Compositions and methods for the detection of bacteria and fungi associated with bacterial and candidal vaginitis - Patents.com

Abstract

A method is described for the rapid detection of the presence or absence of multiple bacterial vaginosis-associated (BV-associated) bacteria and / or vulvovaginal candidiasis (VVC)-associated Candida in a biological or non-biological sample. The method can include performing an amplification step, a hybridization step, and a detection step. Additionally, primers and probes and kits targeted to specific genes designed for the detection of BV-associated bacteria and VVC-associated Candida are provided.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure relates to the field of molecular diagnostics, and more particularly to the detection of various bacterial and fungal strains associated with bacterial and Candida vaginitis. [Background technology]

[0002] 2. Background of the Invention Vaginitis is responsible for as many as 50% of all gynecologic visits in the United States and is a major source of health care costs. Bacterial vaginosis (BV), vulvovaginal candidiasis (VVC), and infectious vaginitis due to trichomoniasis account for up to 90% of these cases. Unlike trichomoniasis, both BV and VVC are due to several pathogens. In cases of VVC, overgrowth of Candida albicans predominates, but other Candida species, including Candida glabrata, may contribute as well. BV is difficult to diagnose because the etiology involves decreased levels of Lactobacillus bacteria with concomitant increased concentrations of BV-associated bacteria such as Gardnerella vaginalis, Mobiluncus spp., and Atopobium vaginae.

[0003] A variety of diagnostic methods are available to identify the underlying cause of vaginitis. In clinicians' offices, a combination of pH, potassium hydroxide (KOH) testing, and microscopy of fresh samples of vaginal secretions is routinely used, despite their relatively poor performance. In the case of BV, diagnosis often relies on the use of either clinical Amsel criteria or Gram stain and Nugent score (considered the gold standard test for the diagnosis of BV). Examination of wet mounts with KOH preparations and / or vaginal cultures for Candida are the most common diagnostic tools for VVC. Although sensitive and specific nucleic acid amplification tests (NAATs) are recommended for the detection of Trichomonas vaginalis, testing of wet mount preparations is still commonly used in clinical practice. However, the use of non-molecular methods is accompanied by several barriers, including lack of clinic equipment, subjectivity of the clinical endpoints used, and inconsistent use among physicians, lack of appropriate training in microscopy, and the overall low sensitivity of the tests. Diagnosis of the underlying cause of vaginitis is further complicated by the common symptomatology reported for BV, VVC, and trichomoniasis; the incidence of mixed or coinfections; and the recurrence of vaginal symptoms.

[0004] There is therefore a need to develop more efficient and rapid methods for detecting vulvovaginal candidiasis and bacterial vaginosis, for example methods that allow for the detection of both vaginal disorders in a single assay in order to effectively deliver appropriate treatment to patients. Summary of the Invention

[0005] In one aspect, a method is disclosed for detecting a plurality of BV-associated bacteria in a biological sample, the plurality of BV-associated bacteria including at least one from Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae, Megasphaera type 1, Eggerthella spp., Prevotella spp., and bacterial vaginosis-associated bacteria BVAB-2. As described herein, the method includes performing an amplification step including contacting the sample with a primer set to produce an amplification product if nucleic acid from BV-associated bacteria is present in the sample; performing a hybridization step including contacting each amplification product with one or more detectable probes; and detecting the presence of each amplification product, the presence of the amplification product indicating the presence of BV-associated bacteria in the sample; and performing a hybridization step including contacting each amplification product with one or more detectable probes; and detecting the presence of each amplification product, the presence of which indicates ...a primer set for producing an amplification product from comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 38, 41 or 44 and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 39, 42 or 45, 47 or 73, and the one or more detectable oligonucleotide probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 40, 43, 46 and 74; a primer set for producing an amplification product from Gardnerella vaginalis comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 1, 4, 5, 8 or 11 and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 2, 6, 9 or 12, and the one or more detectable oligonucleotide probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 3, 7, 10 and 13; A primer set for producing an amplification product from Pseudomonas vaginae comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 14, 17, 20, 67 or 70, and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 15, 18, 21, 68 or 71, and the one or more detectable probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 16, 19, 22, 66, 69 and 72; a primer set for producing an amplification product from Megasphaera type 1 comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 23, 26 or 27, and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 24 or 28, and the one or more detectable probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 25 and 29;a primer set for producing an amplification product from Prevotella spp. comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 84 or 87 and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 85 or 88, and the one or more detectable probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 86 and 89; a primer set for producing an amplification product from Prevotella spp. comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 90, 95, 96 or 97 and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 91, 92, 93, 98, 99 or 100, and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 94, 101, and 102; a primer set for producing an amplification product from BVAB-2 comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 30 or 33, and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 31, 34, or 36, and one or more detectable probes comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 32, 35, and 37.

[0006] In some embodiments, the plurality of BV-associated bacteria are Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae, and the primer set for producing an amplification product from Lactobacillus spp. comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 38, 41 or 44, and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 39, 42 or 45, 47 or 73, and the one or more detectable probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 40, 43, 46, and 74; A primer set for producing an amplification product from Atopobium vaginalis comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 1, 4, 5, 8 or 11, and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 2, 6, 9 or 12, and the one or more detectable oligonucleotide probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 3, 7, 10 and 13; a primer set for producing an amplification product from Atopobium vaginae comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 14, 17, 20, 67 or 70, and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 15, 18, 21, 68 or 71, and the one or more detectable probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 16, 19, 22, 66, 69 and 72.In some embodiments, the primer set for producing an amplification product from Lactobacillus spp. comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 44 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 45, and the one or more detectable probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 46; the primer set for producing an amplification product from Gardnerella vaginalis comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 11 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 12, and the one or more detectable probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 13; the primer set for producing an amplification product from Atopobium vaginae comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 20 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 21, and the one or more detectable probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 72. In other embodiments, the hybridizing step of the method comprises contacting the amplification product with a detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor moiety, and the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, the presence or absence of fluorescence indicating the presence or absence of BV-associated bacteria in the sample. In further embodiments, the amplifying step of the method employs a polymerase enzyme having 5'-3' nuclease activity. In some embodiments, the "contacting" step further comprises contacting the biological sample and the primer with a DNA polymerase, a plurality of free nucleotides including adenine, thymine, cytosine and guanine, and / or a buffer to generate a reaction mixture. The nucleic acid extracted from the biological sample may comprise or consist of double-stranded DNA.The reaction mixture may optionally further comprise divalent cations, monovalent cation potassium ions, one or more detectably labeled probes, and / or any combination thereof. In some embodiments, the step of "generating amplicons" comprises (a) heating the reaction mixture to a first pre-determined temperature for a first pre-determined time to separate strands of double-stranded DNA present in the biological sample or nucleic acid, (b) cooling the reaction mixture to a second pre-determined temperature for a second pre-determined time under conditions in which the primers hybridize to their complementary sequences and the DNA polymerase extends the primers, and (c) repeating steps (a) and (b) at least 10-12 times. In some embodiments, steps (a) and (b) are repeated at least 15, 20, 22 or 25 times. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is collected from the urethra, penis, anus, throat, cervix or vagina. In some embodiments, the biological sample is DNA, RNA or total nucleic acid extracted from a clinical specimen. In some embodiments, the hybridization step includes contacting the amplification product with a detectable genetic probe labeled with a donor fluorescent moiety and a corresponding acceptor moiety, and the detection step includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, where the presence or absence of fluorescence indicates the presence or absence of BV-associated bacteria in the sample. In some embodiments, the amplification and hybridization steps are repeated, where the number of repetitions depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more amplification and hybridization steps will be required to amplify the target sequence enough for detection. In some embodiments, the amplification and hybridization steps are repeated at least about 20 times, but may be repeated at least 25, 30, 40, 50, 60, or even 100 times.Additionally, detection of the presence or absence of amplification product can be performed during or after each amplification and hybridization step, between or after every other amplification and hybridization step, between or after a particular amplification and hybridization step, or between or after a particular amplification and hybridization step, where sufficient amplification product is expected to be present for detection. In some embodiments, the amplification step uses a polymerase enzyme with 5' to 3' nuclease activity. In some embodiments, the donor fluorescent moiety and the corresponding acceptor moiety are within 8 to 20 nucleotides of each other on the probe. In some embodiments, the acceptor moiety is a quencher. In some embodiments, the oligonucleotide comprises or consists of a sequence of nucleotides selected from SEQ ID NOs: 1-47, 66-74, and 84-102, or a complement thereof, and has 100 nucleotides or less, 50 nucleotides or less, 40 nucleotides or less, or 30 nucleotides or less.

