Oligonucleotides for use in determining the presence of Trichomonas vaginalis in a sample

Abstract

Methods for use in multiplex amplification and / or detection of Trichomonas vaginalis. Multiphase amplification provides rapid, quantitative, and highly sensitive detection with low variability even at low analyte concentrations. Detection probes, capture probes, amplification oligonucleotides, nucleic acid compositions, probe mixtures, methods, and kits useful for amplifying and determining the presence of Trichomonas vaginalis in a test sample are described. In some embodiments, the T. vaginalis target nucleic acid sequence comprises a nucleotide sequence containing a portion of the T. vaginalis 16S rRNA nucleotide sequence represented by SEQ ID NO: 173, or its complement.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority under 35 U.S.C. § 119(e) to Provisional Application No. 62 / 870,308, filed on Jul. 3, 2019, the content of which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing The sequence listing described in the file DIA.0106.02_PCT_ST25, which is 38 kilobytes in size, was created on Jun. 25, 2020, and is hereby incorporated by reference in its entirety.

Background Art

[0003] Trichomonas vaginalis is a parasitic protozoan that causes trichomoniasis, one of the most common treatable sexually transmitted infections. Worldwide, T. vaginalis typically infects approximately 180 million people annually through direct person - to - person contact and is a major agent of the most common sexually transmitted diseases (STDs). In the United States, it is estimated that 7 million people are infected with T. vaginalis annually. Despite its prevalence, there is no active management or prevention program. In women, infections are known to cause vaginitis, urethritis, and cervicitis. Complications include preterm birth, low birth weight, premature rupture of membranes, and infections after abortion and hysterectomy. Associations with pelvic inflammatory disease, tubal infertility, and cervical cancer have been reported. Trichomonas vaginalis is also involved as a co - factor in infections in HIV and other STD agents. This organism can also be passed to neonates during passage through the birth canal. In men, symptoms of trichomoniasis include urethral discharge, urethral stricture, epididymitis, urgency, and dysuria. In women, 10 - 50% of T. vaginalis infections are estimated to be asymptomatic. This number is thought to be higher in men.

[0004] Considering its relative prevalence and association with other STDs, there is a growing interest in effectively diagnosing trichomoniasis. Cell culture is considered the current "gold standard" for the clinical detection of T. vaginalis. However, due to its relatively delicate nature, culturing the organism is technically difficult and usually takes up to 7 days to obtain maximum sensitivity. Nevertheless, the sensitivity of the cell culture method is estimated to be about 85 - 95%. SUMMARY OF THE INVENTION MEANS FOR SOLVING THE PROBLEM

[0005] Oligonucleotides and compositions, and methods of using oligonucleotides and compositions for the multiphase (including biphasic) amplification and / or detection of T. vaginalis are described. In some embodiments, the oligonucleotides and compositions, and methods of using oligonucleotides and compositions are described for amplifying and / or detecting T. vaginalis in a sample. In multiphase amplification, at least a portion of the target nucleic acid sequence is subjected to a first-phase amplification reaction under conditions that do not support exponential amplification of the target nucleic acid sequence. The first-phase amplification reaction generates a first amplification product, which is then subjected to a second-phase amplification reaction under conditions that allow exponential amplification of the first amplification product, thereby generating a second amplification product. Multiphase amplification provides improved sensitivity and accuracy at lower limits of analyte concentration compared to single-phase formats. Multiphase amplification exhibits excellent performance in terms of both accuracy and reduced detection time.

[0006] In some embodiments, multiphase amplification of the T. vaginalis target nucleic acid sequence comprises a) contacting a sample containing or suspected of containing the T. vaginalis target nucleic acid sequence with a target capture mixture, the target capture mixture comprising an RNA polymerase promoter-containing oligonucleotide (promoter primer) and optionally a target capture oligonucleotide (TCO) to form a pre-amplification hybrid; b) isolating the pre-amplification hybrid; c) contacting the pre-amplification hybrid with a first-phase amplification mixture, the first-phase amplification mixture comprising a non-RNA polymerase promoter-containing oligonucleotide (non-promoter primer), reverse transcriptase, RNA polymerase, dNTP, and NTP, and the first-phase amplification mixture lacking at least one component necessary for exponential amplification; d) amplifying at least a portion of the target nucleic acid sequence of the pre-amplification hybrid under conditions that support linear amplification and form a first amplification product in a substantially isothermal transcription-related amplification reaction; e) contacting the first amplification product with a second-phase amplification mixture, the second-phase amplification mixture comprising an RNA polymerase promoter-containing oligonucleotide or at least one component necessary for exponential amplification lacking in the first-phase amplification mixture; f) exponentially amplifying the first amplification product to generate a second amplification product in a substantially isothermal transcription-related amplification reaction; g) detecting the second amplification product. In some embodiments, the second-phase amplification mixture contains a detection oligonucleotide.

[0007] In some embodiments, the T. vaginalis target nucleic acid sequence comprises a nucleotide sequence containing a portion of the T. vaginalis 16S rRNA nucleotide sequence represented by SEQ ID NO: 173, or its complement.

[0008] In some embodiments, the target capture oligonucleotide (TCO) comprises a target-specific (TS) sequence complementary to a region of the target nucleic acid sequence and an immobilized capture probe binding region. The immobilized capture probe binding region can be, but is not limited to, a nucleic acid sequence. In some embodiments, the TCO comprises the nucleotide sequence of SEQ ID NO: 39, 40, or 41, or their complements. In some embodiments, the TCO comprises the nucleotide sequence of SEQ ID NO: 1, 2, or 3, or their complements.

[0009] In some embodiments, the promoter primer is an amplification oligonucleotide comprising a 5' promoter sequence that includes a 3' target-specific sequence and an RNA polymerase promoter sequence. The 3' target-specific sequence contains a region complementary to a region of the target nucleic acid (promoter primer binding site) and hybridizes to the target nucleic acid. The promoter primer can bind to its target sequence (promoter primer binding site) in the target nucleic acid and initiate template-dependent synthesis of RNA or DNA by an RNA- or DNA-dependent polymerase. The promoter sequence can be, but is not limited to, a T7 promoter sequence. In some embodiments, the promoter primer comprises the nucleotide sequence of SEQ ID NO: 42, 43, 44, 45, 46, 47, or 48. In some embodiments, the promoter primer comprises SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0010] In some embodiments, the pre-amplification hybrid comprises a target nucleic acid hybridized to a promoter primer. In some embodiments, the pre-amplification hybrid comprises a target nucleic acid hybridized to each of a TCO and a promoter primer. In some embodiments, isolating the pre-amplification hybrid comprises capturing the pre-amplification hybrid using a solid support. In some embodiments, the solid support comprises an immobilized capture probe. The solid support can be, but is not limited to, magnetically attractable particles. In some embodiments, isolating the pre-amplification hybrid comprises removing promoter primers not hybridized to the target nucleic acid.

[0011] In some embodiments, a non-promoter primer (also referred to as an NT7 primer) is an amplification oligonucleotide that specifically binds to its target sequence in the cDNA product of the extension of a promoter primer, downstream of the promoter primer terminus. The promoter primer is configured to be combined with the non-promoter primer to form an amplification pair and together amplify a portion of the target nucleic acid. The non-promoter primer lacks the RNA polymerase promoter sequence of the promoter primer. In some embodiments, the non-promoter primer comprises the nucleotide sequence of SEQ ID NO: 49, 50, 51, 52, 53, 54, or 55. In some embodiments, the non-promoter primer comprises the nucleotide sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19.

[0012] In some embodiments, during the first-phase isothermal transcription-related amplification reaction, the target nucleic acid is used as a template to extend a promoter primer that specifically binds to the target nucleic acid of its target sequence by reverse transcriptase (RT) to produce a cDNA copy. Then, using the cDNA as a template, the non-promoter primer is enzymatically extended to generate double-stranded DNA. Next, the double-stranded DNA functions as a template for RNA transcription from the RNA polymerase promoter provided by the promoter primer. Then, the non-promoter primer binds to the RNA and is extended by reverse transcriptase to yield a first amplification product. In the absence of additional promoter primers, exponential amplification does not occur. Then, the first amplification product is contacted with a second-phase amplification mixture to initiate exponential second-phase amplification.

[0013] In some embodiments, each of the first-phase and second-phase isothermal transcription-related amplification reactions comprises RNA polymerase and reverse transcriptase. In some embodiments, the reverse transcriptase comprises endogenous RNase H activity.

[0014] In some embodiments, the detection oligonucleotide comprises a target-specific (TS) sequence complementary to a nucleic acid base sequence present in the second amplification product. The detection oligonucleotide target-specific sequence is at least 10 nucleic acid bases in length. In some embodiments, the detection oligonucleotide target-specific sequence is from 10 to 30 nucleic acid bases in length. In some embodiments, the detection oligonucleotide contains a detectable molecule. In some embodiments, the detectable molecule comprises a fluorophore. In some embodiments, the detection oligonucleotide contains a fluorophore and a quencher. The detection oligonucleotide can be, but is not limited to, a Torch. The detection oligo can be DNA, RNA, or a combination of DNA and RNA. The detection oligonucleotide can also have one or more modified nucleotides, including but not limited to, methoxy RNA. In some embodiments, the Torch comprises the nucleotide sequence of SEQ ID NO: 56, 57, 58, 59, 60, 61, or 62. In some embodiments, the Torch comprises the nucleotide sequence of SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28.

[0015] In some embodiments, a composition suitable for use in the amplification of the first phase of the multiplex amplification of T. vaginalis comprises (a) an optional target capture oligonucleotide, (b) a promoter primer hybridized to a first portion of the T. vaginalis target nucleic acid sequence, (c) a non-promoter primer, and (d) additional components necessary for the amplification of the target nucleic acid during the linear first-phase amplification reaction but lacking at least one component necessary for the exponential amplification of the target nucleic acid sequence. In some embodiments, what is lacking at least one component necessary for exponential amplification is an additional (free) promoter primer. In some embodiments, the first-phase amplification lacks a promoter primer that is not hybridized to the target nucleic acid. The additional components can include one or more of RNA-dependent DNA polymerase, RNA polymerase, dNTP, NTP, buffer, and salts.

[0016] In some embodiments, a composition suitable for use in the second or subsequent phase amplification of the multiphase amplification of T. vaginalis comprises (a) a first amplification product, (b) a promoter primer, (c) a non-promoter primer, and (d) other necessary components required for amplification of the target nucleic acid during the exponential second-phase amplification reaction. The additional components can include one or more of an RNA-dependent DNA polymerase, an RNA polymerase, dNTPs, NTPs, a buffer, and a salt.

[0017] In some embodiments, methods for the multiphase amplification and / or detection of T. vaginalis are described. The method comprises (a) contacting a sample containing or suspected of containing a T. vaginalis target nucleic acid with a promoter primer specific for a first portion of the target nucleic acid sequence under conditions that allow hybridization of the promoter primer to the first portion of the target nucleic acid sequence, thereby generating a pre-amplification hybrid comprising a first amplification oligonucleotide and the target nucleic acid sequence; (b) isolating the pre-amplification hybrid by target capture on a solid support and subsequent washing to remove any promoter primer that did not hybridize to the first portion of the target nucleic acid sequence in step (a); (c) amplifying at least a portion of the target nucleic acid sequence of the pre-amplification hybrid isolated in step (b) in a first-phase substantially isothermal transcription-related amplification reaction under conditions that support its linear amplification but not its exponential amplification (i.e., the first-phase amplification reaction mixture lacks at least one component required for exponential amplification of the first amplification product), thereby resulting in a reaction mixture comprising the first amplification product; (d) combining the reaction mixture comprising the first amplification product with at least one component that is required for exponential amplification of the first amplification product but is lacking in the reaction mixture comprising the first amplification product to generate a second-phase amplification reaction mixture; (e) In a substantially isothermal transcription-related amplification reaction, exponentially amplifying the first amplification product in the second-phase amplification mixture to generate a second amplification product; (f) Optionally, detecting the second amplification product.

[0018] In some embodiments, at least one component required for the exponential amplification of the first amplification product includes a primer promoter (e.g., a promoter primer in addition to a promoter primer that hybridizes to a target nucleic acid and is isolated as part of a pre-amplified hybrid). In some embodiments, the first amplification product of step (c) is a cDNA molecule having the same polarity as the target nucleic acid sequence in the sample, and the second amplification product of step (e) is an RNA molecule. The second amplification product can be detected using a sequence-specific detection probe. The sequence-specific detection probe can be, but is not limited to, a conformationally sensitive probe that generates a detectable signal when hybridized to the second amplification product. In some embodiments, the sequence-specific detection probe of the step is a fluorescence-labeled sequence-specific hybridization probe. Detection can be performed at regular time intervals. In some embodiments, detection is performed in real time. In some embodiments, detecting the second amplification product includes quantifying the target nucleic acid sequence in the sample using a linear calibration curve.

[0019] In some embodiments, the described oligonucleotides, compositions, and methods can be used to detect T. vaginalis 16S rRNA present in a sample at a copy number of 10 cells / ml or less, 1 cell / ml or less, 0.1 cell / ml or less, or 0.01 cell / ml or less. In some embodiments, the described oligonucleotides, compositions, and methods can be used to detect T. vaginalis 16S rRNA in a sample having 0.002 cells / ml or more. In some embodiments, the detection rate using the described oligonucleotides is 90% or more or 95% or more when T. vaginalis is present in the sample at 0.002 cells / ml or more.

[0020] In some embodiments, the described oligonucleotides, compositions, and methods are suitable for use in the amplification and / or detection of T. vaginalis in a multiplex multi-phase reaction. The multiplex multi-phase reaction can be used to detect T. vaginalis as well as one or more other target sequences and / or organisms. In some embodiments, a CV / TV multiplex assay is described. The CV / TV multiplex assay contains oligonucleotides for the capture, amplification, and detection of C. albicans, C. tropicalis, C. dubliniensis, C. parapsilosis, C. glabrata, and T. vaginalis.

Brief Description of the Drawings

[0021]

Figure 1

DETAILED DESCRIPTION OF THE INVENTION

[0022] For clarity and not limitation, the detailed description of the invention is divided into the following subsections.

