 2007, BioTechniques43: 617-24) may be, and this includes derivatives of purine or pyrimidine bases (e.g., N 4 -methyl deoxyguanosine, deaza- or aza-purine, deaza or aza-pyrimidine, pyrimidine base having a substituent at position 5 or 6, purine base having an altered or substituted substitution at positions 2, 6 and / or 8, e.g., 2-amino-6-methylaminopurine, O 6 -methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine and O 4 -alkyl-pyrimidines and pyrazolo compounds, e.g., unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidines; U.S. Patents US 5,378,825, US 6,949,367 and PCT International Patent Application WO 93 / 13121). The nucleic acid may contain “basic” residues in which the backbone does not contain nitrogenous bases for one or more residues (U.S. Patent US 5,585,481). The nucleic acid may contain only the usual sugars, bases, and bonds found in RNA and DNA, or may contain the usual components and substitutions (e.g., nucleic acids comprising a mixture of a usual base and one or more base analogs linked by a 2' methoxy backbone). Nucleic acids may include "locked nucleic acids" (LNAs) having a double-ring furanose unit in which one or more nucleotide monomers are locked in an RNA-mimicking sugar stereostructure, 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 bases modified to alter the function or behavior of the nucleic acid, for example, the addition of a 3'-terminal dideoxynucleotide that blocks the addition of additional nucleotides to the nucleic acid. Although nucleic acids can be purified from natural sources using routine techniques , in vitro Synthetic methods for producing nucleic acids are well known in the industry.

[0468] As used herein, the term "polynucleotide" refers to a nucleic acid chain. Throughout this application, nucleic acids are designated from the 5' end to the 3' end. Standard nucleic acids, e.g., DNA and RNA, are typically synthesized by adding a nucleotide to the "5' to 3'" end, that is, the 3' end of the growing nucleic acid.

[0469] As used herein, "nucleotide" is a subunit of nucleic acid composed of a phosphate group, a five-carbon sugar, and a nitrogenous base. The five-carbon sugar found in RNA is ribose. In DNA, the five-carbon sugar is 2'-deoxyribose. The term also includes analogs of these subunits, such as a methoxy group at the 2' position of ribose (2'-O-Me).

[0470] The "nucleic acid-based detection assay" used herein is an assay that detects a target sequence within a target nucleic acid and utilizes one or more oligonucleotides that specifically hybridize to the target sequence.

[0471] In a specific embodiment according to the present invention, the nucleic acid-based detection assay is an “amplification-based assay,” that is, an assay utilizing one or more steps of amplifying a nucleic acid target sequence. Various amplification methods used in detection assays are known in the art, some of which are further summarized herein. Clearly, the amplification-based assay may include one or more steps that do not amplify the target sequence, such as steps used in non-amplification-based assays (e.g., hybridization assays or cleavage-based assays).

[0472] In another embodiment, a nucleic acid-based detection assay is a "non-amplification-based assay," that is, an assay that does not rely on any step of amplifying a nucleic acid target sequence. Clearly, a nucleic acid-based detection assay comprising a reaction for primer extension without any corresponding downstream amplification oligomer (e.g., extension of the primer by reverse transcriptase to produce an RNA:DNA duplex, followed by RNase digestion of RNA to produce a single-stranded cDNA complementary to the RNA target without producing a cDNA copy) is understood to be a non-amplification-based assay.

[0473] An exemplary non-amplification-based assay is a "cleavage-based assay," which relies on the specific cleavage by flap endonucleases of linear dichotomous cleavage structures formed by the specific hybridization of overlapping oligonucleotides to target nucleic acids. In such assays, probe oligonucleotides containing non-target-hybridized flap regions are cleaved by flap endonucleases in a overlap-dependent manner, releasing the cleavage products subsequently detected. The principles of cleavage-based assays are well known in the art, and exemplary assays include, for example, Lyamichev et al., Nat. Biotechnol. 17: 292-296, 1999; Ryan et al., Mol. Diagn. 4: 135-144, 1999; Allawi et al., J. Clin. Microbiol. 44: 3443-3447, 2006; Brow et al. U.S. patents US 5,846,717 and US 6,706,471 and Dahlberg et al This is described in U.S. Patent US 5,614,402. Trimming-based tests include, for example, the commercially available Invader® test (Hologic, Inc., Madison, WI).

[0474] As used herein, "target nucleic acid" is a nucleic acid comprising a 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 include sequences other than the target sequence.

[0475] "Isolated" means that the sample containing the target nucleic acid was collected from a natural environment, but this term does not imply any degree of purification.

[0476] As used herein, the term “target sequence” refers to a specific nucleotide sequence of the target nucleic acid to be detected. The “target sequence” includes a complex sequence in which oligonucleotides (e.g., probe oligonucleotides, priming oligonucleotides, and / or promoter oligonucleotides) form a 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). Where the target nucleic acid is originally single-stranded, the term “target sequence” will also refer to a sequence complementary to the “target sequence” present in the target nucleic acid. Where 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 must be selected to distinguish between unrelated and closely related target nucleic acids.

[0477] "Target-hybridization sequence" is used herein to refer to a portion of an oligomer configured to hybridize with a target nucleic acid sequence. Preferably, the target-hybridization sequence is configured to specifically hybridize with the target nucleic acid sequence. The target-hybridization sequence may be 100% complementary to the portion of the target sequence to which they are configured to hybridize, but this is not necessarily the case. The target-hybridization sequence may also include insertions, deletions, and / or substitutions of nucleotide residues relative to the target sequence. Less than 100% complementarity of the target-hybridization sequence to the target sequence is, for example, when the target nucleic acid is from multiple strains within the species, e.g. Lactobacillus This can occur in the case of oligomers configured to hybridize to various strains. It is understood that there are other reasons for configuring target-hybridization sequences to have less than 100% complementarity to the target nucleic acid.

[0478] Lactobacillus bell, A. Virgin or G. Virginalis With respect to regions of nucleic acids, the term "target sequence" as used herein refers to the process in which an oligonucleotide hybridizes to a target sequence in a manner that allows detection as described herein. In one embodiment, the oligonucleotide is targeted Lactobacillus bell, A. Virgin or G. Virginalis It is complementary to the nucleic acid sequence and does not contain mismatches. In another embodiment, the oligonucleotide is targeted Lactobacillus bell, A. Virgin or G. Virginalis It is complementary to the nucleic acid sequence but includes one, two, three, four, or five mismatches. Preferably, the oligonucleotide hybridizing to the target nucleic acid sequence comprises at least 10 to 50 nucleotides complementary to the target sequence. The at least 10 to 50 is understood to be an inclusive range, including 10, 50, and any integer in between. Preferably, the oligomer specifically hybridizes to the target sequence.

[0479] The term "configured to" refers to the actual arrangement of the polynucleotide sequence stereostructure of the reference oligonucleotide target-hybridization sequence. For example, an oligonucleotide configured to hybridize specifically to a target sequence has a polynucleotide sequence that hybridizes specifically to the reference sequence under strict hybridization conditions.

[0480] As used herein, the term "configured to specifically hybridize" refers to the target-hybridization region of an oligonucleotide being referenced Lactobacillus bell, A. Virgin or G. Virginalis It means that it is designed to have a polynucleotide sequence capable of targeting the sequence of the target region. Such oligonucleotides are not limited to targeting only the corresponding sequence, but rather Lactobacillus bell, A. Virgin or G. Virginalis It is useful as a composition in kits or methods for targeting target nucleic acids. Oligonucleotides from a sample Lactobacillus bell, A. Virgin or G. Virginalis It is designed to function as a component of a assay for the detection of, and therefore, when other nucleic acids commonly found in test samples are present Lactobacillus bell, A. Virgin or G. Virginalis It is designed to target. "Specifically hybridize" does not mean exclusively hybridizing, as understood in the industry, because some level of hybridization to non-target nucleic acids may occur. Rather, "specifically hybridize" means that the oligonucleotide is configured to function in an assay that primarily hybridizes to the target, so that the accurate detection of the target nucleic acid in the sample can be determined. The term "configured" refers to the actual arrangement of the polynucleotide sequence stereostructure of the oligonucleotide target-hybridization sequence.

[0481] Lactobacillus bell, A. Virgin or G. VirginalisAs used herein with respect to targeted nucleic acids, the term "fragment" refers to a portion of conjugated nucleic acid. In certain embodiments, the fragment is Lactobacillus bell, A. Virgin or G. Virginalis It contains nucleotides linked from 16S ribosomal RNA, and the number of 16S linked nucleotides in the fragment is less than the number for the entire 16S.

[0482] As used herein, the term “region” refers to a portion of a nucleic acid, said portion being smaller than the entire nucleic acid. For example, when the reference 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, also, as merely an example, when the nucleic acid is 16S ribosomal RNA, the term “region” may be used to refer to a smaller region of the nucleic acid, said smaller region being targeted by one or more oligonucleotides of the present invention. As another non-limiting example, when the reference nucleic acid is an amplicon, the term “region” may be used to refer to a smaller nucleotide sequence identified for hybridization by the target-hybridization sequence of the probe.

[0483] The interchangeable terms “oligomer,” “oligo,” and “oligonucleotide” generally refer to nucleic acids having fewer than 1,000 nucleotide residues, comprising polymers in a range having a lower limit of about 5 nucleotide (nt) residues and an upper limit of about 500 to 900 nt residues. In some embodiments, the oligonucleotide is in a size range having a lower limit of about 12 to 15 nt and an upper limit of about 50 to 600 nt, and in other embodiments, a range having a lower limit of about 15 to 20 nt and an upper limit of about 22 to 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 refer to any specific function of the reagent, but rather is used as a generic term to encompass all of these reagents described herein. Oligonucleotides may impart various different functions. For example, it can function as a primer if it is specific to the complementary strand and can hybridize to it and can be further extended in the presence of a nucleic acid polymerase; it can function as a primer if it contains a sequence recognized by RNA polymerase and allows transcription and can provide a promoter (e.g., a T7 primer); it can function to detect the target nucleic acid if it can hybridize to the target nucleic acid or its amplicon, and additionally provides a detectable moiety (e.g., an acridinium-ester compound).

[0484] As used herein, an oligonucleotide "substantially corresponding" to a specified reference nucleic acid sequence means that the oligonucleotide has hybridization properties similar to the reference nucleic acid sequence in that the oligonucleotide is sufficiently similar to the reference nucleic acid sequence so that the oligonucleotide will hybridize with the same target nucleic acid sequence under strict hybridization conditions. Those skilled in the art will understand that the "substantially corresponding oligonucleotide" may differ from the reference sequence and may still hybridize with the same target nucleic acid sequence. Furthermore, the first nucleic acid corresponding to the second nucleic acid is understood to include RNA and DNA, and to include their complements unless otherwise explicitly stated in the context. Such variation from the nucleic acid may be expressed in terms of the percentage of identical bases within the sequence or the percentage of perfectly complementary bases between the probe or primer and its target sequence. Thus, in certain embodiments, if such percentage of base identity or complementarity is 100% to about 80%, the oligonucleotide is "substantially corresponding" to the reference nucleic acid sequence. In a preferred embodiment, the percentage is 100% to about 85%. In a more preferred embodiment, this percentage is 100% to about 90%; and in another preferred embodiment, this percentage is 100% to about 95%. Similarly, a region of nucleic acid or amplified nucleic acid may be referred to herein as corresponding to a reference nucleic acid sequence. Those skilled in the art will understand various variations of hybridization conditions that may be required at various complementarity percentages to allow hybridization to a specific target sequence without causing unacceptable levels of non-specific hybridization.

[0485] An "amplification oligomer" is an oligomer that has at least a 3' end complementary to the target nucleic acid, hybridizes to the target nucleic acid or its complement, and participates in a nucleic acid amplification reaction. An example of an amplification oligomer is a "primer" that includes a 3' OH end that hybridizes to the target nucleic acid and is extended by a polymerase during the amplification process. Another example of an amplification oligomer is an oligomer that does not extend by a polymerase (e.g., because it has a 3' blocked end) but participates in or facilitates amplification. For example, the 5' region of an amplification oligonucleotide may include a promoter sequence that is not complementary to the target nucleic acid (which may be referred to as a "promoter primer" or "promoter provider"). Those skilled in the art will understand that an amplification oligomer functioning as a primer can 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 may provide an upstream promoter sequence that now hybridizes the target nucleic acid and acts to initiate transcription but does not provide a primer for oligo extension. Such modified oligos are referred herein as "promoter provider" oligomers. The size range of the amplification oligonucleotide is about 10 to about 70 nt in length (not including any promoter sequence or poly-A end) and includes a range containing at least about 10 contiguous bases or even at least 12 contiguous bases that are complementary to a region of the target nucleic acid sequence (or its complementary strand). The contiguous bases are at least 80% or at least 90%, or perfectly complementary, to the target sequence to which the amplification oligomer binds. The amplification oligomer may include optionally modified nucleotides or analogs, or additional nucleotides that participate in the amplification reaction but are not complementary to or included in the target nucleic acid or template sequence.For example, an amplification oligomer may include an additional nucleotide at its 5' end complementary to its 3' end for the purpose of forming a hairpin to adjust priming efficiency (e.g., a promoter primer may include an additional nucleotide upstream of the promoter sequence for this purpose). When referring to a range of lengths for oligonucleotides, amplicons, or other nucleic acids, the range is understood to include all integers (e.g., concatenated nucleotides of length 19 to 25 include 19, 20, 21, 22, 23, 24, and 25).