[0007] The present disclosure also provides a forward primer comprising or consisting of a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 8, 11, 14, 17, 20, 23, 26, 27, 30, 33, 38, 41, 44, 67, 70, 84, 87, 90, 95, 96, and 97, and a reverse primer comprising or consisting of a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 6, 9, 12, 15, 18, 21, 24, 28, 31, 34, 36, 39, 42, 45, 47, 68, 71, 73, 85, 88, 91, 92, 93, 98, 99, and 100. and a probe comprising or consisting of a third detectably labeled oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 7, 10, 13, 16, 66, 69, 19, 22, 72, 25, 29, 32, 35, 37, 40, 43, 46, 74, 86, 89, 94, 101, and 102 or a complement thereof, wherein the third detectably labeled oligonucleotide sequence is configured to hybridize to an amplicon generated by the forward primer and the reverse primer. In some embodiments, the detectably labeled third oligonucleotide sequence comprises a donor fluorescent moiety and a corresponding acceptor moiety. In some embodiments, the kit further comprises nucleoside triphosphates, a nucleic acid polymerase, and a buffer required for the function of the nucleic acid polymerase. In still other embodiments, at least one of the first, second, and third oligonucleotide sequences comprises at least one modified nucleotide. In one embodiment, the BV-associated bacteria include Lactobacillus spp., Gardnerella vaginalis, Atopobium vaginae, Megasphaera type 1, Eggerthella spp., Prevotella spp., and BVAB-2.In some embodiments, the kit comprises primers and probes capable of hybridizing to the 23s rRNA or D-LDH gene of Lactobacillus spp., comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 38-47 and 73-74. In some embodiments, the kit comprises primers and probes capable of hybridizing to the tuf gene or the 23s rRNA gene or the 16s rRNA gene of Gardnerella vaginalis, comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 1-13. In some embodiments, the kit comprises primers and probes capable of hybridizing to the 23s rRNA gene or the tufA gene of Atopobium vaginae, comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 14-22 and 66-72. In some embodiments, the kit comprises primers and probes capable of hybridizing to the 16s rRNA gene of Megasphaera type 1, comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 23-29. In some embodiments, the kit comprises primers and probes capable of hybridizing to the 16s rRNA gene of Eggerthella spp., comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 84-89. In some embodiments, the kit comprises primers and probes capable of hybridizing to the 16s rRNA gene of Prevotella spp., comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 90-102. In some embodiments, the kit comprises primers and probes capable of hybridizing to the 16s rRNA gene of BVAB-2, comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 30-37.

[0008] In another aspect, the disclosure provides methods for detecting vulvovaginal candidiasis (VVC)-associated Candida species in a biological sample, the VVC-associated Candida species including Candida albicans, Candida tropicalis, Candida dubliniensis, and Candida parapsilosis (collectively referred to as Candida spp.). In some embodiments, the VVC-associated species further includes Candida krusei and / or Candida glabrata.As used herein, the method includes performing an amplification step comprising contacting a sample with a set of primers to produce an amplification product if nucleic acid from VVC-associated Candida is present in the sample; performing a hybridization step comprising contacting each amplification product with one or more detectable probes; and detecting the presence of each amplification product, the presence of the amplification product indicating the presence of VVC-associated Candida in the sample; the set of primers for producing an amplification product from Candida spp. comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 48, 51, 54, 57 or 76, and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 49, 52, 55, 58 or 77, and the one or more detectable probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 50, 53, 56, 59, 75 and 78; A primer set for producing an amplification product from Candida krusei comprises a forward primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 60, 79 or 82 and a reverse primer comprising or consisting of an oligonucleotide sequence selected from SEQ ID NO: 61, 80 or 83, and the one or more detectable probes comprise or consist of an oligonucleotide sequence selected from SEQ ID NO: 62 and 81; a primer set for producing an amplification product from Candida glabrata comprises a forward primer comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 63 and a reverse primer comprising or consisting of an oligonucleotide sequence of SEQ ID NO: 64, and the one or more detectable probes comprise or consist of SEQ ID NO: 65.In some embodiments, the primer set for producing an amplification product from Candida spp. comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 76 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 77, and the one or more detectable probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 78; the primer set for producing an amplification product from Candida krusei comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 79 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 80, and the one or more detectable probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 81. In other embodiments, the hybridizing step comprises contacting the amplification product with a detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor moiety, and the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, the presence or absence of fluorescence being indicative of the presence or absence of VVC-associated Candida in the sample. In further embodiments, the amplifying step employs a polymerase enzyme with 5'-3' nuclease activity. In some embodiments, the "contacting" step further comprises contacting the biological sample and the primer with a DNA polymerase, a plurality of free nucleotides including adenine, thymine, cytosine and guanine, and / or a buffer to generate a reaction mixture. The nucleic acid extracted from the biological sample may comprise or consist of double-stranded DNA. The reaction mixture may optionally further comprise divalent cations, monovalent cation potassium ions, one or more detectably labeled probes, and / or any combination thereof.In some embodiments, the step of "generating an amplicon" comprises (a) heating the reaction mixture to a first predetermined temperature for a first predetermined time to separate strands of double-stranded DNA present in the biological sample or nucleic acid, (b) cooling the reaction mixture to a second predetermined temperature for a second predetermined time under conditions in which the primers hybridize to their complementary sequences and the DNA polymerase extends the primers, and (c) repeating steps (a) and (b) at least 10-12 times. In some embodiments, steps (a) and (b) are repeated at least 15, 20, 22, or 25 times.

[0009] The present disclosure also provides a kit for detecting vulvovaginal candidiasis (VVC) associated Candida species in a sample, comprising a forward primer comprising or consisting of a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 48, 51, 54, 57, 76, 60, 79, 82 and 63, a reverse primer comprising or consisting of a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 49, 52, 55, 58, 77, 61, 80, 83 and 64, and a probe comprising or consisting of a third detectably labeled oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 50, 53, 75, 56, 59, 78, 62, 81 and 65 or a complement thereof, wherein the probe is configured to hybridize to an amplicon generated by the forward primer and the reverse primer. In some embodiments, the detectably labeled third oligonucleotide sequence comprises a donor fluorescent moiety and a corresponding acceptor moiety. In some embodiments, the kit further comprises nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase. In yet other embodiments, at least one of the first, second, and third oligonucleotide sequences comprises at least one modified nucleotide. In some embodiments, the VVC-associated Candida spp. include Candida albicans, Candida tropicalis, Candida dubliniensis, and Candida parapsilosis (collectively referred to as Candida spp.), Candida krusei, and Candida glabrata. In some embodiments, the kit comprises primers and probes capable of hybridizing to a ribosomal RNA (rRNA) gene or 25s rRNA of Candida spp., comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 48-59 and 75-78.In some embodiments, the kit comprises a primer and a probe capable of hybridizing to an internal transcribed spacer (ITS) of the rRNA gene of Candida krusei comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 60-62 and 79-83. In some embodiments, the kit comprises a primer and a probe capable of hybridizing to an internal transcribed spacer (ITS) of the rRNA gene of Candida glabrata comprising or consisting of an oligonucleotide sequence selected from SEQ ID NOs: 63-65. In some embodiments, the sample is a biological sample. In some embodiments, the biological sample is collected from the urethra, penis, anus, throat, cervix, or vagina. In some embodiments, the biological sample is DNA, RNA, or total nucleic acid extracted from a clinical specimen.

[0010] Further, the present disclosure provides oligonucleotides comprising a nucleic acid or a complement thereof having at least 70% sequence identity (e.g., at least 75%, 80%, 85%, 90% or 95%) to any one of SEQ ID NOs: 1-102, and having 100 or fewer nucleotides, 50 or fewer nucleotides, 40 or fewer nucleotides or 30 or fewer nucleotides. Generally, these oligonucleotides can be primer nucleic acids, probe nucleic acids, and the like. In some embodiments, the oligonucleotides comprise at least one modified nucleotide, e.g., to alter nucleic acid hybridization stability compared to unmodified nucleotides. In some embodiments, the at least one modified nucleotide is N 6 -Benzyl-dA,N 4 -Benzyl-dC,N 6 -para-tert-butyl-benzyl-dA, and N 4-para-tert-butyl-benzyl-dC. Optionally, the oligonucleotide comprises at least one label and / or at least one quencher moiety. In some embodiments, the oligonucleotide comprises at least one conservatively modified variation. "Conservatively modified variations" or simply "conservative variations" of a particular nucleic acid sequence refer to nucleic acids that code for identical or essentially identical amino acid sequences, or essentially identical sequences if the nucleic acid does not code for an amino acid sequence. Those skilled in the art will recognize that individual substitutions, deletions or additions that alter, add or delete a single amino acid or a small number of amino acids (typically less than 5%, more typically less than 4%, 2% or 1%) in the encoded sequence are "conservatively modified variations" where the modification results in the deletion of an amino acid, the addition of an amino acid, or the replacement of an amino acid with a chemically similar amino acid. In some embodiments, at least one of the first and second target gene primers and the detectable target gene probe comprises at least one modified nucleotide.