[0023] A. Definitions All patents, applications, published applications, and other publications referred to in this specification are hereby incorporated by reference in their entirety. To the extent that different content may be associated with the same citation at different times, it means the content associated with the citation at the effective filing date. The effective filing date means the earliest priority date on which the citation is disclosed. Unless otherwise apparent from the context, any element, embodiment, step, feature, or aspect of the present invention can be implemented in combination with any other. If the definitions set forth in this section of the present invention conflict with or are otherwise inconsistent with the definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth in this section shall control over the definitions incorporated herein by reference.

[0024] As used herein, "a" or "an" means "at least one" or "one or more".

[0025] Throughout this specification and the claims, approximating language may be applied to modify any quantitative or qualitative expression that may vary within a tolerable degree without resulting in a change in the basic function to which it is related. Accordingly, a value modified by a term such as "about" or "approximately" is not limited to the particular exact value and may include values different from the particular value. In some embodiments, about or approximately indicates a slight variation and / or a variation of less than 5%.

[0026] "Sample" refers to a specimen or substance that contains, or is suspected of containing, a target analyte, such as nucleic acids (e.g., target nucleic acids) like microorganisms, viruses, genes, etc., or components thereof that include nucleic acid sequences in the analyte or nucleic acid sequences derived from the analyte. The sample can be of any origin, such as, but not limited to, biological specimens, clinical specimens, and environmental sources. Biological specimens include, but are not limited to, tissues or materials derived from living or dead organisms that may contain the analyte or nucleic acids in the analyte or nucleic acids derived from the analyte. Examples of biological samples include respiratory tissues, exudates (e.g., bronchoalveolar lavage), biopsies, sputum, tracheal aspirates, saliva, mucus, peripheral blood, plasma, serum, lymph nodes, cerebrospinal fluid, gastrointestinal tissues, feces, urine, urogenital organs, body fluids, tissues or materials, and biopsies from genital lesions, anogenital lesions, oral lesions, mucocutaneous lesions, skin lesions, eye lesions or combinations thereof, or derived therefrom. Examples of environmental samples include, but are not limited to, water, ice, soil, slurry, debris, biofilms, airborne particles, and aerosols. The sample may also include samples of in vitro cell culture components, such as conditioned media resulting from the growth of cells and tissues in culture media. The sample may be a processed specimen or material, such as that obtained by treating the sample by filtration, centrifugation, sedimentation, or attachment to a medium such as a matrix or support. Other treatments of the sample may include physically or mechanically disrupting tissues, cell aggregates, or cells to release intracellular components containing nucleic acids into a solution that may contain other components such as enzymes, buffers, salts, surfactants, etc., but are not limited thereto.

[0027] The term "contacting" means bringing two or more components together. Contacting can be achieved by mixing all components in a fluid or semi-fluid mixture. Contacting can also be achieved when one or more components physically contact one or more other components on a solid surface such as a solid tissue section or a substrate.

[0028] "Nucleic acid" refers to a polynucleotide compound containing an oligonucleotide that includes a nucleoside or nucleoside analog having a nitrogen-containing heterocyclic base or base analog covalently bonded by a standard phosphodiester bond or other bond. Nucleic acids include RNA, DNA, chimeric DNA-RNA polymers or their analogs. In nucleic acids, the backbone can be composed of various bonds including a sugar-phosphodiester bond, a peptide-nucleic acid (PNA) bond (PCT Publication No. WO95 / 32305), a phosphorothioate bond, a methylphosphonate bond, or a combination of one or more of these. The sugar moiety of the nucleic acid can be, but is not limited to, ribose, deoxyribose, or a similar compound having a substitution, for example, a 2'-methoxy or 2'-halide (e.g., 2'-F) substitution. The nitrogenous base can be, but is not limited to, a conventional base (A, G, C, T, U), its analog (e.g., inosine, The Biochemistry of the Nucleic Acids 5-36, Adams et al, ed., 11th ed., 1992), which can be a derivative of a purine or pyrimidine base (e.g., N4-methyldeoxyguanosine, deaza- or aza-purine, deaza- or aza-pyrimidine, a pyrimidine or purine having a modified or substituted substituent at any of various chemical positions, e.g., 2-amino-6-methylaminopurine, O6-methylguanine, 4-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O4-alkyl-pyrimidine, or a pyrazolo-compound, e.g., an unsubstituted or 3-substituted pyrazolo[3,4-d]pyrimidine; U.S. Patent Nos. 5,378,825, 6,949,367, and PCT Publication No. WO93 / 13121). The nucleic acid may include "non-basic" positions where the backbone does not have a nitrogenous base at one or more positions (U.S. Patent No. 5,585,481), for example, one or more non-basic positions can form a linker region that binds separate oligonucleotide sequences together.A nucleic acid may contain only conventional sugars, bases, and linkages as found in conventional RNA and DNA, or may contain conventional components and substitutions (e.g., a polymer containing a conventional base linked by a 2'-methoxy backbone, or a mixture of a conventional base and one or more analogs). The term includes "locked nucleic acid" (LNA) containing one or more LNA nucleotide monomers having a bicyclic furanose unit locked in an RNA mimicking sugar configuration, which enhances the hybridization affinity to complementary sequences in ssRNA, ssDNA, or dsDNA (Vester et al., 2004, Biochemistry 43(42):13233-41). Nucleic acids may contain modified bases. Modified bases may alter the function or behavior of the nucleic acid. In particular, references to "the sequence of SEQ ID NO: X" in the claims refer to the nucleotide sequence set forth in the corresponding sequence listing, unless otherwise indicated, and do not require identity of the backbone (e.g., RNA, 2'-O-Me RNA, or DNA) or base modifications (e.g., methylation of cytosine residues).

[0029] A "target nucleic acid" or "target" is a nucleic acid containing a target nucleic acid sequence. A "target nucleic acid sequence", "target sequence", or "target region" is a specific deoxyribonucleotide or ribonucleotide sequence containing the nucleotide sequence of a target organism, such as T. vaginalis, to be amplified. The target sequence or its complement contains a sequence that hybridizes and captures an oligonucleotide, amplification oligonucleotide, and / or detection oligonucleotide used for amplification and / or detection of the target nucleic acid. The target nucleic acid may contain other sequences in addition to the target sequence that may not be amplified. The target nucleic acid may be DNA or RNA, and may be either single-stranded or double-stranded. The target nucleic acid can be, but is not limited to, genomic nucleic acid, transcribed nucleic acid such as rRNA, or nucleic acid derived from genomic nucleic acid or transcribed nucleic acid.

[0030] "Oligonucleotide", "oligomer", or "oligo" is a polymer composed of two or more nucleoside subunits or nucleic acid base subunits joined together. Oligonucleotides can be DNA and / or RNA and their analogs. In some embodiments, the oligonucleotide is in a size range having a lower limit of 5 to 15 nt and an upper limit of 50 to 500 nt. In some embodiments, the oligonucleotide is in a size range of 10 to 100 nt, 10 to 90 nt, 10 to 80 nt, 10 to 70 nt, or 10 to 60 nt. Oligonucleotides are not derived from wild-type chromosomal DNA or its in vivo transcription products. Oligonucleotides can be synthetically produced by using any well-known in vitro chemical or enzymatic method and can be purified after synthesis by using standard methods, such as high-performance liquid chromatography (HPLC). It is described that oligonucleotides include RNA polymerase promoter-containing oligonucleotides (also referred to as promoter primers, e.g., T7 primers), non-RNA polymerase promoter-containing oligonucleotides (e.g., NT7 primers, also referred to as non-promoter primers), detection probe oligonucleotides (also referred to as detection oligos or detection probes, e.g., Torch), and target capture oligonucleotides (TC oligos). The N7 and NT7 primers are priming oligonucleotides and may be referred to as "amplification oligonucleotides".

[0031] The sugar moieties of the nucleoside subunits can be ribose, deoxyribose, and their analogs, including, but not limited to, for example, 2'-substituted ribonucleosides such as methoxy RNA. (Oligonucleotides containing nucleoside subunits having a 2'-substitution and useful as detection probes, capture probes, and / or amplification oligonucleotides are disclosed by Becker et al., "Method for Amplifying Target Nucleic Acids Using Modified Primers", U.S. Patent No. 6,130,038.) The nucleoside subunits can be linked by bonds such as phosphodiester bonds, modified bonds, or by non-nucleotide moieties that do not interfere with the hybridization of the oligonucleotide to its complementary target nucleic acid sequence. Modified bonds include bonds in which the standard phosphodiester bond is replaced by a different bond such as a phosphorothioate bond or a methylphosphonate bond. The nucleobase subunits can be linked, for example, by replacing the natural deoxyribose phosphate backbone of DNA with a pseudopeptide backbone such as a 2-aminoethylglycine backbone that binds the nucleobase subunit to the central secondary amine by a carboxymethyl linker. (DNA analogs having a pseudopeptide backbone are generally referred to as "peptide nucleic acids" or "PNA" and are disclosed by Nielsen et al., "Peptide Nucleic Acids", U.S. Patent No. 5,539,082.) Other non-limiting examples of oligonucleotides or oligomers. Any nucleic acid analog is contemplated by the present disclosure provided that the modified oligonucleotide can hybridize to the target nucleic acid under stringent hybridization conditions or amplification conditions. In the case of a detection probe, the modified oligonucleotide must also be able to preferentially hybridize to the target nucleic acid under stringent hybridization conditions. The described oligonucleotides are configured to specifically hybridize to a T. vaginalis or Candida target nucleic acid or a nucleic acid sequence derived from a T. vaginalis or Candida target nucleic acid.

[0032] Sequence identity can be determined by using algorithms such as BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0 (Genetics Computer Group, 575 Science Dr., Madison, Wis.), aligning the sequences using the default gap parameters, or by inspection and the best alignment (i.e., resulting in the highest percentage of sequence similarity over the entire comparison window). The percentage of sequence identity is calculated by comparing two optimally aligned sequences over the entire comparison window, determining the number of positions at which identical residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions of matched and mismatched positions without counting gaps (i.e., window size) within the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Unless otherwise indicated, the comparison window between two sequences is defined by the full length of the shorter of the two sequences.

[0033] The term "complementarity" refers to the ability of a polynucleotide to hybridize (form hydrogen bonds) with another polynucleotide sequence, either by traditional Watson-Crick or other non-traditional types. The percentage of complementarity indicates the proportion of bases in a continuous strand within a first nucleic acid sequence that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, or 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary). The percentage of complementarity is calculated in a similar manner to the percentage of identity.

[0034] "Stringent hybridization conditions" or "stringent conditions" means conditions that allow an oligonucleotide to preferentially hybridize to a target nucleic acid (e.g., rRNA or rDNA derived from T. vaginalis), but do not allow preferential hybridization to nucleic acids derived from closely related non-target microorganisms. Stringent hybridization conditions can vary depending on the GC content and length of the probe, the degree of similarity between the probe sequence and the sequences of non-target sequences that may be present in the test sample, and factors including the target sequence. Hybridization conditions include temperature and the composition of the hybridization reagents or solutions.

[0035] "Amplification" of a target nucleic acid refers to the process of creating in vitro multiple copies of a target nucleic acid that are identical and / or complementary to at least a portion of the target nucleic acid sequence. Examples of nucleic acid amplification procedures include transcription-mediated amplification (TMA, incorporated herein by reference, U.S. Patent Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246).

[0036] "Single-phase amplification" refers to a nucleic acid amplification reaction in which all of the components necessary for nucleic acid amplification are present in the reaction mixture at the start of amplification. In single-phase amplification, unwanted side reactions that are initiated along with the desired amplification reaction often compete and reduce the overall performance of the desired amplification reaction. In a multiplex single-phase amplification reaction, the amplification of an analyte that is present in large amounts in the reaction mixture, or an analyte with an overall amplification efficiency that is higher than that of other analytes, overly competes with other analytes in the mixture and reduces amplification.

[0037] "Amplification product" is a nucleic acid molecule that is generated in a nucleic acid amplification reaction and that is either derived from a target nucleic acid or the target nucleic acid itself. The amplification product contains all or a portion of the target nucleic acid sequence that can be in the same or opposite direction as the target nucleic acid.

[0038] "Linear amplification" refers to an amplification mechanism designed to generate an increase in target nucleic acid that is linearly proportional to the amount of target nucleic acid during the reaction. For example, transcription-related reactions can be used to generate multiple RNA copies from a DNA target, and the increase in copy number can be described by a linear factor (e.g., starting copy of template × n). In some embodiments, the linear amplification in the first phase of a multiphase amplification procedure increases the starting number of target nucleic acid strands or their complements by at least 10-fold, at least 100-fold, or at least 1,000-fold prior to the initiation of the second-phase amplification reaction. An example of a linear amplification system is "T7-based Linear Amplification of DNA" (TLAD, see Liu et al., BMC Genomics, 4: Art. No. 19, May 9, 2003). Other methods are disclosed herein. Thus, the term "linear amplification" refers to an amplification reaction that does not result in exponential amplification of the target nucleic acid sequence. The term "linear amplification" does not refer to methods that simply produce a single copy of a nucleic acid strand, such as the transcription of an RNA molecule into a single cDNA molecule as in the case of reverse transcription (RT)-PCR.

[0039] "Exponential amplification" refers to nucleic acid amplification designed to generate an increase in target nucleic acid that is geometrically proportional to the amount of target nucleic acid during the reaction. For example, PCR generates one DNA strand for every original target strand and every synthetic strand present. Similarly, transcription-mediated amplification generates multiple RNA transcripts for every original target strand and every strand subsequently synthesized. Since the synthesized strands are used as templates in subsequent amplification rounds, the amplification is exponential. The amplification reaction need not actually generate an exponentially increasing amount of nucleic acid that is considered exponential amplification as long as the amplification reaction is designed to generate such an increase.

[0040] The term "substantially isothermal amplification" refers to an amplification reaction that is carried out at a substantially constant temperature. The isothermal portion of the reaction can be before or after one or more steps at variable temperatures, such as a first denaturation step and a final heat inactivation step or cooling step. This definition is not intended to exclude minor variations in temperature, but rather is used to distinguish isothermal amplification techniques from other amplification techniques known in the art that rely essentially on "cycling temperatures" to produce amplification products. Isothermal amplification is different from, for example, PCR in that PCR relies on a denaturation cycle of heating followed by primer hybridization and polymerization at a low temperature.