[0486] As used herein, "promoter" is a specific nucleic acid sequence recognized by DNA-dependent RNA polymerase ("transcription enzyme") as a signal that binds to nucleic acid and initiates the transcription of RNA at a specific site.

[0487] As used herein, “promoter provider” or “provider” refers to an oligonucleotide modified to include first and second regions and to prevent the initiation of DNA synthesis from its 3’ end. The “first region” of the promoter provider oligonucleotide includes a base sequence that hybridizes to a DNA template, wherein the hybridization sequence is located at 3’ but is not necessarily adjacent to the promoter region. The hybridization portion of the promoter oligonucleotide is typically 10 or more nucleotides long and may be extended to a length of up to 50 or more nucleotides. The “second region” includes a promoter sequence for RNA polymerase. The promoter oligonucleotide is engineered so that it cannot be extended by an RNA- or DNA-dependent DNA polymerase, for example, a reverse transcriptase that preferably includes a blocking moiety at its 3’ end as described above. As mentioned herein, the "T7 provider" is a blocked promoter provider oligonucleotide that provides an oligonucleotide sequence recognized by T7 RNA polymerase.

[0488] “Amplification” refers to any known procedure for obtaining multiple copies of a target nucleic acid sequence, its complements, or fragments thereof. Multiple copies may be referred to as amplicons or amplification products. Known amplification methods include both thermal cycling and isothermal amplification methods. In some embodiments, isothermal amplification methods are preferred. Replication-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand substitution amplification (SDA), and transcription-mediated or transcription-related amplification are non-limiting examples of nucleic acid amplification methods. Replication-mediated amplification uses self-replicating RNA molecules and replication enzymes such as QB-replication enzymes (e.g., U.S. Patent US 4,786,600). PCR amplification synthesizes two complementary strands of dsDNA or multiple copies from cDNA using DNA polymerase, primer pairs, and thermal cycling (e.g., U.S. Patents US 4,683,195, US 4,683,202, and US 4,800,159). LCR amplification amplifies a target and its complementary strand by using four or more different oligonucleotides and multiple cycles of hybridization, ligation, and denaturation (e.g., U.S. Patents US 5,427,930, and US 5,516,663). SDA uses a primer comprising a recognition site for an endonuclease that cuts one strand of a semi-modified DNA duplex containing a restriction endonuclease and a target sequence, thereby causing amplification to occur in a series of primer extension and strand substitution steps (e.g., U.S. Patents US 5,422,252; US 5,547,861; and US 5,648,211). A preferred embodiment uses an amplification method suitable for amplifying RNA target nucleic acids such as transcription-mediated amplification (TMA) or NASBA, but it will be obvious to those skilled in the art that the oligomers disclosed herein can be readily used as primers for other amplification methods.

[0489] "Transcription-mediated amplification," also referred to herein as "transcription-mediated amplification" (TMA), refers to nucleic acid amplification using RNA polymerase to produce multiple RNA transcripts from a nucleic acid template. These methods generally employ RNA polymerase, DNA polymerase, deoxyribonucleoside triphosphate, ribonucleoside triphosphate, and a template-complementary oligonucleotide comprising a promoter sequence and optionally one or more other oligonucleotides. The TMA method is an embodiment of an amplification method used to amplify and detect target sequences as described herein. Variations of transcription-related amplification are well known in the art as previously disclosed in detail (e.g., U.S. patents US 4,868,105; US 5,124,246; US 5,130,238; US 5,399,491; US ​​5,437,990; US 5,554,516; and US 7,374,885; and PCT international patent applications WO 88 / 01302, WO 88 / 10315 and WO 95 / 03430). Those skilled in the art will understand that the disclosed compositions can be used in amplification methods based on the extension of an oligomer sequence by a polymerase.

[0490] As used herein, the term "real-time TMA" refers to transcription-mediated amplification ("TMA") of a target nucleic acid monitored by a real-time detection means.

[0491] The term "amplicon," used interchangeably with "amplification product," refers to a nucleic acid molecule produced during an amplification procedure that is complementary or homologous to a sequence contained within a target sequence. These terms may 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.

[0492] “Probe,” “detection probe,” “detection oligonucleotide,” and “detection probe oligomer” are used interchangeably herein to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence in a nucleic acid or amplified nucleic acid under conditions that promote hybridization to allow detection of the target sequence or amplified nucleic acid. Detection may be direct (e.g., a probe directly hybridized to the target sequence) or indirect (e.g., a probe linked to the target via an intermediate molecular structure). The probe may be DNA, RNA, analogs thereof, or combinations 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 a standard base pair. The probe may include target-specific sequences and other sequences that contribute to the three-dimensional structure of the probe (e.g., U.S. Patents US 5,118,801; US ​​5,312,728; US 6,849,412; US 6,835,542; US 6,534,274; and US 6,361,945; and U.S. Patent Application Publication US 20060068417). In a preferred embodiment, the detection probe includes a 2' methoxy backbone capable of generating a higher signal.

[0493] Term "TaqMan ® A "probe" refers to a detection oligonucleotide containing a fluorescent dye on the 5' base and a non-fluorescent quenching dye (quencher) on the 3' base. When irradiated with light, the excited fluorescent dye transfers energy to a nearby quenching dye molecule rather than fluorescence, generating a non-fluorescent substrate. During amplification, the exonuclease activity of the polymerase cleaves the TaqMan probe to separate the fluorophore from the quencher, thereby causing an unquenched signal to be emitted from the fluorophore as an indicator of amplification.

[0494] As used herein, "label" refers to a moiety or compound directly or indirectly connected to a probe that leads to a detected or detectable signal. Direct labeling may occur through binding or interactions that connect the label to the probe, including covalent or non-covalent interactions, e.g., hydrogen bonding, hydrophobic and ionic interactions, and the formation of chelate or coordination complexes. Indirect labeling may occur through the use of a cross-linking moiety or "linker," such as binding pair members, antibodies, or additional oligomers, which may be directly or indirectly labeled and amplify the detectable signal. Labels include any detectable moiety, such as radionuclides, ligands (e.g., biotin, avidin), enzymes or enzyme substrates, reactive groups or chromophores (e.g., dyes, particles, or beads that impart detectable color), luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent labels), or fluorophores. The label may be detectable in a homogeneous assay in which the bound labeled probe in the mixture exhibits a detectable change, e.g., instability or differential degradation properties, that is different from the change in the unbound labeled probe. A “homogeneous detectable label” may be detected without physically removing it from the unbound form of the label or labeled probe (e.g., U.S. Patents US 5,283,174, US 5,656,207, and US 5,658,737). The label comprises chemiluminescent compounds, e.g., acridinium ester (“AE”) compounds including standard AE and derivatives (e.g., U.S. Patents US 5,656,207, US 5,658,737, and US 5,639,604). Syntheses and methods for attaching the label to nucleic acids and detecting the label are well known (e.g., Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Chapter 10; U.S. Patents US 5,658,737, US 5,656,207, US 5,547,842, US 5,283,174 and US 4,581,333). One or more labels and one or more label types may be present on a particular probe, or detection may use a mixture of probes labeled with a compound in which each probe generates a detectable signal (e.g., U.S. Patents US 6,180,340 and US 6,350,579).

[0495] The structure referred to herein as a “molecular torch” is designed to include distinct regions of self-complementarity (“closure domains”) connected by a binding region (“target binding domain”) and hybridizing to each other under predetermined hybridization assay conditions. All or part of the nucleotide sequence containing the target closure domain may also function as the target binding domain. Thus, the target closure sequence may include a target binding sequence, a non-target binding sequence, and combinations thereof.

[0496] The terms “capture probe,” “capture oligonucleotide,” “target capture oligonucleotide,” and “capture probe oligomer” are used interchangeably herein to refer to a nucleic acid oligomer that specifically hybridizes to a target sequence of a target nucleic acid by a standard base pair and captures the target nucleic acid on a support by linking to a binding partner on an immobilized probe. One example of a capture oligomer comprises an oligonucleotide comprising two binding regions, a target hybridization sequence, and an immobilized probe binding region. As a variation of this example, the two regions may exist in two different oligomers joined together by one or more linkers. In another embodiment of a capture oligomer, the target hybridization sequence is a sequence comprising a random or non-random poly-GU, poly-GT, or poly-U sequence to non-specifically bind to the target nucleic acid and link it to a probe immobilized on a support (see, e.g., PCT International Patent Application WO 2008 / 016988). The immobilized probe binding region may be a nucleic acid sequence referred to as 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 To A 40 ), or about 14 to 33 nt (e.g., T3A 14 T3A 30 It substantially comprises a homopolymer tail of ). Accordingly, non-limiting examples of preferred nucleic acid tails are T in some embodiments. 0-4 A 10-40 It may include a sequence. 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.

[0497] As used herein, "immobilized oligonucleotide," "immobilized probe," or "immobilized nucleic acid" refers to a nucleic acid binding partner that directly or indirectly links a capture oligomer to a support. An immobilized probe linked to a support facilitates the separation of the capture probe-bound target from the unbound material of a sample. One embodiment of the immobilized probe is an oligomer linked to a support that facilitates the separation of the bound target sequence from the unbound material of a sample. The support may comprise known materials such as a matrix and particles that are not in solution, which may be made of nitrocellulose, nylon, glass, polyacrylate, mixed polymer, polystyrene, silane, polypropylene, metal, or other compositions, one embodiment of which is a magnetically attractable particle. 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 interaction) or indirectly (via one or more linkers), and the bonding or interaction between the probe and the support is stable during hybridization conditions.

[0498] Detailed description of the invention

[0499] The present invention generally relates to a method and composition for determining the presence or absence of a selected bacterial organism in a sample. In some embodiments, the present invention provides a method and composition for diagnosing bacterial vaginosis (BV) in a subject. In other, non-exclusive embodiments, the present invention in a sample Lactobacillus bell, A. Virgin and G. Virginalis As any one or more detection methods among, Lactobacillus bell, A. Virgin and G. Virginalis The present invention provides a method comprising the step of performing amplification-based detection of a 16S rRNA target nucleic acid from one or more of the following. The present invention also Lactobacillus bell, A. Virgin and G. VirginalisThe present invention provides a composition (including a reaction mixture) and a kit comprising a combination of oligomers for detecting any one or more of the following: The oligomer combination is generally from a sample Lactobacillus bell, A. Virgin and G. Virginalis It comprises at least two amplification oligomers for detecting one or more of the following, and Lactobacillus bell, A. Virgin and G. Virginalis of To perform amplification-based detection, one or more additional oligomers described herein, such as a capture probe and / or a detection probe, may be further included.

[0500] The method for diagnosing BV is generally from samples from subjects suspected of having BV. Lactobacillus bell, A. Virgin and G. Virginalis The method includes a step of detecting the presence or absence of one or more of them. Specifically, the test Lactobacillus bell, A. Virgin and G. Virginalis It is performed for specific detection in each sample. Based on the results of the detection assay, Lactobacillus bell, A. Virgin and G. Virginalis A quantitative value is assigned to each, and then a single BV score is determined using these quantitative values. Next, the presence or absence of BV is determined based on a comparison of the BV score with a cutoff value, which is typically determined in advance. Generally, a BV score exceeding the cutoff value indicates a BV-positive state, and if the quantitative value is in the units of log copies, the determination of the BV score is A. Virgin and G. Virginalis From the larger of the values LactobacillusIt includes subtracting the quantitative value. The determination of the cutoff value may be based on predictive modeling using results from a clinical sample cohort. The cutoff value may be selected to maximize the match compared to the BV patient infection status established by clinical standard methods, or to aim for a ratio of clinical sensitivity or specificity (e.g., Nugent score with a median value analyzed by Amsel's acceptance criteria). In some embodiments, the minimum quantitative value Lactobacillus species and / or A. Virgin and G. Virginalis A value is assigned to each, and if the quantitative value measured by the detection test is smaller than the assigned minimum value, the minimum value is used as the quantitative value for determining the BV score.