[0011] In another aspect, the disclosure provides a method for simultaneously detecting Lactobacillus spp., Gardnerella vaginalis, Atopobium vaginae, and at least one of the group selected from Candida spp., Candida krusei, and Candida glabrata in a sample, comprising contacting the sample with a primer set to simultaneously detect Lactobacillus spp., Gardnerella vaginalis, Atopobium vaginae, and Candida spp., Candida krusei, and Candida glabrata in a sample. glabrata; performing an amplification step comprising contacting each amplification product with one or more detectable probes to produce an amplification product if a nucleic acid selected from at least one of the group selected from Lactobacillus spp., Gardnerella vaginalis, Atopobium vaginae, and Candida spp., Candida krusei, and Candida glabrata is present in the sample; performing a hybridization step comprising contacting each amplification product with one or more detectable probes; and detecting the presence of each amplification product, the presence of the amplification product indicating the presence in the sample of at least one of the group selected from Lactobacillus spp., Gardnerella vaginalis, Atopobium vaginae, and Candida spp., Candida krusei, and Candida glabrata. The above primer sets and corresponding probes may be used herein. In one embodiment, Lactobacillus spp.a primer set for producing an amplification product from, comprising a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 44 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 45, and one or more detectable probes comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 46; a primer set for producing an amplification product from, Gardnerella vaginalis comprising a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 11 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 12, and one or more detectable probes comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 13; a primer set for producing an amplification product from, Atopobium vaginae comprising a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 20 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 21, and one or more detectable probes comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 72; and / or Candida spp.a set of primers for producing an amplification product from Candida krusei comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 76 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 77, and one or more detectable probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 78; a set of primers for producing an amplification product from Candida krusei comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 79 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 80, and one or more detectable probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 81; a set of primers for producing an amplification product from Candida glabrata comprises a forward primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 63 and a reverse primer comprising or consisting of the oligonucleotide sequence of SEQ ID NO: 64, and one or more detectable probes comprise or consist of the oligonucleotide sequence of SEQ ID NO: 65.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present subject matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0013] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and detailed description, and from the claims. [Brief description of the drawings]

[0014] [Figure 1A]1 shows the growth curves of a PCR experiment to amplify and detect Atopobium vaginae as described in Example 2. [Figure 1B] Calculated Ct values ​​of the same experiments are shown. [Diagram 2] 1 shows the calculated Ct values ​​of PCR experiments to amplify and detect Gardnerella vaginalis as described in Example 3. [Diagram 3] Calculated Ct values ​​for PCR experiments to amplify and detect BVAB-2 as described in Example 4 are shown. [Figure 4] Calculated Ct values ​​for PCR experiments to amplify and detect Megasphaera type 1 as described in Example 5 are shown. [Diagram 5] 1 shows the calculated Ct values ​​of PCR experiments for amplifying and detecting Lactobacillus species as described in Example 6. [Figure 6] 1 shows the calculated Ct values ​​of PCR experiments in the CAPT_rRNA_1 assay for amplifying and detecting Candida spp., as described in Example 9. [Figure 7] FIG. 1 shows the calculated Ct values ​​of PCR experiments in the CVS_rRNA_1 assay for amplifying and detecting Candida spp. and Candida glabrata as described in Example 9. [Figure 8]Figure 1 shows growth curves in channel 1 for detection of Gardnerella vaginalis 16s rRNA in a multiplex PCR assay as described in Example 10. T1-4Ab: Pool of plasmids carrying Gardnerella vaginalis 16s, Lactobacillus crispatus LDH, Atopobium vaginae tufA, Candida albicans 25s rRNA sequences, and human genomic DNA. T1-4Gb: Pool of plasmids carrying Gardnerella vaginalis 16s, Lactobacillus crispatus LDH, Atopobium vaginae tufA, Candida glabrata ITS sequences, and human genomic DNA. HIVD: Sample diluent. [Figure 9] Figure 1 shows growth curves in channel 2 for detection of D-LDH gene of Lactobacillus species in a multiplex PCR assay as described in Example 10. T1-4Ab: Pool of plasmids with Gardnerella vaginalis 16s, Lactobacillus crispatus LDH, Atopobium vaginae tufA, Candida albicans 25s rRNA sequence, and human genomic DNA. T1-4Gb: Pool of plasmids with Gardnerella vaginalis 16s, Lactobacillus crispatus LDH, Atopobium vaginae tufA, Candida glabrata ITS sequence, and human genomic DNA. HIVD: specimen diluent. [Figure 10] Figure 1 shows growth curves in channel 3 for detection of Atopobium vaginae tufA gene in a multiplex PCR assay as described in Example 10. T1-4Ab: Pool of plasmids carrying Gardnerella vaginalis 16s, Lactobacillus crispatus LDH, Atopobium vaginae tufA, Candida albicans 25s rRNA sequence, and human genomic DNA. T1-4Gb: Pool of plasmids carrying Gardnerella vaginalis 16s, Lactobacillus crispatus LDH, Atopobium vaginae tufA, Candida glabrata ITS sequences, and human genomic DNA. HIVD: Sample diluent. [Figure 11] Figure 1 shows growth curves in channel 4 for detection of 25s rRNA of Candida spp. in a multiplex PCR assay as described in Example 10. T1-4Ab: Pool of plasmids with Gardnerella vaginalis 16s, Lactobacillus crispatus LDH, Atopobium vaginae tufA, Candida albicans 25s rRNA sequences, and human genomic DNA. T1-4Gb: Pool of plasmids with Gardnerella vaginalis 16s, Lactobacillus crispatus LDH, Atopobium vaginae tufA, Candida glabrata ITS sequences, and human genomic DNA. HIVD: specimen diluent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Detailed Description of the Invention Methods and compositions for detecting vulvovaginal candidiasis (VVC) and bacterial vaginosis (BV) are provided herein. For example, primers and probes are provided that can bind to specific genes of Candida species associated with VVC and BV-associated bacteria to determine the presence or absence of VVC-associated Candida species and BV-associated bacteria in a sample, such as a biological sample. In some embodiments, multiplex nucleic acid amplification can be performed to allow detection of VVC-associated Candida species and BV-associated bacteria in a single assay.

[0016] The disclosed method may include performing at least one cycling step that includes amplifying one or more portions of a nucleic acid molecule gene target from a sample using one or more primer pairs. As used herein, "primer(s)" refers to an oligonucleotide primer that specifically anneals to a target gene in a Bacteria or Candida species and initiates DNA synthesis therefrom under appropriate conditions to produce a respective amplification product. Each of the contemplated primers anneals to a target within or adjacent to a respective target nucleic acid molecule such that at least a portion of each amplification product contains a nucleic acid sequence corresponding to the target. If one or more of the target gene nucleic acids are present in the sample, one or more amplification products will be generated, and thus the presence of one or more target gene amplification products indicates the presence of a Bacteria or Candida species in the sample. The amplification product must contain a nucleic acid sequence complementary to one or more detectable probes of the target gene. As used herein, "probe(s)" refers to an oligonucleotide probe that specifically anneals to a nucleic acid sequence encoding a target gene. Each cycling step includes an amplification step, a hybridization step, and a detection step in which the sample is contacted with one or more detectable probes to detect the presence or absence of a Bacterium or Candida species in the sample.

[0017] The term "amplifying" as used herein refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule. Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing a primer to the template nucleic acid at a temperature below the melting temperature of the primer, and enzymatically extending from the primer to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and an appropriate buffer and / or cofactors for optimal activity of the polymerase enzyme (e.g., MgCl2 and / or KCl).

[0018] The term "primer" as used herein is known to those skilled in the art and refers to an oligomeric compound, primarily oligonucleotides, but also modified oligonucleotides that are capable of "priming" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group to which further "nucleotides" can be attached by the template-dependent DNA polymerase, which establishes a 3' to 5' phosphodiester bond whereby a deoxynucleoside triphosphate is used, thereby releasing pyrophosphate. Thus, there is no fundamental difference between a "primer", an "oligonucleotide", or a "probe", except perhaps in the intended function.

[0019] The term "hybridizing" refers to the annealing of one or more probes to an amplification product. Hybridization conditions typically include a temperature below the melting temperature of the probe, but which avoids non-specific hybridization of the probe.

[0020] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase typically associated with nucleic acid chain synthesis whereby nucleotides are removed from the 5' end of a nucleic acid chain.

[0021] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, catalyzes the formation of primer extension products complementary to a template, and does not irreversibly denature when exposed to high temperatures for the time required to effect denaturation of the double-stranded template nucleic acid. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have been isolated, for example, from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, non-thermostable polymerases can also be used in PCR assays, provided the enzyme is replenished.

[0022] The term "complement thereof" refers to a nucleic acid that is the same length as and exactly complementary to a given nucleic acid.

[0023] The term "extension" or "lengthening" when used in reference to a nucleic acid refers to when additional nucleotides (or other similar molecules) are incorporated into a nucleic acid. For example, a nucleic acid is optionally extended by a nucleotide incorporating biocatalyst, e.g., a polymerase, which typically adds nucleotides to the 3' end of the nucleic acid.