[0041] References to ranges of values include integers within the range and subranges defined by integers within the range.

[0042] B. Methods of Multiphase Amplification The disclosed methods also use aspects of isothermal amplification systems generally referred to as "transcription-mediated amplification" methods that amplify a target sequence by generating multiple transcripts from a nucleic acid template. Such methods generally use one or more amplification oligonucleotides, one of which provides an RNA polymerase promoter sequence, deoxyribonucleoside triphosphates (dNTPs), ribonucleoside triphosphates (NTPs), and an enzyme having RNA polymerase and DNA polymerase activities to generate a functional promoter sequence near the target sequence and then transcribe the target sequence from the promoter (e.g., U.S. Pat. Nos. 4,868,105, 5,124,246, 5,130,238, 5,399,491, 5,437,990, 5,554,516, and 7,374,,885, and PCT Publications WO1988 / 001302, WO1988 / 010315, and WO1995 / 003430). Examples include transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), and self-sustained sequence replication (3SR).

[0043] To aid in the understanding of some of the embodiments disclosed herein, the previously described TMA method (e.g., U.S. Pat. Nos. 5,399,491, 5,554,516, and 5,824,518) is briefly summarized. In TMA, a target nucleic acid containing the sequence to be amplified is provided as a single-stranded nucleic acid (e.g., ssRNA or ssDNA). Any conventional method for converting double-stranded nucleic acid (e.g., dsDNA) to single-stranded nucleic acid can be used. A promoter primer (e.g., T7 primer) specifically binds to the target nucleic acid at its target sequence, and reverse transcriptase (RT) uses the target strand as a template to extend the 3' end of the promoter primer to create a cDNA copy, resulting in an RNA:cDNA duplex. RNase activity (e.g., RNase H of the RT enzyme) digests the RNA of the RNA:cDNA duplex. A second primer (e.g., NT7 primer) specifically binds to its target sequence in the cDNA downstream of the promoter-primer end. Then, RT synthesizes a new DNA strand by extending the 3' end of the second primer using the cDNA as a template to create a dsDNA containing a functional promoter sequence. RNA polymerase specific for the functional promoter initiates transcription to generate multiple (e.g., 100 to 1000) RNA transcripts (amplified copies or amplicons) complementary to the original target strand. The second primer specifically binds to its target sequence in each amplicon, and RT creates cDNA from the amplicon RNA template to generate an RNA:cDNA duplex. RNase digests the RNA:cDNA duplex amplicon RNA, the target-specific sequence of the promoter primer binds to its complementary sequence of the newly synthesized DNA, and RT extends the 3' ends of the promoter primer and the cDNA to create a dsDNA containing a functional promoter to which RNA polymerase binds and transcribes additional amplicons complementary to the target strand. An autocatalytic cycle that repeatedly uses these steps during the reaction generates amplification of the original target sequence.Amplicons can be detected by using probes that specifically bind to the sequences contained in the amplicons during amplification (real-time detection) or at the end point of the reaction (end-point detection). Detection of the signal arising from the binding probe indicates the presence of the target nucleic acid in the sample.

[0044] Methods for amplifying and / or detecting Trichomonas vaginalis using a multiphase amplification procedure are described. The method includes amplifying a T. vaginalis target nucleic acid sequence in a sample, which includes the following steps. First, the target nucleic acid sequence is subjected to a first-phase amplification reaction under conditions that do not support exponential amplification of the target nucleic acid sequence. The first-phase amplification reaction generates a first amplification product, which is then subjected to a second-phase amplification reaction under conditions that allow exponential amplification of the first amplification product, thereby generating a second amplification product.

[0045] The target nucleic acid sequence of T. vaginalis can be any RNA or DNA sequence. In some embodiments, the target sequence is an RNA sequence such as an mRNA or rRNA sequence. In some embodiments, the T. vaginalis target nucleic acid sequence is a nucleotide sequence comprising a part of the 16S rRNA nucleotide sequence represented by SEQ ID NO: 173, or its complement. In some embodiments, the T. vaginalis target nucleic acid sequence comprises or consists of SEQ ID NO: 174 or its complement. In some embodiments, the T. vaginalis target nucleic acid sequence comprises or consists of SEQ ID NO: 175 or its complement. In some embodiments, the T. vaginalis target nucleic acid sequence consists of the nucleotide sequences present in SEQ ID NO: 173, 174, or 175 or their complements.

[0046] In some embodiments, the portion of the target sequence targeted by the promoter primer (promoter primer binding site) may be different (e.g., non-overlapping) from the portion targeted by the target capture oligonucleotide (if used). The promoter primer binding site may be completely or partially overlapping or identical to the target capture oligonucleotide binding site. In some embodiments, the amplified region of the target sequence partially or completely overlaps with the target capture binding site. In some embodiments, the amplified region of the target sequence does not overlap with the target capture binding site.

[0047] In some embodiments, prior to the first amplification step, the sample is contacted with one or more promoter primers under conditions that allow hybridization of the promoter primer to a portion of the target nucleic acid sequence in the sample. The promoter primer comprises a 3’ target-specific (TS) sequence, an RNA polymerase promoter sequence, and optionally one or more tag sequences. The RNA polymerase promoter sequence is recognized by an RNA polymerase such as T7 RNA polymerase. The tag sequence can be, but is not limited to, an amplification primer binding site, a specific binding site used for capture, or a sequencing primer binding site. The one or more promoter primers may target the same target nucleic acid sequence or different target nucleic acid sequences. The different target nucleic acid sequences may be from the same organism or from different organisms.

[0048] In some embodiments, it may be desirable to isolate the target nucleic acid sequence prior to amplification of the first phase. For this purpose, the sample can be contacted with a target capture oligonucleotide under conditions that allow hybridization of the target capture oligonucleotide to a portion (TCO binding site) of the target nucleic acid sequence. In some embodiments, the target nucleic acid is directly captured on a solid support, for example, by interaction with an immobilized capture probe. In some embodiments, the target nucleic acid is captured on a solid support as a member of a three-molecule complex (pre-amplification hybrid), and the target capture oligonucleotide bridges the target nucleic acid and the immobilized capture probe. In some embodiments, the solid support comprises a plurality of magnetic or magnetizable particles or beads that can be manipulated using a magnetic field. The step of isolating the target nucleic acid sequence can include washing the target capture oligonucleotide:target nucleic acid sequence hybrid to remove unwanted components that may interfere with subsequent amplification. The step of isolating the target nucleic acid sequence can also include washing the target capture oligonucleotide:target nucleic acid sequence hybrid to substantially remove excess promoter primers that are not hybridized to the target nucleic acid.

[0049] In some embodiments, the step of isolating the target nucleic acid sequence comprises contacting the sample with a promoter primer and a TCO under conditions that allow hybridization of the promoter primer and the TCO to the target nucleic acid sequence. The portion of the target sequence targeted by the promoter primer may be different (e.g., non-overlapping) from the portion targeted by the target capture oligonucleotide. The portion of the target sequence targeted by the promoter primer may be completely or partially overlapping or identical to the portion targeted by the target capture oligonucleotide. The promoter primer comprises a 3' target-specific sequence, an RNA polymerase promoter sequence, and optionally one or more tag sequences. In some embodiments, the RNA polymerase promoter sequence is recognized by an RNA polymerase such as T7 RNA polymerase. The tag sequence can be, but is not limited to, an amplification primer binding site, a specific binding site used for capture, or a sequencing primer binding site.

[0050] In some embodiments, one or more target capture oligonucleotides and one or more promoter primers are provided in a target capture reagent (TCR mixture). The one or more promoter primers can be hybridized to one or more target nucleic acid sequences to form a preamplification hybrid (along with the TCO) and isolated along with the one or more target nucleic acid sequences during a target capture step. One advantage of this method is that by hybridizing the promoter primer to the target nucleic acid sequence during target capture, the captured nucleic acid can be washed to remove sample components including unhybridized promoter primers. In a multi-phase reaction, removing unhybridized promoter primers can result in amplification in the first phase without interference from excess promoter primers, thereby substantially reducing or eliminating problems common to multiplex reactions. In a single-phase multiplex amplification reaction, primers can interfere with each other. Excess primers are more likely to misprime (hybridize to non-target nucleic acids) in both uniplex and multiplex reactions. In multiplex reactions where different organisms each have their own rRNA and oligonucleotides, mispriming is a greater concern. Multi-phase amplification addresses these problems by hybridizing the promoter primer to its intended target under stringent conditions and then washing away excess promoter primers. The resulting 1:1 primer / target ratio present in the first phase amplification reaction of multi-phase amplification can drive the population of target nucleic acids to a level that allows for the addition of excess primers later while reducing the level of mispriming or the impact of any mispriming during amplification.

[0051] The amplification reaction of the first phase is carried out under conditions that do not support exponential amplification of the target nucleic acid sequence. In some embodiments, the amplification reaction of the first phase is a linear amplification reaction. The amplification reaction of the first phase typically produces an amplification of about 2-fold to about 10,000-fold. In some embodiments, the amplification reaction of the first phase produces an amplification of about 10-fold to about 10,000-fold of the target nucleic acid sequence. In some embodiments, the amplification reaction of the first phase is substantially isothermal, i.e., it does not involve the thermal cycling characteristic of PCR and other common amplification techniques. The amplification reaction of the first phase can be carried out at 43 ± 2 °C, 43 ± 2 °C, 42 ± 1 °C, 42 ± 0.5 °C, 43 ± 0.5 °C, 44 ± 0.5 °C, 41 to 45 °C, or 42 to 44 °C.

[0052] In some embodiments, the first-phase amplification reaction involves contacting the target nucleic acid sequence with a first-phase amplification reaction mixture (e.g., an AMP mixture) that supports linear amplification of the target nucleic acid sequence and lacks at least one component required for its exponential amplification. In some embodiments, at least one component required for its exponential amplification is an additional or excess promoter primer. In some embodiments, the AMP reaction mixture contains one or more amplification enzymes. The one or more amplification enzymes can be, but are not limited to, DNA polymerase, RNA polymerase, or a combination thereof. The DNA polymerase can be, but is not limited to, an RNA-dependent DNA polymerase (reverse transcriptase), a DNA-dependent DNA polymerase, or a combination thereof. In some embodiments, the AMP mixture contains a ribonuclease (RNase) such as RNase H or a reverse transcriptase having RNase H activity. In some embodiments, the AMP mixture contains a reverse transcriptase having RNase H activity and an RNA polymerase. The RNA polymerase can be, but is not limited to, T7 RNA polymerase. In some embodiments, the AMP mixture contains one or more non-RNA polymerase promoter-containing amplification oligonucleotides (e.g., non-promoter primers (i.e., NT7 primers)). The one or more non-promoter primers can target the same target nucleic acid sequence or different target nucleic acid sequences. The different target nucleic acid sequences can be from the same organism or different organisms. In some embodiments, the AMP mixture contains one or more non-promoter primers, an RNA polymerase, ribonucleotide triphosphates (NTPs), and deoxyribonucleotide triphosphates (dNTPs). The AMP mixture can further contain other components including, but not limited to, a buffer, dNTPs, NTPs, and salts.

[0053] In some embodiments, one or more components necessary for exponential amplification are lacking, there are agents that inhibit exponential amplification, and / or the temperature of the reaction mixture is not conducive to exponential amplification, so the amplification reaction in the first phase cannot support an exponential amplification reaction. Without limitation, the lack of one or more components necessary for exponential amplification, and / or inhibitors, and / or reaction conditions include amplification oligonucleotides (e.g., promoter primers, non-promoter primers, or combinations thereof), enzymes (e.g., polymerases such as RNA polymerase), nucleases (e.g., exonucleases, endonucleases, Cleavase, RNases, phosphorylases, glycosylases, etc.), enzyme cofactors, chelating agents (e.g., EDTA or EGTA), ribonucleotide triphosphates (NTPs), deoxyribonucleotide triphosphates (dNTPs), Mg 2+ , salts, buffers, enzyme inhibitors, blocking oligonucleotides, pH, temperature, salt concentration, and any combination thereof. In some cases, missing components such as agents that reverse the effect of inhibitors of exponential amplification present in the first-phase reaction may be indirectly involved. In some embodiments, the lack of one or more components is a promoter primer (an additional promoter primer beyond the promoter primer hybridized to the target nucleic acid as part of the pre-amplification hybrid).

[0054] The amplification reaction in the second phase (or, if there are three or more phases, a later phase) is carried out under conditions that allow exponential amplification of the target nucleic acid sequence. In some embodiments, the amplification reaction in the second phase is an exponential amplification reaction. In some embodiments, the amplification reaction in the second phase is a substantially isothermal reaction such as, for example, a transcription-related amplification reaction or a strand displacement amplification reaction. In some embodiments, the amplification reaction in the second phase is a transcription-mediated amplification (TMA) reaction. In some embodiments, the amplification reaction in the second phase is carried out at 43 ± 2°C, 43 ± 2°C, 42 ± 1°C, 42 ± 0.5°C, 43 ± 0.5°C, 44 ± 0.5°C, 41 - 45°C, or 42 - 44°C.

[0055] In some embodiments, the amplification of the second (or later) phase involves contacting a first amplification product having an amplification reaction mixture of the second phase (e.g., a PRO mixture) with an amplification reaction mixture of the first phase that supports exponential amplification of the target nucleic acid sequence. Thus, the amplification reaction mixture of the second phase typically includes at least one component necessary for exponential amplification that is lacking in the amplification reaction mixture of the first phase. In some embodiments, the amplification reaction mixture of the second phase includes amplification oligonucleotides (such as promoter primers), reverse transcriptase, polymerase, nuclease, phosphorylase, enzyme cofactors, chelating agents, ribonucleotide triphosphates (NTPs), deoxyribonucleotide triphosphates (dNTPs), Mg 2+ , and one or more components selected from optimal pH, optimal temperature, salts, and combinations thereof. The polymerase can be, but is not limited to, an RNA-dependent DNA polymerase (such as reverse transcriptase), a DNA-dependent DNA polymerase, a DNA-dependent RNA polymerase, and combinations thereof. In some embodiments, the amplification reaction mixture of the second phase includes an RNase such as RNase H or a reverse transcriptase having RNase H activity. In some embodiments, the amplification reaction mixture of the second phase includes a promoter primer, a reverse transcriptase having RNase H activity, and / or an RNA polymerase. In some embodiments, the amplification reaction mixture of the second phase further includes a detection oligo. The detection oligo can be, but is not limited to, a Torch or a molecular beacon.