[0501] Lactobacillus bell, A. Virgin and / or G. Virginalis Quantitative values ​​for may also be standardized and / or weighted. For example, standardization applies to those participating in the BV score calculation. A. Virgin and G. Virginalis It can be used to balance the probability of each quantitative value, and the determination of the BV score is based solely on the maximum of these two values ​​and the corresponding value in the clinical sample population. A. Virgin Observed higher than concentration G. Virginalis This is because only the trend of concentration is used. In some variations, standardization involves adjusting quantitative values ​​measured by detection tests based on population statistics, such as the population median, for example (e.g., dividing the concentration in copies by the population median, or subtracting the log-copy median where the quantitative data is in log-copy units). Weighting is applied to condition values ​​(e.g., Lactobacillus Species quantitative value, A. Beojinae or G. VirginalisThe effect of a change in a quantitative value (or the quantitative value of another term in a prediction formula) can be used to influence the score value. A higher weight can increase the effect of a change in the condition value on the score, whereas a lower ratio can reduce this effect.

[0502] The formula for calculating the BV score may include additional terms. In some variations, the calculation of the BV score additionally involves adding or subtracting an adjustment constant to adjust for a desired cutoff value (e.g., a cutoff value of 0). In some variations, the calculation of the BV score additionally involves adding an internal control (IC) adjustment factor that can be used to compensate for sample inhibition in the detection assay. Specifically, the appropriate IC adjustment factor is based on the ratio of the expected IC value for the detection assay to the observed IC value generated from the detection assay.

[0503] In a detailed variation of the method for diagnosing BV, the BV score is assigned using the following formula:

[0504] BV Score = C0 + W L Maximum value (L S , F LS ) + W GA Maximum value (A S , G S , F GAS ) + W IC log2(IC ratio),

[0505] Here, the formula elements are as described in Table 1.

[0506]

[0507] In some specific embodiment of a method for diagnosing BV in which the BV score is determined by the above formula, A. Virginae / G. Virginalis Clause (F GA The minimum value assigned to ) is set to 0 before standardization.

[0508] Lactobacillus bell, A. Virgin and G. Virginalis Although it can be detected using any suitable method, it is currently preferable to detect these bacteria using a nucleic acid-based detection assay. The nucleic acid-based detection assay according to the present invention generally has minimal cross-reactivity with other nucleic acids suspected to be present in the sample, Lactobacillus bell, A. Virgin or G. Virginalis It uses oligonucleotides that specifically hybridize to the target nucleic acid. Therefore, Lactobacillus bell, A. Virgin or G. Virginalis Oligonucleotides for nucleic acid-based detection of selected species are, for example, Trichomonas bell; Trichomonas vaginalis ; Candida bell; Clostridiales Bacteria from the river; Clostridium -Similar species; Eggerthella Species; Enterobacteria; Peptostreptococcus micros ; Aerococcus christensenii ; Leptotrichia amnionii ; Peptoniphilus bell; Dialister bell; Mycoplasma hominis ; Sneathia sanguinegens ; Anaerococcus tetradius ; Mobiluncus bell; Mobiluncus hominis ; Megaspaera bell; Prevotella bell; Leptotrichia sanguinegens ; and Finegoldia magna It will have minimal cross-reactivity with species within other bacterial genera, including. 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.

[0509] In a specific embodiment, the nucleic acid-based detection assay method Lactobacillus bell, A. Virgin and / or G. VirginalisThe 16S rRNA or the gene encoding 16S rRNA is targeted. A specifically suitable target region of the 16S rRNA or the gene encoding it corresponds to (i) the region of SEQ ID NO. 1 at approximately nucleotide position 40 to approximately nucleotide position 265. L. Crispatus 16S rRNA region; (ii) corresponding to the region of SEQ ID NO. 2 at approximately nucleotide position 43 to approximately nucleotide position 247 L. Genseni 16S rRNA region; (iii) corresponding to the region of SEQ ID NO. 3 at approximately nucleotide position 93 to approximately nucleotide position 298 L. Gasseri 16S rRNA region; (iv) corresponding to the region of SEQ ID NO. 4 at approximately nucleotide position 540 to approximately nucleotide position 625 A. Virgin 16S rRNA region; and (v) corresponding to the region of SEQ ID NO. 5 at about nucleotide position 172 to about nucleotide position 227 G. Virginalis It targets the 16S rRNA region. In a specific variation of a nucleic acid-based detection assay targeting the 16S rRNA region as described above, (a) Lactobacillus The specific oligonucleotide comprises a target-hybridization sequence 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 the sequence of residues 28 through 45 of SEQ ID NO. 10, a sequence substantially corresponding to the sequence of residues 1 through 17 of SEQ ID NO. 11, or a sequence substantially corresponding to the sequence of residues 7 through 23 of SEQ ID NO. 12; and / or (b) A. VirginThe specific oligonucleotide comprises a target-hybridization sequence comprising a sequence substantially corresponding to the sequence of SEQ ID NO. 17, a sequence substantially corresponding to the sequence of residues 28 through 45 of SEQ ID NO. 18, or a sequence substantially corresponding to the sequence of residues 6 through 21 of SEQ ID NO. 19; and / or (c) G. Virginalis The specific oligonucleotide comprises a target-hybridization sequence comprising a sequence substantially corresponding to the sequence of SEQ ID NO. 14, a sequence substantially corresponding to the sequence of residues 36 through 52 of SEQ ID NO. 15, or a sequence substantially corresponding to the sequence of residues 1 through 18 of SEQ ID NO. 16. In some such embodiments, (a) Lactobacillus The specific oligonucleotide comprises the sequence of SEQ ID NO. 7, the sequence of SEQ ID NO. 8, the sequence of SEQ ID NO. 9, the sequence of residues 28 to 45 of SEQ ID NO. 10, the sequence of residues 1 to 17 of SEQ ID NO. 11, or the sequence of residues 7 to 23 of SEQ ID NO. 12, or comprises a target-hybridization sequence composed thereof; (b) A. Virgin The specific oligonucleotide comprises the sequence of SEQ ID NO. 17, the sequence of residues 28 to 45 of SEQ ID NO. 18, or the sequence of residues 6 to 21 of SEQ ID NO. 19, or comprises a target-hybridization sequence consisting of the same; (c) G. Virginalis The specific oligonucleotide comprises a target-hybridization sequence comprising or consisting of the sequence of SEQ ID NO. 14, the sequence of residues 36 through 52 of SEQ ID NO. 15, or the sequence of residues 1 through 18 of SEQ ID NO. 16. In a specific embodiment, the nucleic acid-based detection assay comprises at least two or three Lactobacillus Specific oligonucleotides, at least two or three A. Virgin Specific oligonucleotides and / or at least two or three G. Virginalis A specific oligonucleotide is used, and this may be an oligonucleotide selected from those specified above.

[0510] In some embodiments of the method including the use of a nucleic acid-based detection assay, the amplification-based assay Lactobacillus bell, A. Virgin and / or G. Virginalis It is used to detect. These variations are generally In the exam The method comprises amplifying a target sequence within a bacterial target nucleic acid using a nucleic acid amplification reaction, and detecting the amplified product by specifically hybridizing it with a nucleic acid detection probe that provides a signal indicating the presence of the bacterial target in bacteria, for example, the amplified product. The amplification step comprises contacting a sample with two or more amplification oligomers specific to the target sequence in the target nucleic acid (e.g., the target sequence of 16S rRNA) to produce an amplified product when the target nucleic acid is present in the sample. Amplification is synthesized by using at least one nucleic acid polymerase to extend the sequence from the amplification oligomer (primer) using, for example, a template strand, to produce an additional copy of the target sequence or its complement. One embodiment for detecting the amplified product uses a hybridization step comprising contacting the amplified product with at least one probe specific to the sequence amplified by selected amplification oligomers, for example, a pair of selected amplification oligomers flanking the target sequence. Suitable amplification methods include, for example, replication enzyme-mediated amplification, polymerase chain reaction (PCR), ligase chain reaction (LCR), strand substitution amplification (SDA), and transcription-mediated or transcription-associated amplification (TMA). These amplification methods are well known in the art (see, for example, the discussion of amplification methods in the definition section above) and are readily used according to the methods of the present invention.

[0511] For example, some amplification methods using TMA amplification include 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 recognize that a conventional melting of a double-stranded nucleic acid (e.g., dsDNA) can be used to provide the single-stranded target nucleic acid. A promoter primer specifically binds to the target nucleic acid at its target sequence, and reverse transcriptase (RT) uses the target strand as a template to extend the 3' end of the promoter primer and create a cDNA copy of the target sequence strand to generate an RNA:DNA diploid. RNase digests the RNA strand of the RNA:DNA diploid, 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 generate dsDNA containing the functional promoter sequence. Next, RNA polymerase specific to the promoter sequence initiates transcription, producing RNA transcripts consisting of approximately 100 to 1,000 amplified copies ("amplicons") of the initial target strand in the reaction. Amplification continues as a second primer specifically binds to the target sequence of each amplicon, and RT generates DNA copies from the amplicon RNA template to produce RNA:DNA dimers. The RNase in the reaction mixture digests the amplicon RNA from the RNA:DNA dimers, and the promoter primer specifically binds to the sequence complementary to the newly synthesized DNA. RT extends the 3' end of the promoter primer to generate dsDNA containing a functional promoter to which RNA polymerase binds to transcribe additional amplicons complementary to the target strand. The autocatalytic cycle of producing more amplicon copies is repeated during the reaction, amplifying the target nucleic acid present in the sample by approximately one billionth.The amplified product can be detected in real-time during amplification or at the end of the amplification reaction using a probe that specifically binds to the target sequence contained in the amplified product. Detection of the signal generated from the bound probe indicates the presence of the target nucleic acid in the sample.

[0512] In some embodiments, the method utilizes a “reverse” TMA reaction. In this variation, the initial or “forward” amplification oligomer is a priming oligonucleotide that hybridizes to the target nucleic acid near the 3’ end of the target region. Reverse transcriptase (RT) synthesizes a cDNA strand by extending the 3’ end of the primer using the target nucleic acid as a template. The second or “reverse” amplification oligomer is a promoter primer or promoter provider having a target-hybridization sequence configured to hybridize to the target sequence contained within the synthesized cDNA strand. Where the second amplification oligomer is the promoter primer, RT uses the cDNA strand as a template to extend the 3’ end of the promoter primer to generate a second cDNA copy of the target sequence strand, thereby generating dsDNA containing a functional promoter sequence. Next, amplification continues essentially as described above using RNA polymerase to initiate transcription from the promoter sequence. Alternatively, where the second amplification oligomer is the promoter provider, a termination oligonucleotide hybridizing to a target sequence near the 5' end of the target region is typically used to terminate the extension of the priming oligomer at the 3' end of the termination oligonucleotide, thereby providing the defined 3' end of the initial cDNA strand synthesized by the extension from the priming oligomer. Next, the target-hybridized sequence of the promoter provider hybridizes to the defined 3' end of the initial cDNA strand, and the 3' end of the cDNA strand is extended to add a sequence complementary to the promoter sequence of the promoter provider, thereby forming a double-stranded promoter sequence. Next, the initial cDNA strand is used as a template to transcribe multiple RNA transcripts complementary to the initial cDNA strand, which do not contain the promoter portion, using an RNA polymerase that recognizes the double-stranded promoter and initiates transcription from it.Next, each of these RNA transcripts can be used to act as a template for further amplification from the first priming amplification oligomer.