[0024] The term "identical" or percent "identity" in the context of two or more nucleic acid sequences refers to two or more sequences or subsequences that are identical or have a certain percentage of identical nucleotides, when compared or aligned for maximum correspondence as determined, for example, using one of the sequence comparison algorithms available to those of skill in the art or by visual inspection. Exemplary algorithms suitable for determining percent sequence identity and sequence similarity are the BLAST programs, see, e.g., Altschul et al. (1990) "Basic local alignment search tool," J. Mol. Biol. 215:403-410; Gish et al. (1993) "Identification of protein coding regions by database similarity search," Nature Genet. 3:266-272; Madden et al. (1996) "Applications of network BLAST server," Meth. Enzymol. 266:131-141; Altschul et al. (1997) "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402; and Zhang et al. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation," Genome Genet. Res. 7:649-656.

[0025] "Modified nucleotide" in the context of oligonucleotide refers to the change in which at least one nucleotide of an oligonucleotide sequence is replaced by a different nucleotide that provides the oligonucleotide with desired properties.Exemplary modified nucleotides that can be replaced in the oligonucleotides described herein include, for example, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudomimetic-dU, nitropyrrole, nitroindole, 2'-0-methylriboU, 2'-0-methylriboC, N4-ethyl-dC, N 6 -Methyl-dA,N 6 -Benzyl-dA,N 4 -Benzyl-dC,N 6 -para-tert-butylbenzyl-dA,N 4 -para-tert-butyl-benzyl-dC and the like. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or known in the art. In certain embodiments, modified nucleotide substitutions modify the melting temperature (Tm) of the oligonucleotide compared to the melting temperature of the corresponding unmodified oligonucleotide. To further explain, certain modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation, etc.), increase the yield of the intended target amplicon, and the like. Examples of these types of nucleic acid modifications are disclosed, for example, in U.S. Pat. No. 6,001,611.

[0026] Bacterial and fungal detection As described herein, nucleic acid amplification can be performed to determine the presence, absence and / or levels of Candida species and / or BV-associated bacteria in a sample. Several Candida species are known to be associated with VVC, including, but not limited to, C. albicans, C. tropicalis, C. dubliniensis, C. parapsilosis, C. krusei, and C. glabrata. Many bacteria are also known to be associated with BV, including, but not limited to, Lactobacillus species (e.g., Lactobacillus crispatus (L. crispatus), Lactobacillus jensenii (L. jensenii), and Lactobacillus gasseri (L. gasseri)), Gardnerella vaginalis (G. vaginalis), Atopobium vaginae, Megasphaera type 1 (Megasphaera-1), and BVAB-2. In some embodiments, the presence, absence, and / or levels of VVC-associated Candida species and BV-associated bacteria are determined by detecting one or more target genes of each target organism using methods known in the art, such as DNA amplification. In some embodiments, multiplex PCR can be performed to detect the presence, absence, or levels of each of the target Candida species and / or BV-associated bacteria. In some embodiments, multiplex PCR is performed to detect the presence, absence, and / or levels of each of the target VVC-associated Candida species, L. crispatus, L. jensenii, G. vaginalis, Atopobium vaginae, Megasphaera type 1, and BVAB-2. In some embodiments, the VVC-associated Candida species are C. albicans, C. tropicalis, C. parapsilosis, C. krusei, and C. glabrata.

[0027] In another embodiment, nucleic acid amplification can be performed on the same sample to determine the presence, absence and / or levels of Trichomonas vaginalis (TV). Compositions and methods for rapid detection of the presence or absence of Trichomonas vaginalis in biological or non-biological samples are described in U.S. Patent Application Publication No. 2017 / 0342508.

[0028] Each of the target VVC-associated Candida species and BV-associated bacteria can be detected using separate channels in the DNA amplification. In some cases, it may be desirable to use a single fluorescent channel to detect the presence, absence, and / or level of two or more of VVC-associated Candida species and BV-associated bacteria. For example, a single fluorescent channel can be used to detect the presence, absence, and / or level of two BV-associated bacteria (e.g., BVAB-2 and Megasphaera-1). Such a combination, in some embodiments, can reduce the amount of reagents required to perform an experiment and provide an accurate qualitative metric by which BV determination can be evaluated. Without being bound to a particular theory, it is believed that the use of combined markers can increase the sensitivity and specificity of the assay. In some embodiments, separate fluorescent channels are used to detect the presence, absence and / or levels of Lactobacillus spp. (e.g., L. crispatus, L. jensenii and L. gasseri), G. vaginalis and Atopobium vaginae, respectively, and a single fluorescent channel is used to detect the presence, absence and / or levels of BVAB-2, Megasphaera-1, Eggerthella spp. and Prevotella spp.

[0029] Oligonucleotides (e.g., amplification primers and probes) are provided that can specifically hybridize (e.g., under standard nucleic acid amplification conditions, e.g., under standard PCR conditions and / or stringent hybridization conditions) to target gene regions or their complements in VVC-associated Candida species, L. crispatus, L. jensenii, L. gasseri, G. vaginalis, Atopobium vaginae, Megasphaera type 1 (Megasphaera-1), and BVAB-2. Amplification of the target gene region of an organism in a sample (e.g., a vaginal swab sample) can, in some embodiments, indicate the presence, absence, and / or level of the organism in the sample.

[0030] The target gene region can vary. In some embodiments, oligonucleotides (e.g., amplification primers and probes) are provided that can specifically hybridize (e.g., under standard nucleic acid amplification conditions, e.g., under standard PCR conditions and / or stringent hybridization conditions) to a gene region encoding 16S ribosomal RNA (16S rRNA) in an organism. In some embodiments, the organism is Gardnerella vaginalis. In some embodiments, the organism is BVAB-2. In some embodiments, the organism is Megasphaera type 1. Examples of oligonucleotides that can specifically hybridize to the 16s rRNA gene region of G. vaginalis, Megasphaera-1, and BVAB-2 include, but are not limited to, SEQ ID NOs: 11-13 provided in Table I, SEQ ID NOs: 23-29 provided in Table III, and SEQ ID NOs: 30-37 provided in Table IV, respectively.

[0031] In some embodiments, the 23s rRNA gene is used as a target gene for DNA amplification to detect the presence, absence and / or levels of Gardnerella vaginalis, Atopobium vaginae, and Lactobacillus spp. in a sample. Examples of oligonucleotides capable of specifically hybridizing to the 23s RNA gene region of G. vaginalis, A. vaginae, and Lactobacillus spp. include, but are not limited to, SEQ ID NOs: 5-10 provided in Table I, SEQ ID NOs: 14-16 provided in Table II, and SEQ ID NOs: 38-43 provided in Table V, respectively.

[0032] In some embodiments, oligonucleotides (e.g., amplification primers and probes) are provided that can specifically hybridize (e.g., under standard nucleic acid amplification conditions, e.g., under standard PCR conditions and / or stringent hybridization conditions) to gene regions encoding the elongation factor Tu protein (tuf gene) of G. vaginalis and the tufA gene of A. vaginae. In some embodiments, the tuf (or tufA) gene is used as a target gene for DNA amplification to detect the presence, absence and / or levels of G. vaginalis and A. vaginae in a sample. In some embodiments, primers and probes that can specifically bind to the tuf gene region of G. vaginalis and the tufA gene region of A. vaginae are used to detect the presence, absence and / or levels of G. vaginalis and A. vaginae in a biological sample. Examples of oligonucleotides capable of specifically hybridizing to the tuf gene region of G. vaginalis include, but are not limited to, SEQ ID NOs: 1-4 provided in Table I, and examples of oligonucleotides capable of specifically hybridizing to the tufA gene region of A. vaginae include, but are not limited to, SEQ ID NOs: 17-22 provided in Table II.

[0033] In some embodiments, ribosomal RNA (rRNA) genes are used as target genes for DNA amplification to detect the presence, absence and / or level of VVC-associated Candida species in a sample. In some embodiments, VVC-associated Candida spp. include C. albicans, C. tropicalis, Candida dubliniensis and C. parapsilosis (collectively Candida spp.). In some embodiments, the VVC-associated Candida species is Candida krusei. In some embodiments, the VVC-associated Candida species is Candida glabrata. In some embodiments, the VVC-associated Candida species is C. albicans, C. tropicalis, C. dubliniensis C. parapsilosis, or a combination thereof. Examples of oligonucleotides capable of specifically hybridizing to the rRNA gene region of C. glabrata include, but are not limited to, SEQ ID NOs: 63-65 provided in Table VII. Examples of oligonucleotides capable of specifically hybridizing to rRNA gene regions in C. albicans, C. tropicalis, C. dubliniensis, and C. parapsilosis (collectively Candida spp.) include, but are not limited to, SEQ ID NOs: 48-59 as provided in Table VI. Examples of oligonucleotides capable of specifically hybridizing to rRNA gene regions of C. krusei include, but are not limited to, SEQ ID NOs: 60-62 as provided in Table VII. [Table 1] [Table 2-1] [Table 2-2] [Table 3] [Table 4] [Table 5] [Table 6-1] [Table 6-2] [Table 7] [Table 8] [Table 9]

[0034] In one embodiment, the primer sets and probes described above are used to provide for the detection of bacteria and Candida species associated with vaginitis in biological samples suspected of containing such bacteria and Candida species. The set of primers and probes may comprise or consist of primers and probes specific for the nucleic acid sequences of the respective bacteria and Candida target genes comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1-102. In another embodiment, the primers and probes for the target genes comprise or consist of functionally active variants of any of the primers and probes of SEQ ID NOs: 1-102.