[0056] In some embodiments, the target capture reagent contains one or more target capture oligonucleotides and one or more T7 promoter primers, the AMP reagent contains a buffer, dNTPs, NTPs, salts, and one or more non-T7 primers, the promoter (PRO) reagent contains a buffer, dNTPs, NTPs, salts, a surfactant, one or more T7 promoter primers and one or more torch oligonucleotides, and the enzyme (ENZ) reagent contains a buffer, a detergent, a chelating agent, a reverse transcriptase, and a DNA polymerase.

[0057] Using this method, the T. vaginalis target nucleic acid sequence in a biological sample can be detected and / or quantified. The second-phase amplification reaction can be a quantitative amplification reaction. Methods for detecting the second amplification product are also described. Detection and / or quantification of the second amplification product can be performed using various detection techniques known in the art. Detection and / or quantification can be achieved, for example, by using a detection probe, a sequencing reaction, electrophoresis, mass spectrometry, melting curve analysis, or combinations thereof. In some embodiments, the second amplification product is detected and / or quantified using a detection probe. The detection probe can be, but is not limited to, a molecular torch (Torch as described in U.S. Patent No. 6,534,274), a molecular beacon, a hybridization switch probe, or combinations thereof. In some embodiments, detection and / or quantification can be performed in real time. The detection probe can be included in the first and / or second-phase amplification reactions with substantially equal degrees of success. The detection probe can be supplied into the first and / or second-phase amplification reaction mixture (e.g., the AMP mixture and / or the PRO mixture). In some embodiments, the PRO mixture contains the detection probe. The detection probe can include a Torch.

[0058] In some embodiments, the described method further comprises contacting the second amplification product with another bolus of one or more amplification components selected from, but not limited to, amplification oligonucleotides (promoter primers or non-promoter primers), reverse transcriptase (e.g., reverse transcriptase having RNase H activity), polymerase (e.g., RNA polymerase), nuclease, phosphorylase, enzyme cofactors, chelating agents, ribonucleotide triphosphates (NTPs), deoxyribonucleotide triphosphates (dNTPs), Mg 2+ , salts, and combinations thereof. This additional step can provide a boost to the second-phase amplification reaction because some of the amplification reaction components may become depleted.

[0059] The described method can be used to amplify and / or detect multiple different target nucleic acid sequences in a sample in a multiplex reaction. In some embodiments, in the case of a multiplex reaction, first, the multiple target nucleic acid sequences are subjected to a first-phase amplification reaction under conditions that do not support exponential amplification of any of the target nucleic acid sequences. The first-phase amplification reaction generates multiple first amplification products, which are then subjected to a second-phase (and optionally subsequent-phase) amplification reaction under conditions that allow exponential amplification of the first amplification products, thereby generating second amplification products.

[0060] In some embodiments, a method for amplifying a plurality of different target nucleic acid sequences in a sample is provided, where some but not all of the target nucleic acid sequences are subjected to linear amplification and / or some but not all of the target nucleic acid sequences are amplified exponentially. At least four variations of the first-phase amplification are contemplated: (1) some of the target sequences are subjected to linear amplification and the rest are left unamplified, (2) some of the target sequences are amplified exponentially and the rest are left unamplified, (3) some of the target sequences are subjected to linear amplification, some are subjected to exponential amplification, and the rest are left unamplified, and (4) some of the target sequences are subjected to linear amplification and the rest are subjected to exponential amplification. In some embodiments, the first-phase amplification can result in amplification of all of the target nucleic acid sequences (option 4) or only a subset thereof (options 1-3). The subset of target nucleic acid sequences can represent targets that are known to be present in relatively small amounts and / or targets that are difficult to amplify compared to other targets. The first-phase amplification reaction generates one or more first amplification products. The first amplification products in the sample and any unamplified target nucleic acid sequences are then subjected to a second-phase amplification reaction under conditions that allow their exponential amplification, generating a plurality of second amplification products. In some embodiments, the above conditions 1-4 can be applied to all phases except the final phase, and for the final phase, there can be three or more phases where unamplified or linearly amplified target nucleic acid sequences in the sample are subjected to an amplification reaction under conditions that allow exponential amplification.

[0061] It is understood that any of the various elements and parameters discussed above in connection with multi-phase uniplex (i.e., single target) amplification are also applicable to the multi-phase multiplex amplification modes described herein.

[0062] Composition for multi-phase amplification of C. vaginalis In some embodiments, a TCR mixture for capturing a T. vaginalis target nucleic acid sequence in a sample is described as including the following: (a) a target capture oligonucleotide (TCO) having a region that hybridizes to the target nucleic acid sequence. In some embodiments, the TCR mixture further includes a promoter primer that hybridizes to the target nucleic acid sequence. In some embodiments, the TCR mixture optionally contains an amplification enzyme. The TCR mixture can be used to isolate and / or purify the target nucleic acid sequence from the sample. In some embodiments, the target nucleic acid is isolated as a pre-amplification hybrid that includes the target nucleic acid, the TCO, and the promoter primer.

[0063] A "target capture oligonucleotide" (TCO) includes a nucleic acid oligonucleotide that crosslinks or binds a target nucleic acid and an immobilized capture probe, for example, by using complementary nucleic acid sequences or binding pair members such as biotin and streptavidin. In some embodiments, the target capture oligonucleotide binds nonspecifically to the target nucleic acid and immobilizes it on a solid support. The TCO includes a region of sequence complementarity to the target nucleic acid sequence, i.e., a target-specific (TS) sequence. In some embodiments, among the target capture oligonucleotides, those that specifically bind (hybridize) to the TCO binding sequence in the target nucleic acid. The TCO target-specific sequence includes a 10- to 35-nucleotide sequence having at least 90%, at least 95%, or 100% complementarity to the nucleotide sequence present in the target nucleic acid and hybridizes to a region (TCO binding site) within the target nucleic acid sequence. In some embodiments, the TCO target-specific sequence is 20 to 30 nucleotides in length. In some embodiments, the TCO target-specific sequence is 22 to 26 nucleotides in length and has at least 90% complementarity to the nucleotide sequence present in the target nucleic acid. The TCO target-specific and the TCO binding site may be completely complementary or may have one or more mismatches. In both approaches, the target capture oligonucleotide includes an immobilized capture probe binding region that binds to the immobilized capture probe (e.g., by specific binding pair interactions). Members of a specific binding pair (or binding partners) are portions that specifically recognize each other and bind to each other. The members may be referred to as a first binding pair member (BPM1) and a second binding pair member (BPM2), which represent various portions that specifically bind together. Specific binding pairs are exemplified, for example, by a receptor and its ligand, an enzyme and its substrate, a cofactor or coenzyme, an antibody or Fab fragment and its antigen or ligand, a sugar and lectin, biotin and streptavidin or avidin, a ligand and chelating agent, a protein or amino acid and its specific binding metal (such as histidine and nickel), substantially complementary polynucleotide sequences including completely or partially complementary sequences, and complementary homopolymer sequences.Specific binding pairs can be naturally occurring (e.g., enzymes and substrates), synthetic (e.g., synthetic receptors and synthetic ligands), or a combination of naturally occurring and synthetic BPMs. In some embodiments, both the target-specific sequence and the immobilized capture probe binding region are nucleic acid sequences. The target-specific sequence and the capture probe binding region may be covalently bound to each other or on different oligonucleotides linked by one or more linkers. In some embodiments, the capture probe binding region comprises a polyA sequence, a polyT sequence, or a polyT-polyA sequence. In some embodiments, the polyT-polyA sequence comprises dT3dA30. One or more target capture oligonucleotides may be used in the target capture and / or amplification reaction. The one or more target capture oligonucleotides may bind to the same target sequence or different target sequences. The target sequences may be from the same gene or different genes, and / or from the same organism or different organisms.

[0064] An "immobilized capture probe" provides a means for binding a target capture oligonucleotide to a solid support. In some embodiments, the immobilized capture probe contains a base sequence recognition molecule bound to the solid support, which facilitates the separation of the bound target polynucleotide from unbound material. Any known solid support such as a matrix and particles that are free in solution may be used. For example, the solid support can be nitrocellulose, nylon, glass, polyacrylate, mixed polymers, polystyrene, silane polypropylene, and magnetically attractable particles. In some embodiments, the support comprises monodisperse (i.e., the size is uniform ± about 5%) magnetic spheres. The immobilized capture probe can be bound to the solid support directly (e.g., via covalent bond or ionic interaction) or indirectly. General examples of useful solid supports include magnetic particles or beads.

[0065] The term "target capture" refers to the selective separation or isolation of a target nucleic acid from other components of a sample mixture such as cell fragments, organelles, proteins, lipids, carbohydrates, or other nucleic acids. A target capture system can specifically and selectively separate a given target nucleic acid from other sample components (e.g., by using a sequence specific for the target nucleic acid of interest such as a TCO target specific sequence), or it can non-specifically and selectively separate the target nucleic acid from other sample components by using other characteristics of the target (e.g., the physical properties of the target nucleic acid that distinguish it from other sample components that do not exhibit those physical characteristics). Target capture methods and compositions have been previously described in detail (U.S. Patent Nos. 6,110,678 and 6,534,273, and U.S. Publication No. 2008 / 0286775A1). In some embodiments, target capture utilizes solution-phase target capture oligonucleotides and immobilized capture probes bound to a support to form a complex with the target nucleic acid and separate the captured target from other components.

[0066] The terms "separating," "isolating," or "purifying" generally refer to removing one or more components of a mixture, such as a sample, from one or more other components in the mixture. Sample components can include cell fragments, proteins, carbohydrates, lipids, and generally aqueous solution-phase nucleic acids. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the target nucleic acid is separated or removed from other components in the mixture.

[0067] In some embodiments, the TCO comprises the nucleotide sequence of SEQ ID NO: 39, 40, or 41, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 39, 40, or 41. In some embodiments, the target-specific sequence of the TCO comprises SEQ ID NO: 39, 40, or 41, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 39, 40, or 41. In some embodiments, the TCO comprises SEQ ID NO: 39, 40, or 41, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 39, 40, or 41. In some embodiments, the TCO comprises SEQ ID NO: 39. In some embodiments, the TCO comprises the nucleotide sequence of SEQ ID NO: 1, 2, or 3, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 1, 2, or 3. In some embodiments, the TCO comprises SEQ ID NO: 1, 2, or 3, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 1, 2, or 3. In some embodiments, the nucleotide sequence of the TCO consists of the nucleotide sequence of SEQ ID NO: 1, 2, or 3, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 1, 2, or 3. In some embodiments, the TCO consists of SEQ ID NO: 1, 2, or 3, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 1, 2, or 3.

[0068] An "amplification oligonucleotide" (or more simply, a "primer") is an oligonucleotide that hybridizes to a target nucleic acid, or its complement, and participates in a nucleic acid amplification reaction. The amplification oligonucleotide is complementary to a nucleic acid template (target nucleic acid sequence), forms a complex with the template (by hydrogen bonding or hybridization), and contains at least a 3'-end that is suitable for initiating synthesis by an RNA-dependent or DNA-dependent polymerase, providing a primer:template complex. The amplification oligonucleotide is extended by adding a nucleotide base covalently attached to its 3'-end, and that base is complementary to the template. The result is a primer extension product. The amplification oligonucleotide is at least 10 nucleotides in length. In some embodiments, the amplification oligonucleotide is at least 15 nucleotides in length. In some embodiments, the amplification oligonucleotide is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more nucleotides in length. The amplification oligonucleotide contains a target-specific (TS) sequence at its 3'-end that is at least 90%, at least 95%, or 100% complementary to a region of the target nucleic acid (amplification primer binding site) and hybridizes. The amplification oligonucleotide target-specific sequence can be completely complementary to a region of the target nucleic acid, or it can have one or more mismatches if the amplification oligonucleotide can initiate template-dependent synthesis by an RNA-dependent or DNA-dependent polymerase. In some embodiments, the amplification oligonucleotide target-specific sequence is at least 10 consecutive nucleotides in length. In some embodiments, the amplification oligonucleotide target-specific sequence is at least 15 consecutive nucleotides in length. In some embodiments, the amplification oligonucleotide target-specific sequence is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides in length. The consecutive bases can be at least 90%, at least 95%, or completely (100%) complementary to the target sequence to which the amplification oligonucleotide binds.Nearly all known DNA polymerases (including reverse transcriptases) require an oligonucleotide to hybridize to a single-stranded template (``priming'') to initiate DNA synthesis, while RNA replication and transcription (copying of RNA from DNA) generally do not require a primer.

[0069] In some embodiments, the amplification oligonucleotide comprises an RNA polymerase promoter sequence located 5' of the target-specific sequence. The RNA polymerase promoter sequence can be, but is not limited to, a T7, T3, or SP6 promoter sequence. An amplification oligonucleotide containing a T7 RNA polymerase promoter sequence is referred to herein as a promoter primer. In some embodiments, the RNA polymerase promoter sequence is a T7 promoter sequence (T7 primer). The T7 promoter sequence can be about 25-30 nucleotides in length. Exemplary T7 promoter sequences include, but are not limited to, SEQ ID NO: 65 (5'-AATTTAATACGACTCACTATAGGGAGA-3') and SEQ ID NO: 66 (5'-GAAATTAATACGACTCACTATAGGGAGA-3').

[0070] In some embodiments, the promoter primer is a T7 primer. In some embodiments, the T7 primer comprises a nucleic acid sequence having at least 90% complementarity with the region of SEQ ID NO: 176 or its complement. In some embodiments, the promoter primer contains 15 to 30 consecutive bases having at least 90% complementarity with the region of SEQ ID NO: 176 or its complement. In some embodiments, the T7 promoter primer comprises the nucleotide sequence of SEQ ID NO: 42, 43, 44, 45, 46, 47, or 48, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 42, 43, 44, 45, 46, 47, or 48. In some embodiments, the target-specific sequence of the T7 primer comprises SEQ ID NO: 42, 43, 44, 45, 46, 47, or 48, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 42, 43, 44, 45, 46, 47, or 48. In some embodiments, the T7 promoter primer comprises the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the T7 promoter primer comprises SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the nucleotide sequence of the T7 primer consists of the nucleotide sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the T7 primer consists of SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0071] A promoter primer (e.g., a T7 primer) specifically binds to a target nucleic acid at its target sequence, and reverse transcriptase (RT) uses the target strand as a template to extend the 3’ end of the promoter primer to create a cDNA copy, resulting in an RNA:cDNA duplex. RNase activity (e.g., RNase H of the RT enzyme) digests the RNA of the RNA:cDNA duplex.