[0513] In a specific embodiment comprising an amplification-based detection assay, a combination of at least two amplified oligomers Lactobacillus Species 16S rRNA or Lactobacillus It is used for the detection of genes encoding species 16S rRNA. The oligomer combination corresponds to the region of SEQ ID NO. 1 from approximately nucleotide position 40 to approximately nucleotide position 265. L. Crispatus 16S rRNA region, corresponding to the region of SEQ ID NO. 2 at approximately nucleotide positions 43 to 247 L. Genseni The 16S rRNA region, and / or the region of SEQ ID NO. 3 at about nucleotide positions 93 to 298, corresponding to L. GasseriIt may include first and second amplification oligomers for amplifying a 16S rRNA region. For example, in some embodiments, the first amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 28 through 45 of SEQ ID NO. 10, and the second amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 9; In some of these variations, the first amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 28 through 45 of SEQ ID NO. 10, the second amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 7, and the oligomer combination further comprises a third and a fourth amplification oligomer, wherein the third amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 8, and the fourth amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 9.In a more specific variation, the first amplification oligomer comprises a target-hybridization sequence comprising or consisting of the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10, and the second amplification oligomer comprises a target-hybridization sequence comprising or consisting of the nucleotide sequence of SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 9; in some such embodiments, the first amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 28 to 45 of SEQ ID NO. 10, and the second amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 7, and the oligomer combination further comprises a third and a fourth amplification oligomer, wherein the third amplification oligomer comprises a target-hybridization sequence comprising or consisting of the nucleotide sequence of SEQ ID NO. 8, and the fourth amplification oligomer comprises a target-hybridization sequence comprising or consisting of the nucleotide sequence of SEQ ID NO. 9. In some embodiments as described above, at least one first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence (e.g., a T7 promoter sequence such as the nucleotide sequence of residues 1 to 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 variation, the first amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 10, and the second amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 9; in some such embodiments, the first amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 10, and the second amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 7, and the oligomer combination further comprises third and fourth amplification oligomers, wherein the third amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 8 and the fourth amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 9.

[0514] In a specific embodiment comprising an amplification-based detection assay, a combination of at least two amplified oligomers A. Virgin 16S rRNA or A. Virgin It is used for the detection of genes encoding 16S rRNA. The oligomer combination corresponds to the region of approximately nucleotide positions 540 to 625 of SEQ ID NO. 4. A. VirginIt may include first and second amplification oligomers for amplifying a nucleic acid target region. For example, in some embodiments, the first amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 17 and / or; and the second amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 28 through 45 of SEQ ID NO. 18. In a more specific variation, the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 17 or a target-hybridization sequence composed thereof, and / or the second amplification oligomer comprises the nucleotide sequence of residues 28 through 45 of SEQ ID NO. 18 or a target-hybridization sequence composed thereof. In some embodiments as described above, at least one first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence (e.g., a T7 promoter sequence such as the nucleotide sequence of residues 1 through 27 of SEQ ID NO. 18); in some such embodiments, the first amplification oligomer is a promoter primer or promoter provider. In a more detailed variation, the first amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 17 and / or, or the second amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 18.

[0515] In a specific embodiment including an amplification-based detection assay, a combination of at least two amplified oligomers is G. Virginalis 16S rRNA or G. Virginalis It is used for the detection of genes encoding 16S rRNA. The oligomer combination corresponds to the region of approximately nucleotide positions 172 to 227 of SEQ ID NO. 5. G. VirginalisThe method may include first and second amplification oligomers for amplifying a nucleic acid target region. For example, in some embodiments, the first amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of SEQ ID NO. 14 and / or; and the second amplification oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 36 through 52 of SEQ ID NO. 15. In a more specific variation, the first amplification oligomer comprises the nucleotide sequence of SEQ ID NO. 14 or a target-hybridization sequence composed thereof, and / or the second amplification oligomer comprises the nucleotide sequence of residues 36 through 52 of SEQ ID NO. 15 or a target-hybridization sequence composed thereof. In some embodiments as described above, at least one first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of each target hybridization sequence (e.g., a T7 promoter sequence such as the nucleotide sequence of residues 9 through 35 of SEQ ID NO. 15); in some such embodiments, the first amplification oligomer is a promoter primer or promoter provider. In a more detailed variation, the first amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 14 and / or the second amplification oligomer comprises the nucleotide sequence of residues 9 through 52 of SEQ ID NO. 15 (e.g., the second amplification oligomer is composed of the nucleotide sequence of SEQ ID NO. 15).

[0516] Detection of the amplified product can be achieved by various methods to detect a signal specifically associated with the amplified target sequence. The nucleic acid may be associated with a surface that causes a physical change, such as a detectable electrical change. The amplified nucleic acid can be detected by concentrating it in or on a matrix and detecting the nucleic acid or dye associated with it (e.g., intercalators such as ethidium bromide or cybergreen), or by detecting an increase in the dye associated with the nucleic acid in solution. Other detection methods may be achieved by detecting the presence of a probe:product complex using a nucleic acid detection probe configured to specifically hybridize to the sequence of the amplified product, or by using a complex of probes capable of amplifying a detectable signal associated with the amplified product (e.g., U.S. Patents US 5,424,413; US 5,451,503; and US 5,849,481; each incorporated herein by reference). A directly or indirectly labeled probe that specifically binds to the amplified product provides a detectable signal indicating the presence of the target nucleic acid in the sample. For example, the target nucleic acid Lactobacillus bell, A. Virgin and / or G. Virginalis In the case of 16S rRNA, the amplified product will contain the target sequence of 16S rRNA or a target sequence complementary thereto, and the probe from the tested sample Lactobacillus bell, A. Virgin and / or G. Virginalis It will bind directly or indirectly to the sequence contained in the amplified product to indicate the presence of 16S rRNA.

[0517] The detection probe that hybridizes to the complementary amplified sequence may be a DNA or RNA oligomer, or an oligomer comprising a combination of DNA and RNA nucleotides, or an oligomer synthesized with a modified backbone, e.g., an oligomer comprising one or more 2'-methoxy-substituted ribonucleotides. The probe used for detecting the amplified sequence may be unlabeled and detected indirectly (e.g., by binding of another binding partner to a moiety on the probe), or may be labeled with various detectable labels. In some embodiments of the method for diagnosing BV, such as in a specific embodiment using transcription-mediated amplification (TMA), the detection probe is a linear chemiluminescent labeled probe, e.g., a linear acridinium ester (AE) labeled probe.

[0518] The detection step may also provide additional information about the amplified sequence, such as all or part of a nucleic acid base sequence, for example. Detection may be performed after the amplification reaction is completed, or simultaneously while amplifying the target region, for example, in real time. In one embodiment, the detection step allows for homogeneous detection, for example, detection of hybridized probes without removing unhybridized probes from the mixture (see, for example, U.S. Patents US 5,639,604 and US 5,283,174, each incorporated herein by reference).

[0519] In an embodiment for detecting the amplified product near or at the end of the amplification step, a linear detection probe may be used to provide a signal indicating the hybridization of the probe to the amplified product. One example of such detection uses a luminescently labeled probe that hybridizes to the target nucleic acid. Next, the luminescent label is hydrolyzed on the unhybridized probe. Detection is performed by chemiluminescence using a luminescence detector (see, for example, International Patent Application WO 89 / 002476, incorporated herein by reference). In another embodiment using real-time detection, the detection probe may be, for example, a hairpin probe such as a molecular beacon or molecular torch, or a hybridization switch probe labeled with a reporter moiety that is detected when the probe binds to the amplified product. Such probes may include a target-hybridization sequence and a non-target-hybridization sequence. Various forms of such probes have been previously described (see, e.g., U.S. Patents US 5,118,801; US ​​5,312,728; US 5,925,517; US 6,150,097; US 6,849,412; US 6,835,542; US 6,534,274; and US 6,361,945; and U.S. Patent Applications US 20060068417A1 and US 20060194240A1; each incorporated herein by reference).

[0520] Lactobacillus bell, A. Virgin and / or G. Virginalis 16S rRNA or Lactobacillus bell, A. Virgin and / or G. Virginalis In a specific embodiment comprising an amplification-based detection assay targeting a gene encoding the 16S rRNA of, the method Lactobacillus bell, A. Virgin and / or G. Virginalis One or more detection probes that specifically hybridize to the 16S rRNA amplification product are used. In a specific variant, (A) LactobacillusThe specific detection probe specifically hybridizes to (1) a nucleic acid target region corresponding to the region of SEQ ID NO. 1 at approximately nucleotide position 40 to approximately nucleotide position 265; (2) a nucleic acid target region corresponding to the region of SEQ ID NO. 2 at approximately nucleotide position 43 to approximately nucleotide position 247; and / or (3) a nucleic acid target region corresponding to the region of SEQ ID NO. 3 at approximately nucleotide position 93 to approximately nucleotide position 298; and / or (B) A. Virgin The specific detection probe specifically hybridizes to a nucleic acid target region corresponding to the region of SEQ ID NO. 4 at approximately nucleotide position 540 to approximately nucleotide position 625; and / or; (B) G. Virginalis The specific detection probe specifically hybridizes to the nucleic acid target region corresponding to the region of SEQ ID NO. 5 at approximately nucleotide positions 172 to 227. For example, in some variations, Lactobacillus The first probe for detecting the species amplification product is L. Crispatus and L. Genseni It includes a target-hybridization sequence that specifically hybridizes to the target region of each target nucleic acid, and Lactobacillus The second probe for detecting the species amplification product is L. Gasseri It comprises a target-hybridization sequence that specifically hybridizes to a target region of a target nucleic acid; and in some such embodiments, the first Lactobacillus The specific detection probe substantially comprises a target-hybridization sequence corresponding to the sequence of residues 1 through 17 of SEQ ID NO. 11, and / or the second Lactobacillus A specific detection probe comprises a target-hybridization sequence substantially corresponding to the sequence of residues 7 through 23 of SEQ ID NO. 12 (e.g., a first probe comprising a target-hybridization sequence of residues 1 through 17 of SEQ ID NO. 11 and / or a second probe comprising a target-hybridization sequence of residues 7 through 23 of SEQ ID NO. 12). In some variations, A. VirginA probe for detecting the amplification product comprises a target-hybridization sequence substantially corresponding to the sequence of residues 6 through 21 of SEQ ID NO. 19 (e.g., a probe comprising the target-hybridization sequence of residues 6 through 21 of SEQ ID NO. 19). In some variations, G. Virginalis A probe for detecting an amplification product comprises a target-hybridization sequence substantially corresponding to the sequence of residues 1 through 18 of SEQ ID NO. 16 (e.g., a probe comprising the target-hybridization sequence of residues 1 through 18 of SEQ ID NO. 16). In a specific embodiment, Lactobacillus A first probe for detecting a species amplification product comprises the sequence of SEQ ID NO. 11 or is composed thereof; Lactobacillus A second probe for detecting a species amplification product comprises the sequence of SEQ ID NO. 12 or is composed thereof; A. Virgin A probe for detecting an amplified product comprises the sequence of SEQ ID NO. 19 or is composed thereof; G. Virginalis A probe for detecting an amplified product includes or is composed of the sequence of SEQ ID NO. 16.

[0521] In some embodiments of the method including the use of a nucleic acid-based detection assay, a non-amplification-based assay Lactobacillus bell, A. Virgin and / or G. Virginalis It is used to detect. In some of these embodiments, the non-amplification-based assay is a hybridization assay involving the hybridization of a specific detection probe for the target nucleic acid. Methods for performing polynucleotide hybridization assays are well developed in the industry. The procedures and conditions for the hybridization assay will vary depending on the application, for example, Maniatis et al. , Molecular Cloning: A Laboratory Manual (3rd ed. Cold Spring Harbor, NY, 2002), and Berger and Kimmel, Methods in Enzymology, Vol. 152, Guide to Molecular Cloning TechniquesIt is selected according to known general binding methods, including those mentioned in (Academic Press, Inc., San Diego, Calif., 1987). Generally, the probe and the sample are mixed under conditions that allow for specific nucleic acid hybridization, and then the specific hybridization of the probe for each target is detected. Nucleic acid hybridization is adaptable to various assay formats. One suitable format is the sandwich assay format, which is specifically adaptable to hybridization under non-denaturation conditions. The main component of the sandwich-type assay is a solid support to which an unlabeled immobilized nucleic acid probe, complementary to a portion of the DNA sequence, is adsorbed or covalently bonded. The target nucleic acid is hybridized to the immobilized probe, and a second labeled detection probe, complementary to a different second region of the same DNA strand to which the immobilized unlabeled nucleic acid probe is hybridized, is hybridized to the [target nucleic acid]:[immobilized probe] dimer to detect the target nucleic acid. Another exemplary format utilizes the electrochemical detection of target nucleic acids hybridized to unlabeled detection probes immobilized on electrode surfaces suitable as signal transducers. For example, Drummond et al. , Nat. Biotechnol. 21: 1192, 2003; Gooding, Electroanalysis 14: 1149, 2002; Wang, Anal. Chim. Acta 469: 63, 2002; Cagnin et al. , Sensors 9: 3122, 2009; Katz and Willner, Electroanalysis 15:913, 2003; Daniels and Pourmand, Electroanalysis See 19: 1239, 2007.