[0035] Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1-102 can be identified by using the primers and / or probes in the disclosed methods. Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1-102 relate to primers and / or probes that provide similar or higher specificity and sensitivity in the described methods or kits compared to the respective sequences of SEQ ID NOs: 1-102.

[0036] A variant may vary from the sequence of SEQ ID NO: 1-102 by the addition, deletion or substitution of one or more nucleotides, for example, the addition, deletion or substitution of one or more nucleotides at the 5'-end and / or 3'-end of each of the sequences of SEQ ID NO: 1-102. As detailed above, the primer (and / or probe) may be chemically modified, i.e., the primer and / or probe may comprise modified nucleotides or non-nucleotide compounds. Thus, the probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by some modification, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or a phosphate-like moiety, or a combination thereof. For example, a "label" may be attached to the base portion of a "nucleotide" to obtain a "modified nucleotide". A natural base in a "nucleotide" may be replaced, for example, with a 7-deazapurine, thus also obtaining a "modified nucleotide". The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in this application. A "modified nucleoside" (or "nucleoside analog") differs from a naturally occurring nucleoside by some modification, as outlined above for "modified nucleotides" (or "nucleotide analogs").

[0037] Oligonucleotides, including modified oligonucleotides and oligonucleotide analogs, that amplify nucleic acid molecules encoding target genes can be designed using computer programs such as, for example, OLIGO (Molecular Biology Insights Inc. Cascade, Colorado). Important features in designing oligonucleotides to be used as amplification primers include, but are not limited to, an appropriate size of the amplification product to facilitate detection (e.g., by electrophoresis), similar melting temperatures for the members of the pair of primers, and the length of each primer (i.e., primers must be long enough to anneal with sequence specificity and initiate synthesis, but not so long that fidelity is compromised during oligonucleotide synthesis). Typically, oligonucleotide primers are 8-50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length). In some embodiments, the oligonucleotide primers are 40 nucleotides in length or less.

[0038] In addition to a set of primers, the method may use one or more probes to detect the presence or absence of target bacteria and Candida species. The term "probe" refers to a synthetically or biologically produced nucleic acid (DNA or RNA) that contains a specific nucleotide sequence that allows it to hybridize specifically (i.e., preferentially) to a "target nucleic acid" under a defined, predetermined stringency by design or selection, in this case to a target gene nucleic acid. A "probe" may be referred to as a "detection probe", which means that it detects a target nucleic acid.

[0039] In some embodiments, the disclosed target gene probe can be labeled with at least one fluorescent label.In one embodiment, the target gene probe can be labeled with a donor fluorescent moiety, such as a fluorescent dye, and a corresponding acceptor moiety, such as a quencher.In one embodiment, the probe comprises or consists of a fluorescent moiety, and the nucleic acid sequence comprises or consists of SEQ ID NO: 3, 7, 10, 13, 16, 19, 22, 25, 29, 32, 35, 37, 40, 43, 46, 50, 53, 56, 59, 62 or 65.

[0040] The design of oligonucleotides used as probes can be done in a similar manner to the design of primers. Embodiments can use a single probe or a pair of probes for detection of the amplification product. Depending on the embodiment, the probe(s) used can include at least one label and / or at least one quencher moiety. Like primers, the probes usually have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not so long that fidelity is compromised during synthesis. Oligonucleotide probes are generally 15-40 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides in length.

[0041] The constructs may each comprise a vector that contains one of the target gene primer and probe nucleic acid molecules. The constructs may be used, for example, as a control template nucleic acid molecule. The vectors suitable for use are commercially available and / or produced by recombinant nucleic acid technology methods routine in the art. The target gene nucleic acid molecule may be obtained, for example, by chemical synthesis, direct cloning from CA, or PCR amplification.

[0042] Constructs suitable for use in the present methods typically include, in addition to the target gene nucleic acid molecule (e.g., a nucleic acid molecule comprising one or more of the sequences of SEQ ID NOs: 1-3), a sequence encoding a selection marker (e.g., an antibiotic resistance gene) for selecting the desired construct and / or transformant, and an origin of replication. The choice of vector system usually depends on several factors, including, but not limited to, the choice of host cell, replication efficiency, selectability, inducibility, and ease of recovery.

[0043] The construct containing the target gene nucleic acid molecule can be propagated in a host cell. The term host cell as used herein is meant to include prokaryotic and eukaryotic organisms, such as yeast, plant and animal cells. Prokaryotic hosts include E. coli, Salmonella typhimurium, Serratia marcescens and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, and plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into the host cell using any of the techniques commonly known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and viral-mediated nucleic acid transfer are common methods for introducing nucleic acid into the host cell. Additionally, naked DNA can be delivered directly into the cell (e.g., U.S. Patent Nos. 5,580,859 and 5,589,466).

[0044] Polymerase chain reaction (PCR) U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides that can act as initiation points for nucleic acid synthesis within the target NG gene nucleic acid sequence described. Primers can be purified from restriction digests by conventional methods or can be synthetically produced. Primers are preferably single-stranded for maximum efficiency in amplification, but primers may also be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0045] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturing method, including physical, chemical, or enzymatic means. One method of separating the nucleic acid strands involves heating until the nucleic acid is predominantly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90%, or 95% denatured). The heating conditions required to denature the template nucleic acid depend, for example, on the buffer salt concentration, as well as the length and nucleotide composition of the nucleic acid to be denatured, but typically range from about 90°C to about 105°C, depending on the characteristics of the reaction, such as temperature and nucleic acid length. Denaturation is typically performed for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes and 30 seconds, or 1.5 minutes).

[0046] When the double-stranded template nucleic acid has been denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence on the target NG gene nucleic acid molecule described. The annealing temperature is usually about 35°C to about 65°C, (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature that promotes or optimizes the activity of the polymerase, i.e., a temperature sufficient for extension to occur from the annealed primers to generate products complementary to the template nucleic acid. The temperature must be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but not so high as to denature the extension product from its complementary template (e.g., temperatures for extension generally range from about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). Extension times can be from about 10 seconds to about 5 minutes (e.g., from about 30 seconds to about 4 minutes, from about 1 minute to about 3 minutes, from about 1 minute 30 seconds to about 2 minutes).

[0047] PCR assays can use nucleic acids such as RNA or DNA (cDNA). The template nucleic acid does not need to be purified and can be a minor fraction of a complex mixture such as the nucleic acids contained in human cells. Nucleic acid molecules can be extracted from biological samples by routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al. (eds), 1993, American Society for Microbiology, Washington DC). Nucleic acids can be obtained from any number of sources, such as plasmids, or natural sources including bacteria, yeast, protozoan viruses, organelles, or higher organisms such as plants or animals.

[0048] The oligonucleotide primers are combined with PCR reagents under reaction conditions conducive to primer extension. For example, a chain extension reaction typically contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5-1.0 μg of proto-denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. The reaction typically contains 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0049] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. The steps of strand separation, annealing, and extension can be repeated as many times as necessary to generate a desired amount of amplification product corresponding to the target nucleic acid molecule. The limiting factors of the reaction are the amount of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) are preferably repeated at least once. For detection applications, the number of cycling steps depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence enough for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated 40, 60, or 100 times.

[0050] Fluorescence Resonance Energy Transfer (FRET) FRET technology (e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are placed within a certain distance of each other, energy transfer occurs between two fluorescent moieties that can be visualized or otherwise detected and / or quantified. Typically, the donor transfers energy to the acceptor when excited by light irradiation of a suitable wavelength. Typically, the acceptor re-emits the transferred energy in the form of light irradiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties via a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0051] In one example, an oligonucleotide probe can contain a donor fluorescent moiety and a corresponding quencher that may or may not be fluorescent and dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the donor and acceptor moieties such that the fluorescence emission from the donor fluorescent moiety is quenched by the acceptor moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase, such that the fluorescence emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ), (Biosearch Technologies, Inc., Novato, Calif.), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, Iowa), and BlackBerry® Quencher 650 (BBQ-650) (Berry & Assoc, Dexta, Michigan).

[0052] In another example, two oligonucleotide probes, each containing a fluorescent moiety, can hybridize to the amplification product at a specific position determined by the complementarity of the oligonucleotide probe to the target nucleic acid sequence. When the oligonucleotide probe hybridizes to the amplification product nucleic acid at the appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0053] Fluorescence analysis can be performed, for example, using a photon-counting epifluorescence microscope system (equipped with appropriate dichroic mirrors and filters to monitor a particular range of fluorescence emission), a photon-counting photomultiplier system, or a fluorometer. Excitation to initiate energy transfer or to allow direct detection of the fluorophore can be performed using an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source appropriately filtered for excitation of the desired range.

[0054] As used herein with respect to a donor moiety and a corresponding acceptor moiety, "corresponding" refers to an acceptor fluorescent moiety or dark quencher that has an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Thus, efficient non-irradiation energy transfer can occur between them.