[0072] In some embodiments, the first-phase amplification mixture (AMP mixture) for the linear amplification of the T. vaginalis target nucleic acid sequence comprises a non-RNA polymerase promoter-containing oligonucleotide (also called a non-promoter primer NT7 primer), reverse transcriptase, RNA polymerase, dNTP, and NTP, and the first-phase amplification mixture lacks at least one component necessary for exponential amplification. The RNA polymerase can be T7 RNA polymerase. The AMP mixture further comprises the components necessary to amplify the target nucleic acid during the linear first-phase amplification reaction, provided that at least one component necessary for the exponential amplification of the target nucleic acid sequence is absent. In some embodiments, what is lacking at least one component necessary for exponential amplification is an additional promoter primer.

[0073] In some embodiments, the NT7 primer comprises a nucleic acid sequence having at least 90% complementarity with the region of SEQ ID NO: 177 or its complement. In some embodiments, the NT7 primer contains 15 to 30 consecutive bases having at least 90% complementarity with the region of SEQ ID NO: 177 or its complement. In some embodiments, the non-promoter primer comprises the nucleotide sequence of SEQ ID NO: 49, 50, 51, 52, 53, 54, or 55, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 49, 50, 51, 52, 53, 54, or 55. In some embodiments, the non-promoter primer comprises the nucleotide sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the non-promoter primer comprises the nucleotide sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the nucleotide sequence of the non-promoter primer consists of the nucleotide sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19. In some embodiments, the non-promoter primer consists of the nucleotide sequence of SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19.

[0074] "Detection oligonucleotide", "detection probe", or "probe" is an oligonucleotide that specifically hybridizes to a target sequence such as an amplification product under conditions that promote nucleic acid hybridization for the detection of a target nucleic acid or its amplification product. Detection can be either direct (i.e., a detection oligonucleotide that hybridizes directly to the target) or indirect (i.e., a detection oligonucleotide that hybridizes to an intermediate structure that binds the detection oligonucleotide to the target). The target sequence of a detection oligonucleotide generally refers to a specific sequence within a larger sequence to which the detection oligonucleotide specifically hybridizes. A detection oligonucleotide may contain a target-specific sequence and a sequence that is not complementary to the target. Such non-target-complementary sequences may include sequences that confer a desired secondary or tertiary structure, such as a hairpin structure, that can be used to facilitate detection and / or amplification. (See, e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,849,412, 6,835,542, 6,534,274, and 6,361,945, as well as U.S. Patent Application Publication Nos. 2006 / 0068417A1 and 2006 / 0194240A1). Complementary and non-complementary sequences may be contiguous or linked by a linker. In some embodiments, the linker is a C1, C2, C3, C4, C5, C6, C7, C8, C9, C 10 C 11 C 12 C 13 C 14 C 15 or C 16 linker. In some embodiments, the linker is a C9 linker. A detection oligonucleotide may be RNA, DNA, contain one or more modified nucleotides, or a combination thereof. In some embodiments, the detection oligonucleotide contains one or more 2'-methoxy nucleotides. In some embodiments, the detection oligonucleotide contains all 2'-methoxy ribonucleotides.

[0075] In some embodiments, the detection oligonucleotide contains one or more detectable markers or labels. The detectable marker can be, but is not limited to, a fluorescent molecule. The fluorescent molecule can bind to the 5' or 3' end of the detection oligonucleotide, or anywhere along the oligomer. In some embodiments, the detection oligonucleotide can be a molecular beacon or a torch. In some embodiments, the detection oligonucleotide can be a hydrolysis detection oligonucleotide. The detection oligonucleotide can contain a fluorescent molecule bound to the 5' end and a quencher bound to the 3' end. Alternatively, the fluorescent molecule can be bound to the 3' end of the detection oligonucleotide and the quencher can be bound to the 5' end of the detection oligonucleotide.

[0076] As used herein, "label" or "detectable label" refers to a moiety or compound that is directly or indirectly attached to a detectable oligonucleotide or an oligonucleotide that gives rise to a detectable signal. Direct attachment may use covalent bonds or non-covalent interactions (e.g., hydrogen bonds, hydrophobic or ionic interactions, and chelate or coordination complex formation), while indirect attachment may use a crosslinking moiety or linker (e.g., via an antibody or additional oligonucleotide), which amplify the detectable signal. Any detectable moiety may be used, for example, a radionuclide, a ligand such as biotin or avidin, an enzyme, an enzyme substrate, a reactive group, a chromophore that confers a detectable color such as a dye or particle (e.g., latex or metal beads), a fluorescent compound (e.g., a bioluminescent compound, a phosphorescent compound, or a chemiluminescent compound), and a fluorescent compound (i.e., a fluorophore). Examples of fluorophores include, but are not limited to, FAM™, TET™, CAL FLUOR™ (orange or red), QUASAR™, fluorescein, hexachloro-fluorescein (HEX), rhodamine, carboxy-X-rhodamine (ROX), tetramethylrhodamine, IAEDANS, EDANS, DABCYL, coumarin, BODIPY FL, lucifer yellow, eosin, erythrosin, Texas red, ROX, CY dyes (such as CY5), cyanine 5.5 (Cy5.5), and those known as fluorescein / QSY7 dye compounds. In some embodiments, the detectable oligonucleotide includes a base spacer between the 5' end of the oligonucleotide and the label. The spacer (or linker) may be an alkyl group. The fluorophore may be used in combination with a quencher molecule that absorbs light and reduces background fluorescence when in proximity to the fluorophore.Such quenchers include, but are not limited to, BLACKBERRYY® quencher (BBQ-650®), BLACK HOLE QUENCHER™ (or BHQ™ including but not limited to Black Hole Quencher-2 (BHQ2)), or TAMRA™ compounds. Examples of interaction donor / acceptor label pairs that can be used in connection with the present disclosure include, without attempting to distinguish between FRET and non-FRET pairs, fluorescein / tetramethylrhodamine, IAEDANS / fluorescein, EDANS / DABCYL, coumarin / DABCYL, fluorescein / fluorescein, BODIPY FL / BODIPY FL, fluorescein / DABCYL, CalRed-610 / BHQ-2, lucifer yellow / DABCYL, Quasar750 / BHQ-2, BODIPY / DABCYL, eosin / DABCYL, erythrosin / DABCYL, tetramethyl-rhodamine / DABCYL, texas red / DABCYL, CY5 / BHQ1, CY5 / BHQ2, CY3 / BHQ1, CY3 / BHQ2, and fluorescein / QSY7 dye, but are not limited thereto. In some embodiments, the detection oligonucleotide comprises a label that is detectable in a homogeneous system such that the bound labeled detection oligonucleotide in the mixture exhibits a detectable change compared to the unbound labeled detection oligonucleotide, enabling detection of the label without physically removing hybridization from the unhybridized labeled detection oligonucleotide (e.g., U.S. Pat. Nos. 5,283,174, 5,656,207, and 5,658,737). Detectable labels or detection oligonucleotides known in the art include, but are not limited to, chemiluminescent labels (including acridinium ester compounds, U.S. Pat. Nos. 5,656,207, 5,658,737, and 5,639,604), TaqMan® probes, molecular torches, and molecular beacons. TaqMan® probes contain donor and acceptor labels, and fluorescence is detected when the detection oligonucleotide is enzymatically degraded during amplification to release the fluorophore from the presence of the quencher.The molecular torches and beacons exist in open and closed configurations, where the closed configuration quenches the fluorophore and the open position separates the fluorophore from the quencher to allow fluorescence. Hybridization to a target opens the detection oligonucleotide that would otherwise be closed.

[0077] In some embodiments, the detection probe is Torch. In some embodiments, Torch comprises a nucleic acid sequence having at least 90% complementarity with the region of SEQ ID NO: 178 or its complement. In some embodiments, the promoter primer contains 10 to 30 consecutive bases having at least 90% complementarity with the region of SEQ ID NO: 177 or its complement. In some embodiments, Torch comprises the nucleotide sequence of SEQ ID NO: 56, 57, 58, 59, 60, 61, or 62, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 56, 57, 58, 59, 60, 61, or 62. In some embodiments, Torch comprises the nucleotide sequence of SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28. In some embodiments, Torch comprises SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28. In some embodiments, the nucleotide sequence of Torch consists of the nucleotide sequence of SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28. In some embodiments, Torch consists of SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28, or a nucleic acid sequence having at least 90% identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28. In some embodiments, the torch contains a fluorescent molecule attached to the 5' end and a quencher attached to the 3' end. Alternatively, the fluorescent molecule may be attached to the 3' end of the torch and the quencher may be attached to the 5' end of the detection oligonucleotide. In some embodiments, the torch contains a 5 to 6 nucleotide sequence at the 3' end that is complementary to and hybridizable with a 5 to 6 nucleotide sequence at the 5' end.In some embodiments, the 5-6 nucleotide sequence at the 3' end that is complementary to and capable of hybridizing with the 5-6 nucleotides at the 5' end is linked to the torch via a linker. In some embodiments, the linker is C. 1-16 In some embodiments, the linker is a C9 linker.

[0078] "Detection" of the amplification product can be achieved using any known method. For example, the amplified nucleic acid can be associated with a surface that results in a detectable physical change (e.g., an electrical change). The amplified nucleic acids can be detected in solution phase or by concentrating them within or on a matrix and detecting associated labels (e.g., intercalating agents such as ethidium bromide or SYBR Green). Other detection methods use probes complementary to the sequence of the amplification product to detect the presence of the probe:product complex or use a complex of probes to amplify the signal detected from the amplification product (e.g., U.S. Pat. Nos. 5,424,413, 5,451,503, and 5,849,481). Other detection methods use probes that bind to the target sequence such that signal generation occurs only when the probe bound to the target sequence binds to the amplification product such as molecular beacons, molecular torches, hybridization switch probes, etc. (e.g., U.S. Pat. Nos. 5,118,801, 5,312,728, 5,925,517, 6,150,097, 6,361,945, 6,534,274, 6,835,542, 6,849,412, and 8,034,554; and U.S. Pub. No. 2006 / 0194240A1). Detection can be achieved using a detection oligonucleotide that is present during target amplification and hybridizes to the amplicon in real time. The detection oligonucleotide may contain a fluorophore and a quencher. The torch contains complementary regions at each end. These complementary regions bind to each other to form a "closed" torch. In the closed configuration, the fluorophore and quencher are in proximity and the fluorophore signal is quenched. That is, it does not emit a detectable signal when excited by light. However, when the torch binds to a complementary target, the complementary regions within the torch are forced apart to form an "open" torch. In the open form, the fluorophore and quencher are not in proximity and the fluorophore signal is detectable upon excitation (i.e., it is no longer quenched).The amplicon-torch binding results in the separation of the quencher from the fluorophore, thereby enabling the excitation of the fluorophore in response to light stimulation and signal emission at a specific wavelength. The torch is present during amplification and may bind to the complementary amplicon as it is generated in real time. As more amplicons are produced, more torches bind and more signals are created. The signal will ultimately reach a detectable level above background, and ultimately all available torches will bind to the amplicon and the signal will reach its maximum. At the start of amplification and when the copy number of the amplification sequence is low, most of the detection oligonucleotides are closed (the 3' and 5' ends base pair and the fluorescent signal is quenched). During amplification, more detection oligonucleotides bind to the target sequence, causing the 3' and 5' ends of the detection oligonucleotides to separate and resulting in an increase in fluorescence (a decrease in fluorescence quenching). After further amplification, the fluorescent signal approaches its maximum.

[0079] In some embodiments, detection is performed at time intervals. Detection can be carried out by measuring fluorescence at regular time intervals. The time intervals can be, but are not limited to, 1 - 60 seconds, 1 - 120 seconds, 1 - 180 seconds, 1 - 240 seconds, or 1 - 300 seconds. In some embodiments, the time intervals are 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 seconds. In detection performed at regular time intervals, each interval is referred to as a cycle. Detection can be carried out over 20 - 240 cycles, 30 - 210 cycles, 40 - 180 cycles, 50 - 150 cycles, or 60 - 120 cycles. For example, detection every 30 seconds over 60 minutes constitutes 120 cycles. Detection can occur at the start or end of a cycle. Detection can also be performed continuously.

[0080] In some embodiments, an amplification oligonucleotide (promoter primer or non-promoter primer), a detection oligonucleotide, or a target capture oligonucleotide contains one or more modified nucleotides. The oligonucleotide can have 1, 2, 3, 4, 5, 6, 7, 8 or more modified nucleotides. In some embodiments, more than 50%, more than 60%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or 100% of the nucleotides are modified. Modified nucleotides include nucleotides having modified nucleobases. Modified nucleobases include, but are not limited to, synthetic and natural nucleobases, 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6 substituted purines. Modified nucleotides include, but are not limited to, 2'-modified nucleotides (including but not limited to 2'-halogen nucleotides such as 2'-O-methyl nucleotides and 2'-fluoro nucleotides), nucleotides having modified bases. Modified nucleotides also include nucleotides having modified linkages, such as but not limited to phosphorothioate linkages.

[0081] Any of the oligonucleotides described herein can contain one or more tags. A "tag" can be a nucleotide sequence covalently attached to an oligonucleotide for the purpose of conferring some additional functionality beyond binding to a target sequence. Non-limiting examples of oligonucleotide tags include a 5' promoter for RNA polymerase, a primer binding site, a sequencing tag, a mass tag, a barcode tag, a capture tag, etc. (e.g., U.S. Patent Nos. 5,422,252, 5,882,856, 6,828,098, and PCT Publication No. 05 / 019479). A tag can also be a non-nucleotide molecule covalently attached to an oligonucleotide for the purpose of conferring some additional functionality.

[0082] When multiple amplifications are intended, the composition may include a plurality of different target capture oligonucleotide promoters, and non-promoter primers that hybridize to a plurality of different target nucleic acid sequences. The different target nucleic acid sequences may be from the same organism or from different organisms.