[0522] In a specific embodiment including a hybridization test method, the detection probe is Lactobacillus bell, A. Virgin or G. Virginalis 16S rRNA or Lactobacillus bell, A. Virgin and / or G. VirginalisIt is used for the detection of genes encoding 16S rRNA. In some of these embodiments, (A) Lactobacillus Species 16S rRNA or Lactobacillus A probe for detecting a gene encoding a species 16S rRNA specifically hybridizes to (1) a nucleic acid target region corresponding to the region of SEQ ID NO. 1 at approximately nucleotide position 40 to approximately nucleotide position 265; (2) a nucleic acid target region corresponding to the region of SEQ ID NO. 2 at approximately nucleotide position 43 to approximately nucleotide position 247; and / or (3) a nucleic acid target region corresponding to the region of SEQ ID NO. 3 at approximately nucleotide position 93 to approximately nucleotide position 298; and / or (B) A. Virgin 16S rRNA or A. Virgin A probe for detecting a gene encoding 16S rRNA specifically hybridizes to a nucleic acid target region corresponding to the region of SEQ ID NO. 4 at approximately nucleotide position 540 to approximately nucleotide position 625; and / or (C) G. Virginalis 16S rRNA or G. Virginalis A probe for detecting a gene encoding 16S rRNA specifically hybridizes to a nucleic acid target region corresponding to the region of SEQ ID NO. 5 at approximately nucleotide positions 172 to 227. For example, in some variations, Lactobacillus Species 16S rRNA or Lactobacillus The first probe for the detection of a gene encoding species 16S rRNA is L. Crispatus and L. Genseni It includes a target-hybridization sequence that specifically hybridizes to the target region of each target nucleic acid, and Lactobacillus Species 16S rRNA or Lactobacillus The second probe for the detection of genes encoding species 16S rRNA is L. Gasseri It comprises a target-hybridization sequence that specifically hybridizes to a target region of a target nucleic acid; and in some such embodiments, the first LactobacillusThe specific detection probe substantially comprises a target-hybridization sequence corresponding to the sequence of residues 1 through 17 of SEQ ID NO. 11, and / or the second Lactobacillus A specific detection probe comprises a target-hybridization sequence substantially corresponding to the sequence of residues 7 through 23 of SEQ ID NO. 12 (e.g., a first probe comprising a target-hybridization sequence of residues 1 through 17 of SEQ ID NO. 11 and / or a second probe comprising a target-hybridization sequence of residues 7 through 23 of SEQ ID NO. 12). In some variations, A. Virgin 16S rRNA or A. Virgin A probe for the detection of a gene encoding 16S rRNA comprises a target-hybridization sequence substantially corresponding to the sequences of residues 6 through 21 of SEQ ID NO. 19 (e.g., a probe comprising the target-hybridization sequences of residues 6 through 21 of SEQ ID NO. 19). In some variations, G. Virginalis 16S rRNA or G. Virginalis A probe for detecting a gene encoding 16S rRNA comprises a target-hybridization sequence substantially corresponding to the sequence of residues 1 through 18 of SEQ ID NO. 16 (e.g., a probe comprising the target-hybridization sequence of residues 1 through 18 of SEQ ID NO. 16). In a specific embodiment, Lactobacillus Species 16S rRNA or Lactobacillus A first probe for the detection of a gene encoding a species 16S rRNA comprises the sequence of SEQ ID NO. 11 or is composed thereof; Lactobacillus Species 16S rRNA or Lactobacillus A second probe for the detection of a gene encoding species 16S rRNA comprises the sequence of SEQ ID NO. 12, or is composed thereof; A. Virgin 16S rRNA or A. Virgin A probe for the detection of a gene encoding 16S rRNA comprises the sequence of SEQ ID NO. 19, or is composed of the same; G. Virginalis 16S rRNA or G. VirginalisA probe for detecting a gene encoding 16S rRNA includes or is composed of the sequence of SEQ ID NO. 16.

[0523] In some implementations, Lactobacillus bell, A. Virgin and / or G. Virginalis A non-amplification-based assay for the detection of involves a probe oligonucleotide containing a non-target-hybridizing flap region being cleaved by a flap endonuclease in a overlap-dependent manner, releasing the cleavage product subsequently detected. An exemplary cleavage-based assay reagent is, for example, Lyamichev. et al., Nat. Biotechnol. 17: 292-296, 1999), Ryan et al., Mol. Diagn. 4: 135-144, 1999 and Allawi et al., J. Clin. Microbiol. It is described in 44: 3443-3447, 2006. Appropriate conditions for the flap endonuclease reaction can be easily determined using known or methods known in the art (e.g., Kaiser et al. , J. Biol. Chem. See 274: 2138-721394, 1999). An exemplary flap endonuclease that can be used in the method is hermus aquaticus DNA polymerase I, Thermus thermophilus DNA polymerase I, mammalian FEN-1, Archaeoglobus fulgidus FEN-1, Methanococcus jannaschii FEN-1, Pyrococcus furiosus FEN-1, Methanobacterium thermoautotrophicum FEN-1, Thermus thermophilus FEN-1, Cleavase® (Hologic, Inc., Madison, WI), S. cerevisiae RTH1, S. cerevisiae RAD27, Schizosaccharomyces pombe rad2, bacteriophage T5 5'-3' exonuclease, Pyrococcus horikoshiiIncludes FEN-1, human endonuclease 1, and calf thymus 5'-3' exonuclease, and includes their homologs in euby, eukaryotes, and paleo, such as members of the Class II family of structure-specific enzymes, as well as their mutants or variants. Descriptions of flap endonucleases include, for example, Lyamichev et al. , Science 260: 778-783, 1993; Eis et al. , Nat. Biotechnol. 19: 673-676, 2001; Shen et al. , Trends in Bio. Sci. 23: 171-173, 1998; Kaiser et al. , J. Biol. Chem. 274: 21387-21394, 1999; Ma et al. , J. Biol. Chem. 275: 24693-24700, 2000; Allawi et al. , J. Mol. Biol. 328: 537-554, 2003; Sharma et al. , J. Biol. Chem. 278: 23487-23496, 2003; and Feng et al. , Nat. Struct. Mol. Biol. 11: It can be found in 450-456, 2004.

[0524] In certain variations, the truncation-based test method Lactobacillus bell, A. Virgin and / or G. Virginalis The RNA target nucleic acid detection and cleavage-based assay uses a flap endonuclease capable of cleaving the RNA:DNA linear dimer structure. In some alternative embodiments, the cleavage-based assay Lactobacillus bell, A. Virgin and / or G. Virginalis The DNA target nucleic acid detection and cleavage-based assay uses a flap endonuclease capable of cleaving the DNA:DNA linear dimeric structure. An exemplary flap endonuclease capable of cleaving the RNA:DNA dimeric structure is ThermusPolymerase-deficient 5' nucleases, as well as, for example, Cleavase® BN (Taq polymerase's Bst X- Not I deletion, see U.S. Patent US 5,614,402), Cleavase® II (“ ” mutant of whole-length Taq polymerase, see U.S. Patent US 5,614,402), Cleavase® VII (whole Thermus thermophilus Polymerase, Cleavase® IX ( Tth Polymerase-deficient mutant of DNA polymerase) and Cleavase® XII (Taq DNA polymerase and Tth Includes specific Cleavase® enzymes (Hologic, Inc.), such as polymerase-deficient chimeric polymerases constructed from fragments of DNA polymerase. DNA: Exemplary flap endonucleases capable of cleaving DNA dimers include the flap endonucleases indicated above, as well as Cleavase® 2.0 ( Archaeoglobus fulgidus FEN-1), Cleavase® 2.1 (having 6 histidines at the C-terminus) Archaeoglobus fulgidus FEN-1), Cleavase® 3.0 ( Archaeoglobus veneficus FEN-1) and Cleavase® 3.1 (having 6 histidines at the C-terminus) Archaeoglobus veneficus Includes FEN-1).

[0525] In some embodiments, the truncation-based test method Lactobacillus bell, A. Virgin and / or G. Virginalis The assay detects the RNA target nucleic acid and includes the step of synthesizing the DNA complement of the RNA target region, which then hybridizes to first and second probe oligonucleotides that overlap the cDNA strands to form a linear dichotomous cleavage 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 industry.

[0526] In a specific embodiment using a nucleic acid-based detection assay, the method from other components of the sample Lactobacillus bell, A. Virgin and / or G. Virginalis The method further includes a step of purifying the target nucleic acid. Such purification may include methods for separating and / or concentrating the organism contained in the sample from other sample components. In a specific embodiment, the step of purifying the target nucleic acid includes capturing the target nucleic acid to specifically or non-specifically separate the target nucleic acid from other sample components. Non-specific target capture methods include the selective precipitation of the nucleic acid from a substantially aqueous mixture, the attachment of the nucleic acid to a support that washes away other sample components, or Lactobacillus bell, A. Virgin and / or G. Virginalis Other means for physically separating nucleic acid from a mixture containing nucleic acid and other sample components may be included.

[0527] In some implementations, Lactobacillus bell, A. Virgin and / or G. Virginalis Target nucleic acid (e.g., 16S rRNA target nucleic acid) or a gene encoding 16S rRNA) is separated from other sample components by hybridizing the target nucleic acid to a capture probe oligomer. The capture probe oligomer forms a [target nucleic acid] : [capture probe] complex that is separated from other sample components by including a target-hybridization sequence configured to hybridize specifically or non-specifically to the target nucleic acid. A capture probe including a target-hybridization sequence suitable for the non-specific capture of the target nucleic acid is described, for example, in PCT International Patent Application WO 2008 / 016988, which is incorporated herein by reference. In some variations, the step of separating the target nucleic acid from other sample components involves the sample (i) Lactobacillus A first capture probe oligomer comprising a target-hybridization sequence that specifically hybridizes to a target sequence within a species target nucleic acid (e.g., L. Crispatus , L. Genseni and L. Gasseri (ii) a target-hybridization sequence that specifically hybridizes to the target sequence within each target nucleic acid, and (ii) A father and G. Virginalis It comprises contacting a second capture probe oligomer comprising a target-hybridization sequence that specifically hybridizes to a target sequence within each of the target nucleic acids. In some detailed variations comprising the first and second capture probe oligomers of (i) and (ii), the first capture probe oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 1 through 19 of SEQ ID NO. 6 and / or (e.g., a capture probe comprising a target-hybridization sequence of residues 1 through 19 of SEQ ID NO. 6), a second capture probe oligomer comprises a target-hybridization sequence substantially corresponding to the nucleotide sequence of residues 1 through 20 of SEQ ID NO. 13 (e.g., a capture probe comprising a target-hybridization sequence of residues 1 through 20 of SEQ ID NO. 13). In a preferred variation, the capture probe binds a [target nucleic acid] : [capture probe] complex to an immobilized probe to form a [target nucleic acid] : [capture probe] : [immobilized probe] complex that is separated from the sample and optionally washed to remove non-target sample components (e.g., see U.S. Patents US 6,110,678; US 6,280,952; and US 6,534,273; each incorporated herein by reference). In this modification, the capture probe oligomer further comprises a sequence or moiety that attaches the capture probe to an immobilized probe attached to a solid support, along with the bound target sequence, thereby separating the hybridized target nucleic acid from other sample components.

[0528] In a more specific embodiment, the capture probe oligomer comprises a tail portion (e.g., 3' tail) that is not complementary to the target nucleic acid but specifically hybridizes to a sequence on the immobilized probe and thereby acts as a moiety to separate the target nucleic acid from other sample components, as previously described, for example, in U.S. Patent US 6,110,678 incorporated herein by reference. Any sequence may be used in the tail portion, which is generally about 5 to 50 nt in length, which binds to a complementary immobilized sequence (e.g., poly-T) attached to a solid support, e.g., a matrix or particle, and a preferred embodiment is about 10 to 40 nt (e.g., A 10 To A 40 ), more preferably 14 to 33 nt (e.g., A 14 To A 30 or T3A 14 T3A 30 It includes a substantial homopolymer tail of ). (i) L. Crispatus , L. Genseni and L. Gasseri A first capture probe oligomer comprising a target-hybridization sequence that specifically hybridizes to a target sequence within each 16S rRNA target nucleic acid, and (ii) A father and G. Virginalis In some of these embodiments, the first capture probe oligomer comprises a second capture probe oligomer comprising a target-hybridization sequence that specifically hybridizes to a target sequence within each 16S rRNA target nucleic acid, and / or the second capture probe oligomer comprises or comprises the nucleotide sequence of SEQ ID NO. 6.