[0055] Fluorescent donor moieties and corresponding acceptor moieties are generally selected for (a) high efficiency Forster energy transfer, (b) large final Stokes shift (>100 nm), (c) emission shift as far as possible to the red portion of the visible spectrum (>600 nm); and (d) emission shift to a wavelength higher than the Raman water fluorescence emission caused by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety can be selected that has its maximum excitation wavelength near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap of its fluorescence emission with the excitation spectrum of the corresponding acceptor fluorescent moiety. A corresponding acceptor fluorescent moiety can be selected that has a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm).

[0056] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET technology include fluorescein, lucifer yellow, B-phycoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red 640, LC Red 705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriaminepentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0057] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotide via a linker arm. The length of each linker arm is important since it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm can be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Typically, the linker arm is from about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods of attaching the linker arm to a particular nucleotide base and attaching the fluorescent moiety to the linker arm.

[0058] Acceptor fluorescent moieties such as LC Red 640 can be combined with oligonucleotides containing amino linkers (e.g., C6-amino phosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.) to generate, for example, LC Red 640-labeled oligonucleotides. Linkers commonly used to attach donor fluorescent moieties such as fluorescein to oligonucleotides include thiourea linkers (derived from FITC, e.g., Fluorescein-CPG from Glen Research or ChemGene (Ashland, Mass.)), amide-linkers (derived from Fluorescein-NHS-esters, such as CX-Fluorescein-CPG from BioGenex (San Ramon, Calif.)), or 3'-amino-CPG, which requires attachment of a Fluorescein-NHS-ester after oligonucleotide synthesis.

[0059] Detection by real-time PCR The present disclosure provides a method for detecting the presence or absence of bacterial and fungal target organisms in a biological or non-biological sample. The provided method avoids the problems of sample contamination, false negatives, and false positives. The method includes performing at least one cycling step including amplifying a portion of a target nucleic acid molecule from a sample using one or more primer pairs, and a FRET detection step. The multiple cycling steps are preferably performed in a thermocycler. The method can be performed using primers and probes to detect the presence of the target organism, and detection of the target gene indicates the presence of the target organism in the sample.

[0060] As described herein, the amplification product can be detected using a labeled hybridization probe that utilizes FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of the amplification product, and thus the presence or absence of CA. TaqMan® technology utilizes, for example, a single-stranded hybridization probe labeled with a fluorescent dye and a quencher, which may or may not be fluorescent. When the first fluorescent moiety is excited with light of an appropriate wavelength, the absorbed energy is transferred to a second fluorescent moiety or a dark quencher according to the principles of FRET. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is degraded during the subsequent extension stage, for example, by the 5' to 3' nuclease activity of Taq polymerase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from each other. As a result, upon excitation of the first fluorescent moiety in the absence of the quencher, fluorescent emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for carrying out the methods described herein for detecting the presence or absence of NG in a sample.

[0061] Molecular beacons in combination with FRET can also be used to detect the presence of amplification products using real-time PCR. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and a fluorescent label is typically placed at each end of the probe. Molecular beacon technology uses a probe oligonucleotide with a sequence that allows for secondary structure formation (e.g., a hairpin). The secondary structure formed within the probe results in both fluorescent sites being in spatial proximity when the probe is in solution. After hybridization to the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is disrupted and the fluorescent moieties are separated from each other, so that the emission of the first fluorescent moiety can be detected after excitation with light of an appropriate wavelength.

[0062] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to each other within a target DNA molecule (e.g., an amplification product). A donor fluorescent moiety, e.g., fluorescein, is excited at 470 nm by the light source of the LightCycler® Instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, e.g., LightCycler®-Red 640 (LC Red 640) or LightCycler®-Red 705 (LC Red 705). The acceptor fluorescent moiety then emits light of a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can only occur when the fluorescent moieties are in direct local proximity and the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules (e.g., the number of CA genomes). When amplification of the target nucleic acid occurs and an amplification product is produced, the hybridizing step results in a detectable signal based on FRET between the members of the probe pair.

[0063] Generally, the presence of FRET indicates the presence of target organism in the sample, and the absence of FRET indicates the absence of target organism in the sample.However, insufficient specimen collection, transportation delay, improper transportation conditions, or the use of a specific collection swab (calcium alginate or aluminum shaft) are all conditions that can affect the success and / or accuracy of the test result.Using the method disclosed herein, for example, detection of FRET within 45 cycling steps indicates the presence of target organism(s).

[0064] Representative biological samples that can be used to carry out the method include, but are not limited to, vaginal swabs, fecal specimens, blood specimens, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods for collecting and storing biological samples are known to those skilled in the art. Biological samples can be treated (e.g., by nucleic acid extraction methods and / or kits known in the art) to release target nucleic acid, or in some cases, biological samples can be directly contacted with PCR reaction components and appropriate oligonucleotides.

[0065] Melting curve analysis is an additional step that can be included in the cycle profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which one half of a DNA double strand separates into single strands. The melting temperature of DNA depends mainly on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature(s) of the probe from the amplification product can confirm the presence or absence of the target organism(s) in the sample.

[0066] During each thermocycler run, control samples can be cycled as well. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the described target gene), for example, using control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing a target nucleic acid molecule. Such a plasmid control can be amplified internally (e.g., within the sample) or in a separate sample run alongside the patient sample, using the same primers and probes used to detect the intended target. Such controls are indicative of the success or failure of the amplification, hybridization and / or FRET reaction. Each thermocycler run can also include, for example, a negative control lacking target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false positive signals. Thus, the control reaction can easily determine, for example, the ability of primers to anneal with sequence specificity and initiate extension, and the ability of probes to hybridize with sequence specificity and FRET to occur.

[0067] In one embodiment, the method includes a step to avoid contamination, for example, the enzymatic method utilizing uracil-DNA glycosylase described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycling run and the next.

[0068] This method can be carried out using conventional PCR combined with FRET technology. In one embodiment, the LightCycler® instrument is used. The following patent applications describe real-time PCR used with LightCycler® technology: WO 97 / 46707, WO 97 / 46714, and WO 97 / 46712.

[0069] The LightCycler® can be operated using a PC workstation and utilizes the Windows® NT operating system. Signals from samples are obtained as the machine sequentially positions the capillaries over the optical unit. The software can display the fluorescent signal in real time immediately after each measurement. Fluorescence acquisition times range from 10 to 100 milliseconds (msec). After each cycling step, a quantitative display of fluorescence versus cycle number can be continuously updated for all samples. The data generated can be stored for further analysis.

[0070] As an alternative to FRET, double-stranded DNA binding dyes such as fluorescent DNA binding dyes (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)) can be used to detect the amplification products. Upon interaction with double-stranded nucleic acids, such fluorescent DNA binding dyes emit a fluorescent signal after excitation with light of an appropriate wavelength. Also, double-stranded DNA binding dyes such as nucleic acid intercalating dyes can be used. When using double-stranded DNA binding dyes, a melting curve analysis is usually performed to confirm the presence of the amplification product.

[0071] It is understood that embodiments of the present disclosure are not limited by the configuration of one or more commercially available devices.

[0072] Manufactured products / kits The embodiments of the present disclosure further provide an article of manufacture, composition or kit for detecting bacterial and fungal organisms associated with vaginitis. The article of manufacture may include primers and probes used to detect the target gene, along with suitable packaging materials. The composition may include primers used to amplify the target gene. In certain embodiments, the composition may also include a probe for detecting the target gene. Representative primers and probes for detecting the target organism(s) may hybridize to the target nucleic acid molecule. Furthermore, the kit may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization and detection, such as solid supports, buffers, enzymes and DNA standards. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to the target nucleic acid molecule are provided.

[0073] The article of manufacture may also include one or more fluorescent moieties for labeling the probe, or may label the probe provided with the kit. For example, the article of manufacture may include donor and / or acceptor fluorescent moieties for labeling the probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0074] The article of manufacture may also include a package insert or packaging label with instructions for using the primers and probes to detect target organisms in a sample. The articles of manufacture and compositions may further include reagents (e.g., buffers, polymerase enzymes, cofactors, or agents to prevent contamination) for carrying out the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0075] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the claimed invention. EXAMPLES

[0076] The following examples, tables and figures are provided to aid the understanding of the subject matter, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0077] Example 1 PCR Assay Reagents and Conditions Real-time PCR detection of target bacteria and Candida species was performed using either the cobas® 4800 system or the cobas® 6800 / 8800 system platforms (Roche Molecular Systems, Inc. Pleasanton, Calif.). The final concentrations of amplification reagents were as follows: [Table 10]

[0078] The following table shows a typical thermal profile used in a PCR amplification reaction: [Table 11]

[0079] The Pre-PCR program included incubations at 55°C, 60°C and 65°C for initial denaturation and reverse transcription of the RNA template. The three temperature incubations have the advantageous effect that at the lower temperatures even slightly mismatched target sequences (such as genetic variants of an organism) are transcribed, while at the higher temperatures the formation of RNA secondary structures is suppressed, thus resulting in more efficient transcription. The PCR cycling is divided into two runs, both of which apply a one-stage setup (combining annealing and extension). The first 5 cycles at 55°C allow increased inclusiveness by preamplifying slightly mismatched target sequences, while the 45 cycles of the second run increase specificity by using an annealing / extension temperature of 58°C.