[0083] As described above, the methods and compositions disclosed herein are useful for amplifying a target nucleic acid sequence in vitro to generate an amplified sequence that can be detected to indicate the presence of the target nucleic acid in a sample. The methods and compositions are useful for synthesizing amplified nucleic acids to provide useful information for diagnosing and / or determining the prognosis of a medical condition, for detecting the purity or quality of environmental and / or food samples, or for investigating forensic evidence. The methods and compositions are advantageous for providing highly sensitive assays over a broad dynamic range that are relatively quick and inexpensive to perform, making them suitable for use in high-throughput and / or automated systems. The methods and compositions can be used in assays that analyze a single target sequence, i.e., a uniplex amplification system, and are particularly useful in assays that simultaneously analyze multiple different target sequences, i.e., a multiplex amplification system. In some embodiments, the composition and reaction mixture are provided in a kit that includes defined assay components that are useful because they enable a user to efficiently perform a method of using the components together in an assay to amplify a desired target.

[0084] An oligonucleotide composition for the multiplex amplification and detection of D. vaginalis. In some embodiments, the TCO comprises the nucleotide sequence of SEQ ID NO: 41, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 47, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 51, and the Torch comprises the nucleotide sequence of SEQ ID NO: 58.

[0085] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 3, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 11, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 15, and Torch comprises the nucleotide sequence of SEQ ID NO: 24.

[0086] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 41, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 42, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 50, and Torch comprises the nucleotide sequence of SEQ ID NO: 56.

[0087] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 3, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 4, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 14, and Torch comprises the nucleotide sequence of SEQ ID NO: 20.

[0088] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 41, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 42, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 50, and Torch comprises the nucleotide sequence of SEQ ID NO: 57.

[0089] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 3, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 4, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 14, and Torch comprises the nucleotide sequence of SEQ ID NO: 21.

[0090] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 41, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 42, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 49, and Torch comprises the nucleotide sequence of SEQ ID NO: 57.

[0091] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 3, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 4, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 13, and Torch comprises the nucleotide sequence of SEQ ID NO: 21.

[0092] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 41, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 45, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 51, and Torch comprises the nucleotide sequence of SEQ ID NO: 58.

[0093] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 3, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 9, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 15, and Torch comprises the nucleotide sequence of SEQ ID NO: 23.

[0094] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 40, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 42, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 50, and Torch comprises the nucleotide sequence of SEQ ID NO: 56.

[0095] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 2, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 4, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 14, and Torch comprises the nucleotide sequence of SEQ ID NO: 20.

[0096] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 40, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 42, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 50, and Torch comprises the nucleotide sequence of SEQ ID NO: 57.

[0097] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 2, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 4, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 14, and Torch comprises the nucleotide sequence of SEQ ID NO: 21.

[0098] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 40, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 42, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 49, and Torch comprises the nucleotide sequence of SEQ ID NO: 57.

[0099] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 2, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 4, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 13, and Torch comprises the nucleotide sequence of SEQ ID NO: 21.

[0100] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 40, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 45, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 51, and Torch comprises the nucleotide sequence of SEQ ID NO: 58.

[0101] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 2, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 9, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 15, and Torch comprises the nucleotide sequence of SEQ ID NO: 23.

[0102] In some embodiments, TCO comprises the nucleotide sequence of SEQ ID NO: 40, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 42, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 49, and Torch comprises the nucleotide sequence of SEQ ID NO: 56.

[0103] In some embodiments, the TCO comprises the nucleotide sequence of SEQ ID NO: 2, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 4, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 13, and the Torch comprises the nucleotide sequence of SEQ ID NO: 20.

[0104] Additional oligonucleotides are provided in Table 1.

Table 1-1-1

Table 1-1-2

Table 1-1-3

Table 1-1-4

Table 1-1-5

[0105] E. Compositions and Kits The present disclosure provides oligomers, compositions, and kits useful for the amplification, detection, and / or quantification of T. vaginalis in a sample. The oligomers, compositions, and kits can be used in uniplex or multiplex multiphase amplification methods.

[0106] Also described are reaction mixtures for determining the presence or absence of, or quantifying the amount of, T. vaginalis target nucleic acid in a sample. Various reaction mixtures include, but are not limited to, a target capture (TCR) mixture, an amplification (AMP) mixture, a promoter primer (PRO) mixture, and an enzyme (ENZ) mixture. According to the present disclosure, the mixtures independently include one or more of the promoter primers (e.g., T7 primers), non-promoter primers (NT7 oligonucleotides), TCOs, detection oligonucleotides, reverse transcriptase, RNA polymerase, dNTPs, NTPs, buffers, salts, and combinations thereof described herein for amplification and / or detection of T. vaginalis target nucleic acid in a sample. In some embodiments, any combination of oligonucleotides described herein can be provided in a kit. The compositions, kits and / or reaction mixtures can further include any of several optional components. In some embodiments, the kit includes one or more test sample components, which may or may not contain T. vaginalis target nucleic acid. In some embodiments, the kit includes one or more control oligonucleotides, including but not limited to, a control TCO, a control promoter primer, a control non-promoter primer, a control detection oligonucleotide, and combinations thereof. The kit may include oligonucleotides for amplification and detection of T. vaginalis, or may include oligonucleotides for amplification and detection of one or more other organisms including, but not limited to, T. vaginalis and Candida species.

[0107] In some embodiments, the composition or kit comprises a detection oligonucleotide comprising one or more detection oligonucleotides. The detection oligonucleotide independently comprises a fluorescent label and a quencher. In some embodiments, the composition or kit comprises one or more Torch detection oligonucleotides. In some embodiments, the composition or kit comprises two or more Torch detection oligonucleotides. Two or more Torch oligonucleotides can detect amplification products from different organisms and can be detectable in different channels.

[0108] In some embodiments, the kit, composition, or reaction mixture further comprises one or more of DNA polymerase, deoxyribonucleotides, positive control nucleic acid, negative control nucleic acid, control nucleic acid, dNTP (e.g., dATP, dTTP, dGTP, and dCTP), NTP (e.g., ATP, UTP, GTP, and CTP), Cl, MgCl2, potassium acetate, buffer, BSA, sucrose, trehalose, DMSO, betaine, formamide, glycerol, polyethylene glycol, nonionic surfactant, ammonium ion, EDTA, and other reagents or buffers suitable for isothermal amplification and / or detection. The DNA polymerase can be, but is not limited to, reverse transcriptase. The buffer can be, but is not limited to, Tris-HCl and Tris-acetate. The nonionic surfactant can be, but is not limited to, Tween®-20 and Triton® X-100.

[0109] In some embodiments, the described primers and detection oligonucleotides for T. vaginalis have a shelf life of at least 3 months, at least 6 months, at least 9 months, at least 12 months, at least 15 months, at least 18 months, or at least 24 months from the date of manufacture.

[0110] Every method disclosed herein should also be understood as a disclosure of the corresponding use of materials involved in the method for the purpose of the method. Any of the oligonucleotides comprising T. vaginalis sequences, as well as any combination (e.g., kits and compositions) comprising such oligonucleotides, are disclosed for use in detecting and / or quantifying T. vaginalis or amplifying T. vaginalis nucleic acid sequences, and should also be understood as being disclosed for use in the preparation of compositions for detecting and / or quantifying T. vaginalis or amplifying T. vaginalis nucleic acid sequences.

[0111] In certain embodiments, the kit further comprises a set of instructions for carrying out the methods according to the present disclosure, which instructions may be associated with the accompanying documents and / or the packaging of the kit or its components.

[0112] Embodiments of the compositions and methods described herein can be further understood by the following examples. The method steps used in the examples are described herein, and the following information describes more specifically the typical reagents and conditions used in the methods. Other reagents and conditions may be used as long as they do not substantially affect the process or results, following the guidance provided in the above description. Further, the disclosed methods and compositions may be implemented manually or in a system that performs one or more steps (e.g., pipetting, mixing, incubation, etc.) in an automated device, and may be used in any type of known device (e.g., multi-well devices such as test tubes, multi-tube unit devices, 96-well microtiter plates, etc.).

Examples

[0113] Exemplary reagents used in the methods described in the examples include the following.

[0114] "Sample Transport Medium" or "STM" is a phosphate buffer (pH 6.7) containing EDTA, EGTA, and lithium lauryl sulfate (LLS).

[0115] "Target Capture Reagent" or "TCR" is a HEPES buffer (pH 6.4) containing lithium chloride and EDTA, together with 250 μg / ml of magnetic particles (1 micron SERA-MAG™ MG-CM particles, Seradyn, Inc., Indianapolis, IN) to which (dT)14 oligonucleotides are covalently attached.

[0116] "Target Capture Wash Solution" or "TC Wash Solution" is a HEPES buffer (pH 7.5) containing sodium chloride, EDTA, 0.3% (v / v) absolute ethanol, 0.02% (w / v) methylparaben, 0.01% (w / v) propylparaben, and 0.1% (w / v) sodium lauryl sulfate.

[0117] "Amplification Reagent" or "AR" is a HEPES buffer (pH 7.7) containing magnesium chloride, potassium chloride, four deoxyribonucleotide triphosphates (dATP, dCTP, dGTP, and dTTP), and four ribonucleotide triphosphates (rATP, rCTP, rGTP, and rUTP). Primers and / or probes may be added to the reaction mixture in the amplification reagent or separately from the reagent (primerless amplification reagent).

[0118] When used in an amplification or pre-amplification reaction mixture, "Enzyme Reagent" or "ENZ" is a HEPES buffer (pH 7.0) containing MMLV reverse transcriptase (RT), T7 RNA polymerase, salts, and cofactors.

[0119] Example A. Multiphase Amplification / Detection The T7 primer is hybridized to the target sequence during target capture, followed by removal of the excess T7 primer.

[0120] In the first phase, the NT7 primer, except for the additional T7 primer, is introduced along with all the necessary amplification, detection, and enzyme reagents. In the presence of reverse transcriptase, the T7 primer hybridized to the captured target is extended to create a cDNA copy, and the target RNA template is degraded by the RNase H activity of the reverse transcriptase. Subsequently, the NT7 primer hybridizes to the cDNA, extends, fills in the promoter region of the T7 primer, and creates an active double-stranded DNA template. Then, T7 polymerase generates multiple RNA transcripts from the template. Subsequently, the NT7 primer hybridizes to the RNA transcript, extends, and generates a promoterless cDNA copy of the target RNA template. The RNA strand is degraded by the RNase activity of the reverse transcriptase. Since there is no free T7 primer available in the one-phase amplification mixture, the reaction does not proceed further. The second phase is then initiated by the addition of the T7 primer and optionally a detection oligonucleotide, thus starting the exponential amplification of the cDNA pool generated in the first phase.

[0121] For multiplex amplification and detection, one or more of each of the TCO, T7 primer, NT7 primer, and Torch oligonucleotide are used. The oligonucleotides may amplify different sequences in the same target nucleic acid, different sequences in different target nucleic acids, or combinations thereof. The different target nucleic acids may be from the same organism or from different organisms.

[0122] Plate setup: In some embodiments, four different plates are set up for use with two automated KingFisher devices. 1. Plate 1 (TCR plate) contains the lysed sample. To this plate, the target capture reagent (100 μL) is added. The TCO and T7 primer hybridize to the target nucleic acid (400 μL of sample). Using a magnet and magnetic beads (capture probes on a solid support), the TCO:target nucleic acid:T7 primer (pre-amplification hybrid) is captured. 2. Plate 2 is a deep well plate and holds 500 μL / well of APTIMA wash buffer. The Aptima wash buffer contains surfactants and alcohol used to wash away excess proteins and lipids remaining from cell lysis. 3. Plate 3 contains 200 μL / well of APTIMA wash buffer and is used to provide a second wash of the pre-amplified hybrid. 4. Plate 4 contains 50 μL / well of AMP reagent. In some embodiments, the AMP reagent contains buffer, salts, dNTPs, NTPs, and one or more non-T7 primers.

[0123] Target capture and isolation: TCO and T7 primers are added to a sample containing (or suspected of containing) the target nucleic acid. The T7 primer is added at a ratio of approximately one T7 primer per one target nucleic acid. The TCO and T7 primers are incubated with the target nucleic acid for a period of time to allow hybridization of the TCO and T7 primers to the target nucleic acid. The pre-amplified hybrid is then purified to remove excess or unhybridized T7 primers. The pre-amplified hybrid is then isolated using magnetic particles having a poly(dT) binding partner in the case of TCO. 1. Place Plate 1 (TCR plate) in a heat block and heat to 62 °C for 30 minutes, followed by incubation at room temperature for 20 minutes to 2 hours. In some embodiments, cover the TCR plate with a 65 °C lid to prevent condensation from forming on the top of the wells. The captured pre-amplified hybrid is then transferred to Plate 2. 2. After the first wash (about 10 minutes), add a deep well comb / magnet cover to Plate 2 to capture the pre-amplified hybrid. The captured pre-amplified hybrid is transferred to Plate 3. 3. After the second wash, add a small comb (magnet cover) to Plate 3 to capture the pre-amplified hybrid. The washed pre-amplified hybrid is captured and transferred to Plate 4. The fourth plate is transferred to a thermal cycler for isothermal amplification and detection in real time.

[0124] Multiphase transcription-mediated amplification and real-time detection. Amplification in the first phase: NT7 primers, enzymes, dNTPs, and NTPs (AMP mixture) are present together with the purified target nucleic acid containing the pre-amplification hybrid. The mixture is incubated for a period of time to allow the formation of the first amplification product. 1. Incubate an AMP plate containing NT7 primers and the purified target nucleic acid with the hybridized T7 primers at 44°C for 5 minutes. 2. Add 25 μL of the ENZ mix containing reverse transcriptase, T7 RNA polymerase, dNTPs, and NTPs to each well of the plate, seal it, mix at 1400 rpm for 1 minute, and incubate at 44°C for 5 minutes in a thermal cycler. Amplification in the second phase: T7 primers are added to the first amplification product and incubated for a period of time to allow the formation of the second amplification product. 3. Add 25 μL of the PRO mixture to each well, seal it, and mix at 1400 rpm for 1 minute. In some embodiments, the PRO mixture contains buffer, salts, surfactant, dNTPs, NTPs, one or more T7 primers, and a Torch probe. 4. Reaction program: 120 cycles of 30 seconds at 43°C, with labeled detection (collection) performed at the end of each cycle.