[0529] Target capture is typically performed under hybridization conditions using the T of the [tail sequence] : [immobilized probe sequence] duplex. mIt occurs in a solution-phase mixture containing one or more capture probe oligomers that hybridize to the target nucleic acid at a higher temperature. In the case of an embodiment containing 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 attached [immobilized probe] : [capture probe] : [target nucleic acid] is washed one or more times to further remove other sample components. A preferred embodiment uses a particulate solid support, such as a paramagnetic bead, so that the particle having the attached [target nucleic acid] : [capture probe] : [immobilized probe] complex is suspended in a washing solution and, preferably, recovered from the washing solution by using magnetic attraction. In an embodiment of the method including the use of an amplification-based detection assay, to limit the number of handling steps, the target nucleic acid may be amplified simply by mixing the target nucleic acid of the complex on the support with the amplification oligomer and proceeding to the amplification step.

[0530] In some implementations of a method for diagnosing BV, Lactobacillus bell, A. Virgin and / or G. VirginalisWhere detection indicates BV in the subject, the method further includes the step of treating BV in the subject. Treatment regimens for BV are generally known in the art and include, for example, the administration of antibiotics such as metronidazole (e.g., Plagill, Metrogel-Virginal), clindamycin (e.g., Cleocin, Clindesse), and tinidazole (e.g., Tindamax). In certain variations, the subject has not previously been diagnosed with BV. In other embodiments, the subject has previously been diagnosed with BV and is receiving treatment for BV at the time the diagnostic method of the present invention is performed. These variations are particularly useful for monitoring treatment of BV in the subject. For example, if the method indicates that BV is still present in the subject, the subject may continue treatment. In some embodiments, the same treatment regimen (i.e., the same treatment the subject is receiving at the time the diagnostic method of the present invention is performed) is re-administered to the subject. Alternatively, the continued presence of BV in a subject undergoing treatment may indicate that a change in ongoing treatment is necessary, and a different treatment regimen (e.g., different dosage, or increased dose and / or frequency of the drug) is administered to the subject.

[0531] According to the present invention, Lactobacillus bell, A. Virgin and / or G. VirginalisDetecting the presence or absence of is performed separately for each target (e.g., in separate reaction vessels, sequentially or simultaneously) or together as a multiple complex reaction system. Accordingly, in some embodiments, the method described herein (e.g., a method for diagnosing BV) uses a multiple complex reaction, wherein the reaction mix comprises reagents for simultaneously testing a plurality of (e.g., at least two, three, or four or more) different targets. In such cases, the reaction mix may comprise a plurality of different target-specific oligonucleotides for performing a detection assay. For example, in a method using an amplification-based detection assay, the multiple complex reaction may comprise a plurality of sets of amplification oligomers (e.g., a plurality of pairs) (e.g., a plurality of PCR primer pairs or a plurality of TMA amplification oligomer pairs (e.g., a plurality of promoter primer and non-promoter primer pairs for TMA or a plurality of promoter provider and non-promoter primer pairs)). In another embodiment using a cleavage-based detection assay, the multiple complex reaction may include a plurality of probe oligonucleotides having different flaps once cleaved, a plurality of different overlapping probe oligonucleotides, and a plurality of different FRET cassettes detecting different flaps.

[0532] Additional microbial detection assays may similarly be performed to determine the presence and / or relative amounts of multiple microorganisms involved in BV. By way of example, these multiple microorganisms are anaerobic Gram-positive cocci; Eggerthella bell; Clostridiales Bacteria from the river; Clostridium -Similar species; Enterobacteria; Peptostreptococcus micros ; Aerococcus christensenii ; Leptotrichia amnionii ; Peptoniphilus bell; Dialister bell; Mycoplasma hominis ; Sneathia sanguinegens ; Anaerococcus tetradius ; Mobiluncus bell; Mobiluncus hominis ; Eggerthella hongkongensis ; Prevotella bell; Megaspaera bell; Leptotrichia sanguinegens and Finegoldia magna It may include one or more of the above. The assay may be performed separately or multiple complex. Accordingly, the diagnosis of BV may involve identifying multiple microorganisms in a sample and optionally determining their relative concentrations.

[0533] In a specific embodiment, the method for diagnosing BV comprises the detection of 10 or fewer bacterial genera associated with BV. In another embodiment, the method comprises 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 Lactobacillus , Atopobium , Gardnerella It does not include the detection of other BV-related bacterial genera.

[0534] In addition, in the present invention, from the sample Lactobacillus bell, A . Virgin and G . Virginalis An oligomer or a combination thereof is provided for determining the presence or absence of any one or more of the following. In various embodiments, the oligomer or a combination thereof is provided in a sample Lactobacillus bell, A . Virgin and G . Virginalis The method for determining the presence or absence of comprises an oligomer as presented herein. In some variations, the oligomer combination comprises at least one Lactobacillus Specific oligonucleotides (e.g., at least two or three that each bind to a different target sequence) Lactobacillus specific oligonucleotide); at least one A . VirginSpecific oligonucleotides (e.g., at least two or three that each bind to a different target sequence) A . Virgin specific oligonucleotide); and / or at least one G . Virginalis Specific oligonucleotides (e.g., at least two or three that each bind to a different target sequence) G . Virginalis It includes specific oligonucleotides. In some variations, the oligomer combination consists of at least two Lactobacillus Specific oligonucleotides (e.g., at least three that each bind to a different target sequence) Lactobacillus Specific oligonucleotides); at least two A . Virgin Specific oligonucleotides (e.g., at least three that each bind to a different target sequence) A . Virgin Specific oligonucleotide); and / or at least two G . Virginalis Specific oligonucleotides (e.g., at least three that each bind to a different target sequence) G . Virginalis It includes specific oligonucleotides. In some embodiments, the oligomer combination is Lactobacillus At least two species nucleic acid target regions for amplifying Lactobacillus Specific amplification oligonucleotide; A. Virgin At least two nucleic acid target regions for amplifying A. Virgin Specific amplification oligonucleotides; and / or G. Virginalis At least two nucleic acid target regions for amplifying G. Virginalis It includes a specific amplification oligonucleotide. In some embodiments, the composition of the present invention is Lactobacillus Species target nucleic acid, A. Virgin Target nucleic acid and / or G. VirginalisIt comprises one or more detection probe oligomers for detecting target nucleic acids. The oligomers or combinations thereof may be in the form of a reaction mixture or a kit containing the oligomer(s). The reaction mixture or kit may further include many optional components, such as, for example, capture probe nucleic acids or capture probe nucleic acid arrays. In the case of an amplification reaction mixture, the reaction mixture typically comprises, for example, buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP) and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase), In vitro It will include other reagents suitable for performing amplification, typically Lactobacillus bell, A . Virgin and / or G . Virginalis It will include test sample components in which the target nucleic acid may or may not be present. In addition, Lactobacillus bell, A . Virgin and / or G . Virginalis Oligomer combinations for the amplification of one or more target nucleic acids include, for example, buffers, salt solutions, appropriate nucleotide triphosphates (e.g., dATP, dCTP, dGTP, dTTP, ATP, CTP, GTP, and UTP) and / or enzymes (e.g., reverse transcriptase and / or RNA polymerase), In vitro Other reagents suitable for performing amplification may be included. In the case of an oligomer combination (e.g., reaction mixture or kit) comprising a detection probe along with an amplification oligomer combination targeting a common target nucleic acid, the selection of the amplification oligomer and the detection probe oligomer is linked by a common target region (i.e., the combination will include a probe that binds to a sequence amplifiable by the amplification oligomer combination). Specific details for implementing the invention

[0535] The present invention is further explained by the following non-limiting embodiments.

[0536] Example 1: BV Multivariate Algorithm

[0537] 1. Summary of Result Interpretation

[0538] To determine whether BV is positive or negative, Lactobacillus Species ("L.spp", negatively correlated with BV) and Gardnella Virginalis and Atopobium virginiana ("Gvag" and "Avag", respectively, positively correlated with BV) are combined into a single numerical BV score using a multivariate linear predictor algorithm. This score is compared to a single cutoff value, where a negative value indicates a value below the cutoff and a positive value indicates a value above the cutoff.

[0539] In the simplified version of this algorithm, the quantified L.spp value (in log copies) is subtracted from the Gvag or Avag value (the maximum of the two).

[0540] Simplified score = max (Gvag, Avag) - L.spp

[0541] As actually applied to the interpretation of APTIMA B results, the formula becomes more complex. A negative constant is added (making the score of the clinical decision point zero), an additional factor (IC term) is added (compensating for observed inhibition), a minimum value is assigned to L.spp (improving reproducibility), the concentrations of the three analytes are standardized (balancing the participation of Gvag and Avag in the algorithm), and weights are assigned to each term.

[0542] Applicable formula:

[0543] Score = C0 + W L *Maximum value (LM L , F L -M L ) + W AG *Maximum value (GM) G , AM A , F GA -M G) + WIC*log2(IC / calIC)

[0544] 2. Details of result interpretation

[0545] Sample interpretation consists of evaluating validity criteria, calculating BV scores, and interpreting the scores. The description of this embodiment begins with the T-period values ​​of the internal control (IC) signal and the quantified log copies / mL values ​​for L.spp, Gvag, and Avag (L, G, A) for each sample to be evaluated.

[0546] 2.1 Calculation of BV score

[0547] For samples that meet the validity criteria (not described herein), a BV score is calculated. 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 a universal formula.

[0548] 2.1.1 Standardization of analytes

[0549] In the population of clinical samples, observed Gvag concentrations tend to be higher than Avag concentrations. Since the linear predictor formula uses only the maximum value (Gvag log C or Avag log C), conditions are leveled to balance the probability of each analyte participating in the algorithm. A robust leveling method is to adjust the term by the population statistic (median). If the concentrations were in copy units, they would have been divided by the median and the relative concentrations provided as standardized values. Since the quantitative data used herein is in log copy units, the equivalent operation is to subtract the median log C value.

[0550] Standardization provides two main functions:

[0551] 1) Level the concentration values ​​(as described above),

[0552] 2) Improve the confidence interval of the trained constant.

[0553] This form of standardization applies to all terms existing on a log-copy basis. This applies to the three sample analyte values ​​(L, G, A) and the base value F. L and F GA It includes. Each of these is the median population value (M prior to the use of the linear predictor formula). L , M G , M A It is standardized by subtracting ). The standardized value is indicated by the subscript "S" in the universal formula.

[0554]

[0555] 2.1.2 Calculation of IC ratio

[0556] The IC ratio is used in the universal formula as part of the term compensating for sample suppression to reduce the probability of false negative indications. The IC ratio is calculated by dividing the observed value for the internal control (IC) time T by the expected value for the IC time T. If the response is suppressed (a slower-than-expected response), the IC ratio is greater than 1. The expected IC time T is calculated as the average observed IC time T of valid kit correction factor repetitions.

[0557] For samples that do not generate an IC T-hour, the IC ratio is defined as the constant ICd. The default IC T-hour can be calculated by multiplying the average observed IC T-hour of valid kit correction factor repetitions by ICd. For all IC T-hours not less than the default IC T-hour, ICd is used as the IC ratio.

[0558]

[0559] 2.1.3 Universal formula

[0560] The formula coded with PANTHER® software for the interpretation of APTIMA® BV results is referred to as the general formula. In this formula, the subscript "S" refers to a value standardized by subtracting the population median for the relevant analyte.

[0561] BV Score = C0 + W L Maximum value (L S , F LS ) + W A Maximum value (A S , F AS ) + W G Maximum value (G S , F GS ) + W GA Maximum value (A S , G S , F GAS ) + W IC log2(IC ratio)

[0562] This general-purpose formula was implemented before the algorithm was completed. Subsequently, W A and W G Both were set to 0. Therefore, the formula (as applied) can be simplified as follows.

[0563] BV Score = C0 + W L Maximum value (L S , F LS ) + W GA Maximum value (A S , G S , F GAS ) + W IC log2(IC ratio)

[0564] In this formula, the four terms are added together to make a score.

[0565]

[0566] 2.2 Interpretation of BV scores

[0567] To determine the BV positive or BV negative status (reported indication), the numerical BV score is compared to a single cutoff value (currently set to 0). If the sample's BV score is less than the cutoff value, the sample is reported as BV negative. If the score is greater than or equal to the cutoff value, the sample is reported as BV positive.

[0568] 3. Application of Multivariate Algorithms to the Interpretation of Clinical Sample Results

[0569] To interpret the BV clinical results, "true results" were determined using the Nugent score with the median value determined by the Amsel judgment criteria as shown in Table 4.

[0570]

[0571] The constants used in the algorithm are described in Table 5 and are represented by the numerical values ​​used in this analysis.