[0080] Example 2: Amplification and detection of Atopobium vaginae Amplification and detection of the target genes, 23s ribosomal RNA and tufA, of Atopobium vaginae were performed using the conditions described in Example 1. For the AVAG_23s_1 assay, forward primer SEQ ID NO: 14, reverse primer SEQ ID NO: 15 and probe SEQ ID NO: 16 were used. For the AVAG_TufA_2 assay, forward primer SEQ ID NO: 17, reverse primer SEQ ID NO: 18 and probe SEQ ID NO: 19 were used. Forward primer SEQ ID NO: 20, reverse primer SEQ ID NO: 21 and probe SEQ ID NO: 22 were used for the AVAG_TufA_3 assay. These primers and probes were tested with genomic A. vaginae DNA present at concentrations of 1e5, 1e4 and 1e3 copies per PCR reaction. The results of the experiment are shown as growth curves in Figure 1A and calculated Ct values ​​in Figure 1B. All three assays showed good linearity for the detection of A. vaginae.

[0081] Example 3: Amplification and detection of Gardnerella vaginalis Five assays with three target genes were performed for the amplification and detection of Gardnerella vaginalis. In the GVAG_Tuf_1 assay (forward primer SEQ ID NO: 1, reverse primer SEQ ID NO: 2, probe SEQ ID NO: 3) and GVAG_Tuf_2 assays (forward primer SEQ ID NO: 4, reverse primer SEQ ID NO: 2, probe SEQ ID NO: 3), the tuf gene was used as the target gene. The 23s rRNA gene was the target in the GVAG_23s_5 assay (forward primer SEQ ID NO: 5, reverse primer SEQ ID NO: 6, probe SEQ ID NO: 7) and GVAG_23s_7 assay (forward primer SEQ ID NO: 8, reverse primer SEQ ID NO: 9, probe SEQ ID NO: 10). For the GVAG_16s_1 assay, the forward primer SEQ ID NO: 11, reverse primer SEQ ID NO: 12 and probe SEQ ID NO: 13 were used to amplify and detect the 16s rRNA gene. These primers and probes were tested with genomic G. vaginalis DNA present at concentrations of 1e5, 1e4 and 1e3 copies per PCR reaction and the results, expressed as Ct values, are shown in FIG.

[0082] Example 4: Amplification and detection of BVAB-2 The 16s rRNA gene was used as the target gene for amplification and detection of BVAB-2 and three assays were performed using the following primers and probes: BVAB2_16s_1: forward primer SEQ ID NO: 30, reverse primer SEQ ID NO: 31, probe SEQ ID NO: 32; BVAB2_16s_3: forward primer SEQ ID NO: 33, reverse primer SEQ ID NO: 34, probe SEQ ID NO: 35; BVAB2_16s_4: forward primer SEQ ID NO: 33, reverse primer SEQ ID NO: 36, probe SEQ ID NO: 37. These assays used 16s rRNA plasmid as template present at concentrations of 1e5, 1e4 and 1e3 copies per PCR reaction and the results are presented as Ct values ​​and are shown in FIG. 3.

[0083] Example 5: Amplification and detection of Megasphaera type 1 The 16s rRNA gene was used as the target gene for the amplification and detection of Megasphaera type 1 and three assays were performed with the following primers and probes: MEGA1_16s_2: forward primer SEQ ID NO: 23, reverse primer SEQ ID NO: 24, probe SEQ ID NO: 25; MEGA1_16s_4: forward primer SEQ ID NO: 26, reverse primer SEQ ID NO: 24, probe SEQ ID NO: 25; MEGA1_16s_6: forward primer SEQ ID NO: 27, reverse primer SEQ ID NO: 28, probe SEQ ID NO: 29. These assays used the 16s rRNA plasmid as template present at concentrations of 1e5, 1e4 and 1e3 copies per PCR reaction and the results are presented as Ct values ​​and are shown in Figure 4.

[0084] Example 6: Amplification and detection of Lactobacillus spp. Amplification and detection of Lactobacillus spp. utilized two target genes, 23s ribosomal RNA (23s rRNA) and D-lactate dehydrogenase (LDH), and four assays. The following primers and probes were used in each assay: LBS_23s_1: forward primer SEQ ID NO: 38, reverse primer SEQ ID NO: 39, probe SEQ ID NO: 40; LBS_23s_2: forward primer SEQ ID NO: 41, reverse primer SEQ ID NO: 42, probe SEQ ID NO: 43; LBS_LDH_1: forward primer SEQ ID NO: 44, reverse primer SEQ ID NO: 47, probe SEQ ID NO: 46; LBS_LDH_2: forward primer SEQ ID NO: 44, reverse primer SEQ ID NO: 45, probe SEQ ID NO: 46. These primers and probes were tested with genomic Lactobacillus DNA present at concentrations of 1e5, 1e4 and 1e3 copies per PCR reaction, and the results, expressed as Ct values, are shown in FIG. 5.

[0085] Example 7 Amplification and detection of Eggerthella spp. 16s ribosomal RNA (16s rRNA) from Eggerthella lenta was used as a target for DNA amplification and detection of Eggerthella spp. Selection of candidate primer and probe sequences was based on coverage for Eggerthella lenta, Eggerthella sinensis, Eggerthella timonensis and unclassified Eggerthella sp., as well as coverage for Atopobium spp., Coriobacterium glomerans, Collinsella vaginalis, Slackia exigua, Olsenella spp., Acetomicrobium faecale, Lacotbacillus spp., Bifodobacterium longum, and other species. longum, Mobiluncus spp., and Burkholderia. As a result, primer sequences of SEQ ID NOs: 84, 85, 87, and 88 and probe sequences of SEQ ID NOs: 86 and 89 (Table VIII) were selected.

[0086] Example 8 Amplification and detection of Prevotella species. 16s ribosomal RNA (16s rRNA) from Prevotella bivia was used as a target for DNA amplification and detection of Prevotella spp. The selection of candidate primer and probe sequences was based on the inclusion of the following Prevotella species: Prevotella amnii, Prevotella bivia, Prevotella buccalis, Prevotella corporis, Prevotella disiens, and Prevotella intermedia. Prevotella melaninogenica, Prevotella nigrescens, Prevotella oris, Prevotella timonensis, Prevotella denticola, Prevotella leoscheii, Prevotella corporis, Prevotella massiliensis, Prevotella bergensis, Prevotella oralis, and Prevotella lascolaii.Candidate primer and probe sequences were also based on exclusivity to Rifkennellaceae, Bacterioidaceae, Porphyromonas, Fusobacterium nucleatum, Sneathia amnii, Mycoplasma genitalium, Lactobacillus spp., Bifidobacterium longum and Burkholderia. As a result, primer sequences of SEQ ID NOs: 90-93 and 95-100 and probe sequences of SEQ ID NOs: 94, 101-102 (Table IX) were selected.

[0087] Example 9: Amplification and detection of Candida spp. Ribosomal RNA (rRNA) genes are used as target genes for DNA amplification to detect the presence, absence and / or levels of VVC-associated Candida species in samples. In the CAPT_rRNA_1 and CAPT_rRNA_6 assays, VVC-associated Candida species include C. albicans, C. parapsilosis, C. dubliniensis and C. tropicalis (collectively Candida spp.). The CAPT_rRNA_1 assay uses a forward primer of SEQ ID NO: 54, a reverse primer of SEQ ID NO: 55 and a probe of SEQ ID NO: 56. The CAPT_rRNA_6 assay uses a forward primer of SEQ ID NO: 57, a reverse primer of SEQ ID NO: 58 and a probe of SEQ ID NO: 59. Both assays were tested with genomic DNA from C. albicans, C. parapsilosis and C. tropicalis at concentrations of 2.5e6, 2.5e5 and 2.5e4 copies per PCR reaction and the results are presented as Ct values ​​and are shown in FIG. 6.

[0088] The rRNA genes were also used as target genes in the CVS_rRNA_1 and CVS_rRNA_2 assays targeting C. albicans, C. parapsilosis, C. tropicalis, and Candida glabrata. The CVS_rRNA_1 assay uses a forward primer of SEQ ID NO: 48, a reverse primer of SEQ ID NO: 49, and a probe of SEQ ID NO: 50. The CVS_rRNA_2 assay uses a forward primer of SEQ ID NO: 51, a reverse primer of SEQ ID NO: 52, and a probe of SEQ ID NO: 53. Both assays were tested with genomic DNA from C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata at concentrations of 2.5e6, 2.5e5, and 2.5e4 copies per PCR reaction, and the results, expressed as Ct values, are shown in Figure 7.