[0125] Detection: The amplification of the target nucleic acid sequence is detected in real time by recording the fluorescence signal from the detection oligonucleotide at regular intervals.

[0126] Example 1. Two-phase real-time TMA oligonucleotide screening of T. vaginalis. Multiphase amplification was performed as described above using the following conditions.

Table 1-1

Table 1-2

[0127] Results: None of the combinations resulted in a curve strong enough to allow amplification and / or detection of T. vaginalis.

[0128] Example 2. T. vaginalis biphasic real-time TMA oligo screen. Alternative target captures will be screened and the T. vaginalis T7 primers titrated to see if they improve assay performance. Polyphasic amplification was performed as described above using the following conditions: [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

[0129] Results: None of the combinations resulted in a curve strong enough to allow amplification and / or detection of T. vaginalis.

[0130] Example 3. T. vaginalis biphasic real-time TMA oligonucleotide screening. Using the following conditions, the polyphase amplification was performed as described above. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6]

[0131] Results: AMP1 / PRO1, AMP1 / PRO3, AMP2 / PRO3, and AMP3 / PRO5 resulted in good amplification and / or detection of T. vaginalis. [Table 3-7]

[0132] Example 4. T. vaginalis biphasic real-time TMA oligonucleotide screening. Using the following conditions, the polyphase amplification was performed as described above. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

Table 4-5

Table 4-6

[0133] Some of the systems showed amplification, but none of the systems generated a strong curve. The oligos shown could be candidates for a viable system, but none of the combinations worked well for the amplification / detection of T. vaginalis.

[0134] Example 5. Cross-reactivity of T. tenax with the T. vaginalis amplification system. Using the following conditions, multiplex amplification was performed as described above.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

[0135] Result: 1×10 5The presence of T. Tenax cells / reaction did not interfere with the detection of T. vaginalis using the indicated oligonucleotides. T. vaginalis was detected at the same point of appearance and reached the same RFU regardless of the presence of T. Tenax. The indicated oligonucleotides detected T. Tenax, despite having substantially slower appearance times (about 8 minutes later vs. about 14 minutes) and lower RFUs (about 22,000 vs. about 7300 with 15 pmol of Torch). Torch SEQ ID NO: 64 showed a very low background with T. Tenax.

[0136] Example 6. Cross-reactivity of T. Tenax and Pentatrichomonas hominis with the T. vaginalis amplification system. Multiplex amplification was performed as described above using the following conditions. For the specificity of amplification of T. vaginalis vs. T. tenax and P. hominis, N7 oligonucleotide SEQ ID NO: 9 was compared to N7 oligonucleotide SEQ ID NO: 11, and Torch SEQ ID NO: 23 was compared to Torch SEQ ID NO: 64. The duplex amplification reaction was performed as described using TCO SEQ ID NO: 3 and NT7 primer SEQ ID NO: 15. Torch SEQ ID NO: 23 provided a stronger amplification curve (Tables 5 - 7). N7 oligonucleotide SEQ ID NO: 11 had less background due to a late TTime and a low RFU range (Tables 5 - 8).

Table 6-1

Table 6-2

[0137] No cross-reactivity was observed between P. hominis, a closely related non-target species, and T. vaginalis.

[0138] The performance of the T7 primers of T. vaginalis with SEQ ID NO: 9 and SEQ ID NO: 11 and SEQ ID NO: 24 was confirmed in multiplex format using all assay oligonucleotides including Candida species and C. glabrata. T7 primer SEQ ID NO: 11 had a lower T. tenax background compared to SEQ ID NO: 9 in the CV / TV multiplex assay.

[0139] T7 primer SEQ ID NO: 11 had a lower T. tenax background by the RFU range (5,992 vs. 4,921) and the later-occurring T time (14.88 vs. 6.30) compared to SEQ ID NO: 9 using the same torch in the CV / TV multiplex amplification assay (Tables 5 - 9).

Table 6 - 3

[0140] T7 primer SEQ ID NO: 11 had a lower T. tenax background by the RFU range (5,992 vs. 4,921) and the later-occurring T time (14.88 vs. 6.30) compared to SEQ ID NO: 9 using the same torch in the CV / TV multiplex amplification assay (Tables 5 - 9).

[0141] Example 7. Multiplex amplification of T. vaginalis and Candida species. Two-phase amplification was performed as described above using the following conditions.

Table 7 - 1

Table 7 - 2

Table 7 - 3

Table 7 - 4

Table 7 - 5

Table 7-6

[0142] Result: Torch of both C. albicans and T. vaginalis was read in the FAM channel.

[0143] 1×10 4 C. albicans at 1×10 cells / reaction, when present during the reaction, the T. vaginalis oligo of S1 partially inhibited C. albicans amplification, and 1×10 6 C. albicans at 1×10 cells / reaction, when present during the reaction, did not inhibit. None of the combinations of the five T. vaginalis oligos affected the amplification of C. albicans at 1×10 6 cells / reaction when present during the reaction. Furthermore, none of the combinations of the five T. vaginalis oligos had an adverse effect on the amplification of C. glabrata.

[0144] The T. vaginalis cell / reaction system S4 at 0.1 amplified and detected T. vaginalis. The T. vaginalis cell / reaction systems S1, S2, and S3 at 1 amplified and detected T. vaginalis. The amplification of T. vaginalis was not significantly inhibited by the presence of Candida oligos.

[0145] Example 8. Optimization of the multiplex assay. Using the following conditions, multiplex multiphase amplification was performed as described above. A multiplex assay was performed for the detection of T. vaginalis using Torch SEQ ID NO: 22 and SEQ ID NO: 23 containing carboxy-X-rhodamine (ROX). The TCO of T. vaginalis was SEQ ID NO: 3, the NT7 primer was SEQ ID NO: 15, and the T7 primer was SEQ ID NO: 11. The multiplex assay further contained oligonucleotides for the detection of C. albicans and other Candida species (detected in the FAM channel respectively) as well as the detection of C. glabrata (detected in the HEX channel). The control Torch was detected in the Cy5.5 channel. The Candida oligonucleotides are listed in Table 9-5.

[0146] Four targets in combination with competing controls were tested in multiplex format. Titrations of the oligonucleotide concentrations in the Candida species and C. glabrata channels were performed to find a balance between all amplification systems. Formulations with increased amounts of Candida species oligonucleotides for T7 in TCR and NT7 were tested and verified. Next, the oligonucleotide concentration of Candida species was tested by increasing T7 of C. glabrata in TCR and NT7. In both test sets, no inhibition of other channels was shown.

[0147] Optimization of the oligonucleotide concentration of Candida species showed an improvement in the FAM channel when comparing the original concentration of System 1 (6 pmol / rxn SEQ ID NO: 35, 5 pmol / rxn SEQ ID NO: 36) with the increased oligonucleotide concentration of System 2.

[0148] In the second optimization of the C. glabrata amplification system, the oligonucleotide concentration increased with an increase in the C. albicans oligonucleotide concentration. A faster TTime in the HEX channel for C. glabrata was observed without changing the amplification efficiency of Candida species in FAM. The increase in the new C. glabrata oligonucleotide concentration also improved the competing control.

[0149] When testing the combination of targets, a significant negative interaction was discovered during the amplification of C. glabrata in the presence of high-titer T. vaginalis. For both C. glabrata and T. vaginalis, a four-factor characterization design strategy of high, medium, and low concentrations of T7 of TCR and NT7 of AMP was selected, and for each analyte, it was determined which factor had the greatest impact at T time. The high concentration was set as the current concentration. The experiment consisted of 20 runs. It was found that when the concentration of TV T7 in TCR was low, the amplification of C. glabrata became faster.

[0150] Using Torch accession number 23, T. vaginalis was detected multiplexly at 0.001 cells / mL.

[0151] Example 9. Analytical sensitivity. Serial dilutions of culture lysates of each Candida species (C. albicans, C. tropicalis, C. dubliniensis, C. parapsilosis, and C. glabrata) and T. vaginalis in Aptima Transport Media (STM) were tested in the CV / TV multiplex assay. For each species, a 1 / 2 log titration from 1000 CFU / mL to 30 CFU / mL for C. albicans, C. tropicalis, and C. dubliniensis, from 300 CFU / mL to 3 CFU / mL for C. parapsilosis, from 100 CFU / mL to 10 CFU / mL for C. glabrata, and from 0.01 cells / mL to 0.0001 cells / mL for T. vaginalis was repeated 15 times and performed. Multiplex amplification was carried out as described using the following conditions.

[0152] The percent positive, mean TTime, mean RFU range, and mean T gradient of the Candida species are shown in Table 9-7. The Candida species were detected in the FAM channel, C. glabrata in the HEX channel, T. vaginalis in the ROX channel, and the competitive control Torch of C. glabrata in the Cy5.5 channel.

[0153] Table 9-8 shows the positive percentage, average TTime, average RFU range, and average T gradient for T. vaginalis. The detection limit for reaching 100% positive signal of T. vaginalis was 0.001 cells / ml. [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8]

[0154] Using the normal probit model, the probability of detecting T. vaginalis present at 0.0004 (0.0003 - 0.0005) cells / mL is 50% (95% confidence level), and the probability of detecting T. vaginalis present at 0.001 (0.0007 - 0.0003) cells / mL is 95% (95% confidence level). Using the Gompertz probit model, the probability of detecting T. vaginalis present at 0.00004 (0.0003 - 0.0006) cells / mL is 50% (95% confidence level), and the probability of detecting T. vaginalis present at 0.0008 (0.0006 - 0.0016) cells / mL is 95% (95% confidence level). Table 9-9 and 9-10 show the types of probit values.

Table 9-9

Table 9-10

[0155] Example 10. In-silico specificity analysis. In-silico analyses of T. vaginalis, Candida, and control oligonucleotides (Table 9.5) and control oligonucleotides (Table 9.5) were performed to evaluate whether the system cross-reacts with unwanted targets or forms unwanted intermolecular or intramolecular interactions. The oligonucleotides were also subjected to interaction analysis using the OLIGO7 and OligoAnalyzer applications. Potential interactions with forward and reverse primer pairs with a target start position of 300 bp or less were examined regardless of the presence of the internal Torch sequence. Matches were filtered for forward primers in the same direction as the target sequence, reverse primers in the opposite direction to the target sequence, and Torch sequences in the same direction as the target sequence. The results of BLAST using T. vaginalis and control oligonucleotides as queries against the human and GenBank databases were examined for targets that might be amplified and detected by the ACV / TV system. Among all datasets (bacteria, fungi, viruses, human) queried by BLAST, only one interaction of potential interest was identified: between HIV-1 (accession number AF254708) and oligonucleotide numbers 36 and 11. HIV-1 was tested in panel 11 of the cross-reactivity test (see below) and showed no signs of cross-reactivity or interference. Thus, amplification of HIV by these two oligos is minimal.

[0156] Example 11. Cross-reactivity test. T. vaginalis was added as a target for the ROX channel, and cross-reactivity against various organisms was evaluated with a quadruple assay panel. Multiplex amplification was performed as described above using the described T. vaginalis oligos. The panel and results are shown in Table 10-1. Five replicates of each panel were tested to determine if cross-reactivity occurred. (Note: Panels 10 and 12 are not listed because the target species was included.)

Table 11-1

Table 11-2

[0157] One replicate of Panel 7 was positive in the FAM channel. All other replicates were negative in all channels. The cross-reactivity against the organisms in Panel 7 was re-evaluated. When these organisms were retested, no cross-reactivity was observed and all replicates were negative. The false positive replicate found in Panel 7 was concluded to be due to a random contamination event.

[0158] Example 12. Interference in the ROX channel for T. vaginalis detection. Five replicates of the cross-reactivity panel were tested at 3 times the detection limit (0.003 cells / mL) in the presence of Trichomonas vaginalis. Multiplex amplification was performed as described. When the panel was present in the ROX channel, no interference was observed and all replicates were positive as expected. The control Torch (Cy5.5, RTF2) was effective for all replicates. The results showed that the T. vaginalis oligos were able to detect T. vaginalis in the presence of various organisms in Panels 1-9, 11, and 13 of the above examples.

Table 12-1

[0159] Five replicates of the cross-reactive panel were tested at three times the detection limit (0.003 cells / mL) in the presence of T. vaginalis. Multiphase amplification was performed as described. No interference was observed when the panel was present in the ROX channel and all replicates were positive as expected. The control Torch (Cy5.5, RTF2) was effective for all replicates. The results showed that the T. vaginalis oligo was able to detect T. vaginalis in the presence of various organisms in panels 1 - 9, 11, and 13 of the above examples.

[0160] Example 13. T. vaginalis Clinical Sample Testing Seventeen vaginal swab clinical specimens initially determined to be positive by the Aptima Trichomonas assay were cleanly tested by the Aptima CV / TV multiplex assay. Multiphase amplification was performed as described using the T. vaginalis oligo TCO SEQ ID NO: 3, NT7 primer SEQ ID NO: 15, T7 primer SEQ ID NO: 11, and Torch SEQ ID NO: 24. One replicate of each undiluted sample was taken for testing. 15 / 17 (88%) of the samples yielded valid results by the CV / TV multiplex assay and were all positive for T. vaginalis. 3 / 15 (20%) of the valid samples were positive for both Candida species and T. vaginalis. The invalid samples were determined to be invalid due to no signal in all channels and there were recorded instrument errors, which could be due to insufficient sample volume.

Table 13 - 1

[0161] Next, following the initial test, serial dilutions by STM were created and comparative tests were conducted against the Aptima CV / TV multiplex and Aptima Trichomonas Vaginalis IVD assays. Dilutions with STM in the range of 1:5 to 1:10,000 were performed on clinical samples according to the T time of the undiluted sample test. Samples 11207 and 13023, which were determined to be invalid from the undiluted sample test, were diluted 1:10. Each dilution was run using the CV / TV multiplex assay and retested with the Aptima Trichomonas Vaginalis assay. Previously invalid samples were valid when retested at a 1:10 dilution. All samples, including previously invalid samples, were consistent with the interpretation of the Aptima Trichomonas Vaginalis assay.