[0572]

[0573] Two sets of data were collected from the BV RT-TMA PANTHER test results for the APTIMA product under development. The training set consists of 1,204 test results from one test per subject. This dataset was used to derive the constant values ​​shown in Table 5. The validation set consists of 1,076 test results from one test per subject. Although the subject populations were identical, there were no common test results between the training and validation sets. (In this context, "training set" and "validation set" refer to sets of clinical samples tested with the same assay design to optimize algorithms for generating clinical indications (positive or negative). The training set is a sample group used to design a provisional algorithm, while the validation set is a separate sample set used to test the accuracy of the algorithm's performance.)

[0574] Using constants from Table 5 in the general formula provides clinical performance (sensitivity / specificity) estimates for the training and validation sets shown in Table 6.

[0575]

[0576] BV may vary across different racial and ethnic groups. Therefore, the training and validation sets are categorized by race and ethnicity using the following categories: Black or African Americans regardless of ethnicity (Black), Non-Hispanic White (White-NH), Hispanic White (White-Hisp), Asians regardless of ethnicity (Asian), and Others. The Others category consists of Irish, Indian, Middle Eastern, Native Americans, and other people of unknown or mixed ancestry.

[0577]

[0578]

[0579] Example 2: Amplification Interference

[0580] Preferably, the amplification oligo specifically detects the 16S rRNA of the targeted species with minimal interference from other organisms that may be present at high titers in the desired sample type. Other qualitative organisms and G. Virginalis The 16S rRNA sequence alignment of G. Virginalis It showed similarities between and Bifidobacteria, and interference by Bifidobacteria or other related organisms in some samples G. Virginalis of This suggests that it may affect detection. Additional G. Virginalis The amplification oligo is designed to minimize possible interference.

[0581] The initial design targeted the amplification of SEQ ID NO. 5 at approximately bases 964 through 1033 (Region 1). Additional oligos targeting sites with potentially greater specificity were screened (SEQ ID NO. 5, approximately bases 160 through 277 (Region 2)).

[0582] To test for interference, the primer set is of higher concentrations present in the sample Bifidobacterium breve 1E4 CFU / mL with or without G. VirginalisIt was tested in the form of passive RT-TMA on samples containing. Each set is B. breve Tested on the average values ​​(RFU range, denormalized T-time and denormalized T-slope) shown for each condition compared to the absence of 1E4 CFU / mL Gvag condition.

[0583]

[0584] G. Virginalis Test Results on Amplification Interference Both sets of Zone 2 have high potency. B. breve Relatively stable when present G. Virginalis It provided detection. In contrast, Set 1 (of Region 1) is increasing B. breve Providing a reduced RFU range, reduced T slope, and increased T time in the presence of concentration G.vag Amplification interference was proven.

[0585] Lactobacillus bell ( L. Gasseri, L. Genseni and L. Crispatus The initial oligo combination for detection is low concentration in the presence of high concentrations of various different organisms L. Gasseri It showed a reduction in detection. To reduce interference and improve specificity, three targeted Lactobacillus Separate forward primers were designed for each species. The native oligosets and modified oligosets were used individually or N. gonorrhoeae 1E6 CFU / mL, C. difficile t 1E6 CFU / mL, A. lwoffii 1E6 CFU / mL, M. hominis 5E9 copy / mL or L. iners In the presence of 1E6 CFU / mL, L. Gasseri It was used in a passive RT-TMA format for the detection of 1E4 CFU / mL. Both conditions used the same promoter primer and torch combination (SEQ No. 10, SEQ No. 259, and SEQ No. 11). The forward primers used for each condition are shown in the table below. To evaluate interference, the average T time of each sample L. GasseriDivided by the average T-hour for 1E4 CFU / mL. A T-hour ratio exceeding 100% indicates a T-hour delay (amplification interference).

[0586]

[0587] For Lactobacillus species amplification interference Test results . Initial conditions ( L. Gasseri , L. Genseni and L. Crispatus (Use of a single forward primer for) C. difficile , A. lwoffi and M. hominis While it showed amplification interference, the modified condition (using separate forward primers for each targeted species) did not.

[0588] Example 3: Lactobacillus Target capture, raising, and screening

[0589] Lactobacillus To improve the initial design (sequence number 257) for a species-targeted capture oligo, L. Crispatus , L. Gasseri or L. Genseni In samples containing 1E4 CFU / mL, additional designs were created and screened simultaneously with the existing design. . The amplification and detection oligos (common to all conditions) were SEQ ID NO. 258, SEQ ID NO. 10, SEQ ID NO. 259, and SEQ ID NO. 11. Screening was performed in manual RT-TMA format with 3 repetitions per condition, with the mean normalized T-time values ​​displayed.

[0590]

[0591] Since the conditions vary solely due to the target capture oligo, slower amplification (higher T-time) may be attributed to a decrease in target capture efficiency. In Condition 1, the target capture oligo was not used, and the T-time was delayed or absent. The use of the target capture oligo is expected to improve this by increasing capture efficiency. Under different conditions, three types LactobacillusA single TCO was used to capture the species. Under conditions 4 to 8, the TCO was Lactobacillus It was designed to specifically capture 16S rRNA and preferred this sequence over sequences from other genera. In conditions 2 and 3, TCO was designed to capture rRNA from Lactobacillus as well as other bacteria.

[0592] result. Conditions 5, 6, and 7 are the initial Lactobacillus Three targeted TCO designs (Condition 4) and less specific TCOs (Conditions 2 and 3) Lactobacillus T-time is faster for all species (more efficient capture). 3 desirable Lactobacillus Condition 7 (TCO sequence number 6) in the TCO design exhibited the highest efficiency (lowest T time) in this experiment.

[0593] Example 4: Cross-reactivity of initial multicomplexing

[0594] G.vag, A.vag and Lactobacillus The multiple complex stereostructure of the RT-TMA reagent for species detection was assembled using the oligos in Table 12.

[0595]

[0596] The reagents were tested on a PANTHER instrument with 5 replicates per sample for pools containing 1 to 5 untargeted organisms at high titers (1E6 CFU / mL in most cases). An unexpected signal in the FAM channel was observed in pool #7, which corresponds to the following 4 organisms: Mobiluncus curtisii (5E9 copy rRNA / mL), Mycoplasma genitalium (1E6 CFU / mL), Mycoplasma hominis (5E9 rRNA copy / mL) and Neisseria gonorrhoeaeIt consisted of (1E6 CFU / mL). Tracking tests were performed on samples containing individual organisms from Pool #7. The reagents were tested on a PANTHER instrument with 2 replicates per control condition and 4 replicates per test condition. The mean normalized T-times are shown in Table 13 below.

[0597]

[0598] An unexpected FAM signal M. genitalium It was observed only in [specific case]. To determine whether oligomers were responsible for the unexpected signal generation, experiments were conducted to omit individual oligomers from the multicomplex mixtures for each condition. The oligomers in Table 12 were used for the multicomplexes, and SEQ ID NO. 23 was omitted. Each of the eight reagent sets (a control condition containing all oligomers and seven conditions with a single oligomer omitted) was tested in the form of manual RT-TMA against Specificity Pool #7, with three repetitions per condition. The normalized FAM T-times are shown in Table 14 below.

[0599]

[0600] This experiment indicated that SEQ ID NO. 259, SEQ ID NO. 10, and SEQ ID NO. 45 are required for the generation of unexpected FAM signals. In FAM M. genitalium RT-TMA reagents were constructed to test the sufficient amount of these oligos for detection, and these three oligomers (SEQ No. 259, SEQ No. 10, and SEQ No. 45) were the only torch, promoter primer, and primer. M. genitalium While was detected on FAM in all four tested iterations, A. vag , G. vag , L. gas and the negative control was not detected with this oligomer combination (see Table 15). Therefore, this set of oligomers is sufficient.

[0601]

[0602] For the multicomplexes described in Table 12 M. genitalium Cross-reactivity risk was not indicated in the initial bioinformatics assessment. Sequence No. 10 and Sequence No. 259 are M. genitalium The initial acceptance criteria for the predicted hybridization were not met. Therefore, the observed cross-reactivity was unexpected.

[0603] Example 5

[0604] A.vag To increase sensitivity, the multiple complex test has two types A.vag It was executed simultaneously with the system: (1) double image with long amplicon ("LAG-5") A. vag using the system Lacto - A.vag - G.vag Multicomplex and (2) non-double phase with short amplicon ("LAG-6") A. vag using the system Lacto - A.vag - G.vag Multicomplex. Such A.vag The system started with the oligo shown in Table 16.

[0605]

[0606] A.vag The dual-phase system is an IVT-equivalent clinically relevant high concentration A.vag It was too rapid to provide T hours from (≥1E11 cp / mL). Previously designed A.vag T7 oligos were re-screened. One T7 with a predicted secondary structure slowed down the T time.

[0607] A.vag Improvements to non-duplex systems are A.vag The focus was on improving the curve slope. A number of A.vag Torch repeats were designed within the same region and screened as a single complex. Having the nucleotide sequence of SEQ ID NO. 19 A.vag The torch was selected due to improved sensitivity in a multicomplex context.

[0608] The analytical specificity result is two types A.vag The systems (LAG5 and LAG6) were similar.

[0609] improved A.vag Oligo sets are shown in Table 17 below.

[0610]

[0611] LAG5 and LAG6 multicomplex oligo combinations (as shown in Table 17 above) A.vag The clinical performance of the design oligo (different from the design) was compared in two small vaginal clinical swab sample sets. The data were based on two types of algorithms, namely, as described in Example 6. L. crisp , G. vag and A. vag It was interpreted using a decision tree algorithm based on the quantification of the standard curve and an uncorrected T-time ratio algorithm. In the uncorrected T-time ratio algorithm, a sample was marked as positive if it met both of the two acceptance criteria (otherwise, it was negative): 1) G. VAG T hours or A. VAG The ratio of the internal control T time to the T time exceeds the cutoff value, and 2) G. vag T hours or A. vag T-hour comparison L. The ratio of spp T-times exceeded other cutoff values. Absence time was assigned a fixed value. Although both LAG5 and LAG6 showed similar clinical performance, LAG6 had a pre-established log c / mL cutoff value ( Lacto 8.3, G.vag 10.5 and A.vag It showed slightly better performance in 7.4). This project was conducted using a LAG6 multicomposite design, and all systems are non-dual-phase, and A.vag The amplicon was short.

[0612] Example 6

[0613] The oligomer having the nucleotide sequence shown in Table 18 L. Crispatus , L. Genseni , L. Gasseri , G. Virginalis , and A. Virgin And it was evaluated in the multiple complex APTIMA® bacterial vaginosis (ABV) assay for amplification and detection of the general internal control (GIC).

[0614]

[0615] The oligomers designed and screened for the bacterial vaginosis assay are shown in Table 34. Based on this screening, the oligomers shown in Table 16 were selected for further evaluation on the PANTHER® system for linearity, cross-reactivity, clinical performance, and strain tolerance.

[0616] The following large-volume liquid reagents were constructed:

[0617] 1) TCR: HEPES free acid dihydrate (250 mM), lithium hydroxide monohydrate (310 mM), lithium chloride (1.883 M), EDTA free acid (100 mM), poly dT14 magnesium particles (0.03% w / v), pH with lithium hydroxide and hydrochloric acid, volume with water;

[0618] 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 isodium dehydrate (0.39 mM), N-acetyl-L-cysteine ​​(49.58 mM), D(+) trehalose dehydrate (0.03 w / v), cloned MMLV enzyme (224 MR / L), T7 RNA polymerase (140 MU / L), volume with water;

[0619] 3) Amplification and promoter reagents: Potassium chloride (23.3 mM), glycerol anhydride (3.33%), zinc acetate dehydrate (0.05 mM), Proclean 300 preservative (0.02%), Tween-20 (1%), Trisma base (11.61 mM), Trisma 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 (volume).

[0620] The Amp and Pro reagents in Table 19 were prepared by spiking oligonucleotides into aliquots of the amplification and promoter high-dose reagents. The TCR reagent in Table 19 consisted of oligonucleotides and an internal control. In vitro Transcripts were prepared by spiking with a TCR high-dose reagent. No oligos were added to the enzyme reagent.

[0621] To improve torch stability by reducing aggregation, the surfactant Tween-20 was added to the amp / promoter buffer at a final concentration of 1%.

[0622] The test was conducted on the PANTHER system, and the results were analyzed using in-house developed software for real-time analysis of curve properties.