[0089] Example 10 Multiplex PCR Assay A multiplex PCR single-well assay to simultaneously detect three BV-associated bacteria, Lactobacillus spp., Gardnerella vaginalis, Atopobium vaginae, and Candida spp., including Candida krusei and Candida glabrata, was performed using four different detection channels. The first channel detects the 16s rRNA of Gardnerella vaginalis. The second channel detects the D-LDH gene of Lactobacillus spp. The third channel detects the tufA gene of Atopobium vaginae. The fourth channel detects multiple Candida species, including 25s rRNA of Candida spp., as well as rRNA ITS segments of Candida krusei and Candida glabrata. The selected combination set of primers and probes used in the multiplex assay is shown in Table XII. [Table 12]

[0090] PCR assay reagents and conditions were used as described in Example 1, and the results of experiments testing various template samples at 10,000 (10K) and 1,000 (1K) copies are shown as growth curves in Figures 8, 9, 10, and 11 (for Candida spp. only) for channels 1, 2, 3, and 4, respectively.

[0091] Although the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be apparent to those skilled in the art upon reading this disclosure that various changes in form and detail can be made therein without departing from the true scope of the invention. For example, all of the techniques and devices described above can be used in various combinations.

Claims

1. 1. A method for detecting a plurality of bacterial vaginosis-associated (BV-associated) bacteria in a sample, the plurality of BV-associated bacteria being at least one of the group selected from Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae, Megasphaera type 1, Eggerthella spp., Prevotella spp., and bacterial vaginosis-associated bacteria BVAB-2, the method comprising: a) performing an amplification step comprising contacting the sample with a primer set to produce an amplification product if nucleic acid from the BV associated bacteria is present in the sample; b) performing a hybridization step, which comprises contacting each amplification product with one or more detectable probes; and c) detecting the presence of each amplification product, the presence of which indicates the presence of the BV-associated bacteria in the sample. Including, The primer set for producing an amplification product from Lactobacillus spp. comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 38, 41 or 44, and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 39, 42 or 45, 47 or 73, and the one or more detectable probes comprise an oligonucleotide sequence selected from SEQ ID NO: 40, 43, 46 and 74; the primer set for producing an amplification product from Gardnerella vaginalis comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 1, 4, 5, 8 or 11, and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 2, 6, 9, or 12, and the one or more detectable oligonucleotide probes comprise an oligonucleotide sequence selected from SEQ ID NO: 3, 7, 10, and 13; the primer set for producing an amplification product from Atopobium vaginae comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 14, 17, 20, 67 or 70, and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 15, 18, 21, 68 or 71, and the one or more detectable probes comprise an oligonucleotide sequence selected from SEQ ID NOs: 16, 19, 22, 66, 69 and 72; the primer set for producing an amplification product from Megasphaera type 1 comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 23, 26 or 27 and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 24 or 28, and the one or more detectable probes comprise an oligonucleotide sequence selected from SEQ ID NO: 25 and 29; The primer set for producing an amplification product from Eggerthella spp. comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 84 or 87 and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 85 or 88, and the one or more detectable probes comprise an oligonucleotide sequence selected from SEQ ID NO: 86 and 89; the primer set for producing an amplification product from Prevotella spp. comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 90, 95, 96 or 97, and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 91, 92, 93, 98, 99 or 100, and the one or more detectable probes comprise an oligonucleotide sequence selected from SEQ ID NOs: 94, 101 and 102; 20. The method of claim 19, wherein the primer set for producing an amplification product from BVAB-2 comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 30 or 33 and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 31, 34 or 36, and the one or more detectable probes comprise an oligonucleotide sequence selected from SEQ ID NO: 32, 35 and 37.

2. The BV-associated bacteria are Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae; The primer set for producing an amplification product from Lactobacillus spp. comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 38, 41 or 44, and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 39, 42 or 45, 47 or 73, and the one or more detectable probes comprise an oligonucleotide sequence selected from SEQ ID NO: 40, 43, 46 and 74; the primer set for producing an amplification product from Gardnerella vaginalis comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NO: 1, 4, 5, 8 or 11, and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NO: 2, 6, 9, or 12, and the one or more detectable oligonucleotide probes comprise an oligonucleotide sequence selected from SEQ ID NO: 3, 7, 10, and 13; 2. The method of claim 1, wherein the primer set for producing an amplification product from Atopobium vaginae comprises a forward primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 14, 17, 20, 67 or 70 and a reverse primer comprising an oligonucleotide sequence selected from SEQ ID NOs: 15, 18, 21, 68 or 71, and the one or more detectable probes comprise an oligonucleotide sequence selected from SEQ ID NOs: 16, 19, 22, 66, 69 and 72.

3. The primer set for producing an amplification product from Lactobacillus spp. comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 44 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 45, and the one or more detectable probes comprise the oligonucleotide sequence of SEQ ID NO: 46; the primer set for producing an amplification product from Gardnerella vaginalis comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:11 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:12, and the one or more detectable probes comprise the oligonucleotide sequence of SEQ ID NO:13; 3. The method of claim 2, wherein the primer set for producing an amplification product from Atopobium vaginae comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:20 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:21, and the one or more detectable probes comprise the oligonucleotide sequence of SEQ ID NO:

72.

4. 4. The method of claim 1, wherein the hybridizing step comprises contacting the amplification product with a detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor moiety, and the detecting step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, the presence or absence of fluorescence indicating the presence or absence of the BV-associated bacteria in the sample.

5. The method according to any one of claims 1 to 4, wherein the amplification step uses a polymerase enzyme having 5'-3' nuclease activity.

6. 1. A method for simultaneously detecting Lactobacillus spp., Gardnerella vaginalis, and Atopobium vaginae in a sample, comprising: a) performing an amplification step comprising contacting the sample with a primer set to produce an amplification product if nucleic acid from Lactobacillus spp., Gardnerella vaginalis, Atopobium vaginae is present in the sample; b) performing a hybridization step, which comprises contacting each amplification product with one or more detectable probes; and c) detecting the presence of each amplification product, the presence of said amplification products being indicative of the presence of Lactobacillus spp., Gardnerella vaginalis, Atopobium vaginae in said sample. Including, The method according to claim 1, wherein the primer set for producing an amplification product from Lactobacillus spp. comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 44 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 45, and the one or more detectable probes comprise the oligonucleotide sequence of SEQ ID NO: 46; the primer set for producing an amplification product from Gardnerella vaginalis comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 11 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 12, and the one or more detectable probes comprise the oligonucleotide sequence of SEQ ID NO: 13; the primer set for producing an amplification product from Atopobium vaginae comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 20 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 21, and the one or more detectable probes comprise the oligonucleotide sequence of SEQ ID NO:

72.

7. The method of claim 6, further comprising detecting at least one selected from the group consisting of Candida spp., Candida krusei, and Candida glabrata in the sample, the amplifying step further comprises contacting the sample with a primer set to produce an amplification product if at least one nucleic acid of the group selected from Candida spp., Candida krusei, and Candida glabrata is present in the sample; and The method, wherein detecting the presence of the amplification product indicates the presence of Candida spp., Candida krusei and Candida glabrata in the sample.

8. The method of claim 7, wherein the primer set for producing an amplification product from Candida spp. comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 76 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 77, and the one or more detectable probes comprise the oligonucleotide sequence of SEQ ID NO: 78; and / or the primer set for producing an amplification product from Candida krusei comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO: 79 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO: 80, and the one or more detectable probes comprise the oligonucleotide sequence of SEQ ID NO: 81; and / or 8. The method of claim 7, wherein the primer set for producing an amplification product from Candida glabrata comprises a forward primer comprising the oligonucleotide sequence of SEQ ID NO:63 and a reverse primer comprising the oligonucleotide sequence of SEQ ID NO:64, and the one or more detectable probes comprise the oligonucleotide sequence of SEQ ID NO:

65.

9. 1. A kit for detecting bacterial vaginosis-associated (BV-associated) bacteria in a sample, comprising: a forward primer comprising a first oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 4, 5, 8, 11, 14, 17, 20, 23, 26, 27, 30, 33, 38, 41, 44, 67, 70, 84, 87, 90, 95, 96 and 97; a reverse primer comprising a second oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 6, 9, 12, 15, 18, 21, 24, 28, 31, 34, 36, 39, 42, 45, 47, 68, 71, 73, 85, 88, 91, 92, 93, 98, 99 and 100; a probe comprising a third detectably labeled oligonucleotide sequence selected from the group consisting of SEQ ID NOs: 3, 7, 10, 13, 16, 66, 69, 19, 22, 72, 25, 29, 32, 35, 37, 40, 43, 46, 74, 86, 89, 94, 101, and 102, or a complement thereof; wherein the probe is configured to hybridize to an amplicon generated by the forward primer and the reverse primer.

10. The kit of claim 9 , wherein the third detectably labeled oligonucleotide sequence comprises a donor fluorescent moiety and a corresponding acceptor moiety.

11. 11. The kit of claim 9 or 10, further comprising nucleoside triphosphates, a nucleic acid polymerase, and buffers necessary for the function of the nucleic acid polymerase.

12. 12. The kit of any one of claims 9 to 11, wherein at least one of the first oligonucleotide sequence, the second oligonucleotide sequence, and the third oligonucleotide sequence comprises at least one modified nucleotide.

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