[0623]

[0624] Linearity / Dynamic Range Test / Analysis Sensitivity

[0625] 5 test targets ( A.vag, G.vag, L.crisp , L.gas and L.jenLinearity tests were performed for each. Two sets of panels were prepared for each analyte. One panel was prepared by spiking the culture lysate into sample transport medium, STM (sodium phosphate monobasic monohydrate (2.07 g / L), lithium lauryl sulfate (30 g / L), EDTA isodium dehydrate (0.372 g / L)), EGTA free acid (0.38 g / L), dibasic sodium phosphate (2.13 g / L)). The second panel In vitro Transcripts (IVTs) were prepared by spiking them into an STM. Each panel was tested in 5 replicates on a PANTHER system. Panel concentrations are listed in Table 20.

[0626]

[0627] Table 21 shows a summary of the positivity rates for all five analyses. A.vag and 3 types Lacto All species have 100% IVT positivity up to 1E + 07 c / mL. G.vag It has 100% IVT positivity at 1E + O8 c / mL. All lysates have 100% positivity at 1E + O3 CFU / mL.

[0628] Detailed linearity results for all analytes are shown in Tables 22 through 26. The results shown include positivity, mean RFU range, mean T-hour, mean T-slope, and standard deviation. All responses were valid as indicated by the general internal control.

[0629]

[0630]

[0631]

[0632]

[0633]

[0634]

[0635] Cross-reactivity to microorganisms - Ordered (analytical specificity) and spike (inhibition) tests

[0636] Seventeen pools consisting of bacterial or yeast material representing the normal genitourinary flora or organisms closely associated with APTIMA BV targets were constructed to support cross-reactivity testing (see Table 27). The concentration of each organism targeted was 1.00E + 06 CFU / mL or cell equivalent / mL, which is above the maximum value of the expected concentration.

[0637] To verify analytical specificity, the pool was organized and tested (no spikes from the test targets). The pool was also tested for each test target ( A.vag , G.vag and 3 types Lacto It was tested by spiking with a lysate exemplifying the lysate, and tested for inhibition by other microorganisms. Each condition was tested in 5 repetitions.

[0638]

[0639]

[0640] Organized cross-reactivity results

[0641] FAM channel ( Lacto Species) is grass #16 ( Lacto acidophilus It provided a positive signal for ). These positive results L.crisp It was expected due to the substantial sequence identity of the test organism. . L. acidophilus This cross-reactivity is acceptable because it is found in the upper gastrointestinal tract and is unlikely to exist in the vaginal flora.

[0642] HEX Channel ( G.vag ) provided voice results for all 17 panels.

[0643] ROX Channel ( A.vag ) is Pool #13 ( AtopobiumIt provided a positive signal for the species. The three constituting pool #13 Atopobium bell ( A.rimae, A.minimum, A.parvulum ) was separated and tested again. The component test consisted of 3 types. Atopobium All species provided a positive signal in the ROX channel.

[0644] The Cy5.5 channel (GIC) provided positive results for all 17 pools.

[0645] Spiked cross-reactivity results

[0646] Target test analysis ( L.crisp , L.jen , L.gas , A.vag or G.vag ) was spiked into each of the 17 pools in Table 27. Positive analytes were spiked at approximately 3xLOD concentrations, and five replicate tests were performed per panel per pool. The analyte spike concentrations are shown in Table 28. Spiked STM (Pool 0) was tested as a control for comparison.

[0647]

[0648] A set of high-volume reagents was constructed for the spiked cross-reactivity test. The high-volume reagents were aliquoted into three 100-test reagent kits for testing. Individual vials of the enzyme were reconfigured separately for each of the three kits. When the T-hour results were plotted, it was observed that Reagent Kit #2 had slower T-hours for all four channels compared to Reagent Kits #1 and #3. This appears to have been caused by enzyme recognition variability and thus represents the variance that may arise from inter-kit variability. Dividing the analyte T-hours by the GIC T-hours normalizes the data and accounts for inter-kit variability. The T-hour results are listed in Table 29.

[0649] FAM channel ( Lacto When compared to the control group (Pool #0), Pool #16 ( L. acidophilusWhen ) exists L.crisp , L.jen and L.gas Faster T-hour results from and Pool #06 ( L.bucalis, M.curtisii, M.genitalium, M.hominis It showed a slightly delayed T-time for ). Pool #16 exhibited cross-reactivity even during the ordered test, and in the 16S design region of the test organism. L.crisp It was expected due to characteristics closely related to. Pool #06 was divided into sub-components and retested. The retest is M.hominis When existing L.gas It showed a 1.6-minute delay in T hours.

[0650] HEX Channel ( G.vag ) is Pool #12 ( Candida tropicalis, krusei and lusitaniae When testing in the presence of ), it showed a loss of sensitivity in 4 out of 5 iterations. The 3 types constituting Pool #12 Candida The species were isolated and tested again. In the component test C. krusei It was found that it caused a loss of sensitivity. This potential interference C. krusei It is found in the vaginal flora, but caution is required because it is uncommon. (BD Max TM In a US clinical study on the vaginal panel C. krusei The ratio was 4 / 1647 or 0.2% (BD PI reference number 443710).

[0651] ROX Channel ( A.vag ) compared to the control group (Pool 0) Pool #13 ( Atopobium It showed a slightly faster T time for the species. This is the same pool that caused cross-reactivity in the ordered test, and the APTIMA BV target of the test organism. A.vag It was expected due to characteristics closely related to it.

[0652] Cy5.5 channel (GIC) is full #6 ( L.bucalis, M.curtisii, M.genitalium, M.hominis It showed a slightly delayed T time for ). The component tests of these four organisms are M.hominisIt shows that it delays the GIC T time by only 1 minute and slightly changes the shape of the GIC emergence curve. Mycoplasma hominis is commonly found in BV subjects. Using the BV multivariate algorithm described in Example 1, the T-time lag of GIC increases the BV score through the IC ratio term, and thus increases the probability of a BV positive indication. This M. hominis The inhibitory effect of the reaction caused by the presence of organisms associated with BV, including [the virus], can be mitigated.

[0653]

[0654] Evaluation of clinical samples

[0655] Clinical sample testing was performed using a set of 100 clinical vaginal swab samples collected from the STM. Samples from 10 clinical sites of symptomatic women were tested using the APTIMA BV assay on a PANTHER instrument. One replicate was tested per sample.

[0656] The clinical sample has 20 correction factors ( L.crisp , A.vag and G.vag It was developed with 5 single analyte correction factor levels and 5 mixed analyte correction factor levels including all 3 analytes. Each single analyte correction factor was tested in 4 replicates, and each mixed analyte correction factor was tested in 5 replicates.

[0657] Of the 100 clinical sample replicates tested, 93 were valid. The remaining 7 replicates were invalid due to insufficient sample volume.

[0658] Clinical sample quantification is L.crisp , G.vag and A.vag It was calculated based on the standard curve.

[0659] The diagnosis of BV status was based on quantification and decision tree algorithms. The decision tree algorithm can be summarized as follows (where L, A, and G are respectively L.crisp , A.vag and G.vag It is the measured log copy value for , and X, Y, and Z are, respectively L.crisp , A.vag and G.vag (This is the cutoff value for): If L > X, BV is negative; otherwise, if A > Y or G > Z, BV is positive, and otherwise, BV is negative. In this test method L.crisp, G.vag and A.vag The following cutoff values ​​were used for each: 8.3, 10.4, and 7.4 log c / mL, respectively.

[0660] The clinical standard used in this embodiment is a Nugent score having the median value determined by 3 of the 4 Amsel judgment criteria.

[0661] The single analyte and mixed analyte calibration factors provided identical sensitivity results (90.2%) and similar specificity results (88.5% and 86.5%, respectively), with one sample changed due to differences in quantification (see Table 30).

[0662]

[0663] Inclusiveness test

[0664] Inclusiveness tests were performed for each of the five assay targets using one control strain and all available polymorphic IVT strains. Tests were conducted at low and medium concentrations of each strain. Low panel results were used to determine the effect of polymorphism on analyte sensitivity, and medium panel results were analyzed to determine the effect of polymorphism on analyte time T.

[0665] Table 31 details the number of strains tested, as well as the low and high concentrations targeting each analyte. The results are shown in Table 32.

[0666] A.vag It showed a loss of sensitivity at low copy levels for 3 of the 4 polymorphic strains tested. Medium copy levels showed a T-hour lag of 2 to 3.5 minutes for 2 of the 4 strains. The 2 strains showing a T-hour lag are A.vag It has two mismatches with T7 oligo. The two strains showing a T-hour delay have prevalence rates of 21.95% and <4.87%.

[0667] G.vag It did not show a loss of sensitivity to the tested polymorphic strains. At the intermediate copy level, the polymorphic strains showed a T-hour lag of approximately 2 minutes. These polymorphisms have one mismatch in TCO and one mismatch in T7. This G.vag The prevalence of the strain is very low (2.04%).

[0668] L.crisp One of the two polymorphic strains tested showed only a slight loss of sensitivity at low copy levels. Both polymorphic strains showed a difference in T-time. Compared to the control, one strain was 3.5 minutes faster in T-time, while the other strain was 2 minutes slower. Both of these strains have two mismatches for T7, and both have a prevalence of less than 2%.

[0669] L.gas It had 100% sensitivity for all tested strains. The median copy level showed the minimum change over T hours for polymorphic strains compared to the control.

[0670] L.jen It had 100% sensitivity for all tested strains. The median copy level showed the minimum change over T hours for polymorphic strains compared to the control.

[0671]

[0672]

[0673]

[0674]

[0675]

[0676]

[0677]

[0678]

[0679]

[0680]

[0681]

[0682]

[0683]

[0684]

[0685] From the foregoing, it will be understood that while specific embodiments are described herein for illustrative purposes, various modifications may be made without departing from the spirit and scope of the express invention. Accordingly, the invention is not limited to the appended claims. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety for all purposes. In the event that any material incorporated herein by reference does not conform to the express content of the invention, the express content shall prevail.

[0686] The present application includes a list of sequences submitted electronically in ASCII format, the full text of which is incorporated herein by reference. The above ASCII copy, produced on August 20, 2019, is named 2019-08-20_01159-0039-60PCT_Seq List_ST25.txt and has a size of 66.8 kilobytes.

Claims

Claim 1 (1) G. vaginalis A sample suspected of containing, G. Virginalis A step of contacting first and second amplification oligomers for amplifying a target region of a target nucleic acid, wherein (i) the first oligomer comprises a first target-hybridization sequence consisting of a nucleotide sequence of residues 36 to 52 of SEQ ID NO. 15; and (ii) the second amplification oligomer comprises a second target-hybridization sequence consisting of a nucleotide sequence of SEQ ID NO. 14; (2) In vitro As a step of performing a nucleic acid amplification reaction, wherein an arbitrary G. Virginalis If the target nucleic acid is present in the sample, G. Virginalis Step (3) used as a template for generating one or more amplification products corresponding to a target region; and (3) detecting the presence or absence of one or more amplification products in the sample, thereby [determining] the G. Virginalis In a sample, comprising the step of determining the presence or absence of G. Virginalis A method for determining the presence or absence of. Claim 2 The method according to claim 1, wherein the first amplification oligomer is a promoter primer or promoter provider further comprising a promoter sequence located at the 5' of the target hybridization sequence. Claim 3 In paragraph 2, the method wherein the promoter sequence is a T7 promoter sequence. Claim 4 In paragraph 1, prior to step (2), the sample G. Virginalis When target nucleic acid is present G. Virginalis The method further includes a step of purifying the target nucleic acid from other components, wherein the purification step comprises a sample G. Virginalis A method comprising contacting a capture probe oligomer comprising a target-hybridization sequence that specifically hybridizes to a target sequence within a target nucleic acid, wherein the capture probe target-hybridization sequence is covalently attached to a sequence or moiety that binds to an immobilized probe. Claim 5 A method according to claim 4, wherein the probe target-hybridization sequence consists of the nucleotide sequence of residues 1 to 20 of SEQ ID NO.

13. Claim 6 In paragraph 1, the detection step (3) is (i) G. Virginalis A target-hybridization sequence comprising a target region that specifically hybridizes to the target region G. Virginalis A step of contacting a specific detection probe with one or more amplification products; and (ii) any target-hybridized G. Virginalis A method comprising the step of detecting the presence or absence of a specific detection probe. Claim 7 In paragraph 6, G. Virginalis A method in which the specific detection probe target-hybridization sequence consists of the nucleotide sequence of residues 1 to 18 of SEQ ID NO.

16. Claim 8 In paragraph 6, the detection step (3) occurs during the amplification step (2). Claim 9 In paragraph 8, G. Virginalis A specific detection probe comprising a fluorescent label and a quencher, method. Claim 10 In Paragraph 9, G. Virginalis A specific detection probe is a molecular torch or a molecular beacon.

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