Table 13-2

[0162] Embodiment Embodiment 1. An amplification oligonucleotide for use in amplifying a T. vaginalis target nucleic acid sequence in a sample comprising a promoter primer containing 15 to 30 adjacent bases having at least 90% complementarity with the region of SEQ ID NO: 176 or its complement. Embodiment 2. The amplification oligonucleotide according to Embodiment 1, wherein the promoter primer comprises a 5' promoter sequence of T7 RNA polymerase. Embodiment 3. The amplification oligonucleotide according to Embodiment 2, wherein the promoter sequence of T7 RNA polymerase comprises SEQ ID NO: 65 or 66. Embodiment 4. The amplification oligonucleotide according to Embodiment 2, wherein the promoter primer comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 42, 43, 44, 45, 46, 47, or 48. Embodiment 5. The amplification oligonucleotide according to Embodiment 4, wherein the promoter primer comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12. Embodiment 6. A set of amplification oligonucleotides, comprising the amplification oligonucleotide according to any one of Embodiments 1 to 5 and one or more additional amplification oligonucleotides suitable for use in amplifying one or more additional target nucleic acids. Embodiment 7. Use or an amplification oligonucleotide in amplifying a T. vaginalis target nucleic acid sequence in a sample, the amplification oligonucleotide comprising a non-promoter primer containing 15 to 30 adjacent bases having at least 90% complementarity with the region of SEQ ID NO: 177 or its complement. Embodiment 8. The amplification oligonucleotide according to Embodiment 6, wherein the non-promoter primer comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 49, 50, 51, 52, 53, 54, or 55. Embodiment 9. The amplification oligonucleotide according to Embodiment 7, wherein the non-promoter primer comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19. Embodiment 10. A set of amplification oligonucleotides, comprising the non-promoter primer according to any one of Embodiments 7 to 9 and one or more additional non-promoter primers suitable for use in amplifying one or more additional target nucleic acids. Embodiment 11. A detection oligonucleotide for detecting a T. vaginalis target nucleic acid amplification product, the detection oligonucleotide comprising a nucleic acid sequence having at least 90% identity with SEQ ID NO: 56, 57, 58, 59, 60, 61, or 62. Embodiment 12. The detection oligonucleotide according to Embodiment 11, wherein the detection oligonucleotide is a three-dimensional configuration sensitive hybridization probe that generates a detectable signal when hybridized to an amplification product of a T. vaginalis target nucleic acid. Embodiment 13. The detection oligonucleotide according to Embodiment 12, wherein the detection oligonucleotide contains a fluorophore and optionally a quencher. Embodiment 14. The detection oligonucleotide according to Embodiment 13, wherein the detection oligonucleotide is a molecular torch. Embodiment 15. The detection oligonucleotide according to Embodiment 11, wherein the detection oligonucleotide contains a nucleic acid sequence having at least 90% identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28. Embodiment 16. A set of detection oligonucleotides, comprising the detection oligonucleotide according to any one of Embodiments 11 to 15 and one or more additional detection oligonucleotides suitable for use in detecting amplification products of one or more additional target nucleic acids. Embodiment 17. A target capture oligonucleotide (TCO) for use in capturing a T. vaginalis target nucleic acid in a sample, wherein the TCO comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 39, 40, or 41 and an immobilization capture probe binding region that binds to an immobilized capture probe. Embodiment 18. The TCO according to Embodiment 17, wherein the immobilization capture probe binding region comprises a nucleic acid sequence capable of stably hybridizing to an oligonucleotide that binds to the capture probe under assay conditions. Embodiment 19. The TCO according to Embodiment 18, wherein the TCO comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 1, 2, or 3. Embodiment 20. A set of TCOs, comprising the TCO according to any one of Embodiments 17 to 19 and one or more additional TCOs for use in capturing one or more additional target nucleic acids. Embodiment 21. A composition for detecting T. vaginalis in a sample, (a) a promoter primer comprising the amplification oligonucleotide according to any one of Embodiments 1 to 5, (b) a non-promoter primer comprising the amplification oligonucleotide according to any one of Embodiments 7 to 9, [[ID=**********]] (c) a detection oligonucleotide comprising the detection oligonucleotide according to any one of Embodiments 11 to 15, (d) optionally, a TCO comprising the target capture oligonucleotide (TCO) according to any one of Embodiments 17 to 19. Embodiment 22. The composition according to embodiment 21, wherein the promoter primer is present in the target capture mixture, the non-promoter primer is present in the first-phase amplification mixture, and the promoter primer and the detection oligonucleotide are present in the second-phase amplification mixture. Embodiment 23. The composition according to embodiment 22, wherein the target capture mixture further comprises TCO. Embodiment 24. The composition according to embodiment 22, wherein the first-phase amplification mixture contains one or more of reverse transcriptase, RNA polymerase, deoxyribonucleotide triphosphates, and ribonucleotide triphosphates. Embodiment 25. The composition according to any one of embodiments 21 to 24, further comprising an immobilized capture probe, wherein the immobilized capture probe contains a first binding pair member that binds to a second binding pair member present on TCO. Embodiment 26. The composition according to embodiment 25, wherein the immobilized capture probe is a magnetically attractable particle. Embodiment 27. The composition according to embodiment 22, wherein the first-phase amplification reaction mixture lacks the promoter primer. Embodiment 28. The target capture mixture contains one or more additional promoter primers, the first-phase amplification mixture contains one or more additional non-promoter primers, the second amplification mixture contains one or more additional further promoter primers and one or more detection oligonucleotides, and the one or more additional promoter primers, non-promoter primers, and detection oligonucleotides are suitable for the amplification and detection of species other than T. vaginalis. The composition according to embodiment 21. Embodiment 29. The method according to embodiment 24, wherein at least one of the species other than T. vaginalis is a Candida species. Embodiment 30. The composition according to embodiment 21, wherein TCO contains the nucleotide sequence of SEQ ID NO: 3, the T7 primer contains the nucleotide sequence of SEQ ID NO: 11, the NT7 primer contains the nucleotide sequence of SEQ ID NO: 15, and Torch contains the nucleotide sequence of SEQ ID NO: 24. Embodiment 31. The composition according to Embodiment 21, wherein the TCO contains the nucleotide sequence of SEQ ID NO: 3, the T7 primer contains the nucleotide sequence of SEQ ID NO: 4, the NT7 primer contains the nucleotide sequence of SEQ ID NO: 14, and Torch contains the nucleotide sequence of SEQ ID NO: 20. Embodiment 32. The composition according to Embodiment 21, wherein the TCO contains the nucleotide sequence of SEQ ID NO: 3, the T7 primer contains the nucleotide sequence of SEQ ID NO: 4, the NT7 primer contains the nucleotide sequence of SEQ ID NO: 14, and Torch contains the nucleotide sequence of SEQ ID NO: 21. Embodiment 33. The composition according to Embodiment 21, wherein the TCO contains the nucleotide sequence of SEQ ID NO: 3, the T7 primer contains the nucleotide sequence of SEQ ID NO: 4, the NT7 primer contains the nucleotide sequence of SEQ ID NO: 13, and Torch contains the nucleotide sequence of SEQ ID NO: 21. Embodiment 34. The composition according to Embodiment 21, wherein the TCO contains the nucleotide sequence of SEQ ID NO: 3, the T7 primer contains the nucleotide sequence of SEQ ID NO: 9, the NT7 primer contains the nucleotide sequence of SEQ ID NO: 15, and Torch contains the nucleotide sequence of SEQ ID NO: 23. Embodiment 35. The composition according to Embodiment 21, wherein the TCO contains the nucleotide sequence of SEQ ID NO: 2, the T7 primer contains the nucleotide sequence of SEQ ID NO: 4, the NT7 primer contains the nucleotide sequence of SEQ ID NO: 14, and Torch contains the nucleotide sequence of SEQ ID NO: 20. Embodiment 36. The composition according to Embodiment 21, wherein the TCO contains the nucleotide sequence of SEQ ID NO: 2, the T7 primer contains the nucleotide sequence of SEQ ID NO: 4, the NT7 primer contains the nucleotide sequence of SEQ ID NO: 14, and Torch contains the nucleotide sequence of SEQ ID NO: 21. Embodiment 37. The composition according to Embodiment 21, wherein the TCO contains the nucleotide sequence of SEQ ID NO: 2, the T7 primer contains the nucleotide sequence of SEQ ID NO: 4, the NT7 primer contains the nucleotide sequence of SEQ ID NO: 13, and Torch contains the nucleotide sequence of SEQ ID NO: 21. The composition according to embodiment 21, wherein the TCO comprises the nucleotide sequence of SEQ ID NO: 2, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 9, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 15, and Torch comprises the nucleotide sequence of SEQ ID NO: 23. The composition according to embodiment 21, wherein the TCO comprises the nucleotide sequence of SEQ ID NO: 2, the T7 primer comprises the nucleotide sequence of SEQ ID NO: 4, the NT7 primer comprises the nucleotide sequence of SEQ ID NO: 13, and Torch comprises the nucleotide sequence of SEQ ID NO: 20. Embodiment 40. A method for detecting T. vaginalis in a sample, (a) contacting the sample with a promoter primer under conditions that allow hybridization of the promoter primer to a first portion of the T. vaginalis target nucleic acid sequence, thereby generating a pre-amplification hybrid comprising the promoter primer and the target nucleic acid sequence, wherein the promoter primer comprises a nucleic acid sequence having at least 90% complementarity to the region of SEQ ID NO: 176 or its complement, generating; (b) isolating the pre-amplification hybrid by target capture on a solid support and subsequent washing to remove any promoter primers that did not hybridize to the first portion of the target nucleic acid sequence in step (a); (c) amplifying at least a portion of the target nucleic acid sequence of the pre-amplification hybrid isolated in step (b) in the first-phase substantially isothermal transcription-related amplification reaction under conditions that support its linear amplification but not its exponential amplification, thereby yielding a reaction mixture comprising a first amplification product, wherein the first-phase amplification reaction mixture comprises a non-promoter primer, the non-promoter is complementary to a portion of the extension product of the promoter primer, and comprises a nucleic acid sequence having at least 90% complementarity to the region of SEQ ID NO: 177 or its complement, wherein the first amplification product is not a template for nucleic acid synthesis during the first-phase substantially isothermal transcription-related amplification reaction, yielding; (d) Combining the reaction mixture containing the first amplification product with an additional promoter primer to generate a second-phase amplification reaction mixture, wherein the second-phase amplification reaction mixture further comprises a detection oligo; (e) In the second phase, performing a transcription-related amplification reaction substantially isothermal in the second-phase amplification reaction mixture to perform exponential amplification of the first amplification product, thereby synthesizing a second amplification product; (f) Detecting the synthesis of the second amplification product in the second-phase amplification reaction mixture using a detection oligonucleotide at regular time intervals; (g) Using the results of step (f) to quantify the target nucleic acid sequence in the sample. Embodiment 41. The method according to embodiment 40, wherein the promoter primer comprises a 5' promoter sequence of T7 RNA polymerase. Embodiment 42. The method according to embodiment 41, wherein the promoter sequence of T7 RNA polymerase comprises SEQ ID NO: 65 or 66. Embodiment 43. The method according to embodiment 41, wherein the promoter primer comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 42, 43, 44, 45, 46, 47, or 48. Embodiment 44. The method according to embodiment 43, wherein the promoter primer comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 4, 5, 6, 7, 8, 9, 10, 11, or 12. Embodiment 45. The method according to embodiment 40, wherein the non-promoter primer is enzymatically extended in the first-phase isothermal transcription-related amplification reaction. Embodiment 46. The method according to embodiment 45, wherein the non-promoter primer comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 49, 50, 5, 52, 53, 54, or 55. Embodiment 47. The method according to embodiment 46, wherein the non-promoter primer comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 13, 14, 15, 16, 17, 18, or 19. Embodiment 48. The method according to Embodiment 40, wherein isolating the pre-amplification hybrid comprises contacting the sample with a target capture oligonucleotide (TCO), and the pre-amplification hybrid comprises a target nucleic acid sequence hybridized to each of the TCO and the promoter primer. Embodiment 49. The method according to Embodiment 48, wherein the TCO comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 39, 40, or 41. Embodiment 50. The method according to Embodiment 48, wherein the TCO comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 1, 2, or 3. Embodiment 51. The method according to Embodiment 40, wherein the solid support comprises an immobilized capture probe. Embodiment 52. The method according to Embodiment 51, wherein the immobilized capture probe is a magnetically attractable particle. Embodiment 53. The method according to Embodiment 40, wherein each of the isothermal transcription-related amplification reactions in the first and second phases comprises an RNA polymerase and a reverse transcriptase, and the reverse transcriptase comprises an endogenous RNase H activity. Embodiment 54. The method according to Embodiment 40, wherein the amplification reaction mixture in the first phase lacks a free promoter primer. Embodiment 55. The method according to Embodiment 40, wherein the first amplification product in step (c) is a cDNA molecule having the same polarity as the target nucleic acid sequence in the sample, and the second amplification product in step (e) is an RNA molecule. Embodiment 56. The method according to Embodiment 40, wherein the detection oligonucleotide in step (d) is a three-dimensional configuration sensitive hybridization probe that generates a detectable signal when hybridized to the second amplification product. Embodiment 57. The method according to Embodiment 56, wherein the detection oligonucleotide in step (d) is a fluorescence-labeled sequence-specific hybridization probe. Embodiment 58. The method according to Embodiment 57, wherein the detection oligonucleotide contains a region having at least 90% complementarity with the region of SEQ ID NO: 178 or its complement. Embodiment 59. The method according to embodiment 58, wherein the detection oligonucleotide comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 56, 57, 58, 59, 60, 61, or 62. Embodiment 60. The method according to embodiment 59, wherein the detection oligonucleotide comprises a nucleic acid sequence having at least 90% identity with SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, or 28. Embodiment 61. The method according to embodiment 40, wherein step (g) comprises quantifying the target nucleic acid sequence in the sample using a calibration curve and the results from step (f). Embodiment 62. The method according to embodiment 40, wherein the method comprises two or more different promoter primers and two or more different non-promoter primers, and the two or more different promoter primers and the two or more different non-promoter primers amplify different target nucleic acids to generate two or more different amplification products. Embodiment 63. The method according to embodiment 62, further comprising detecting the two or more different amplification products using two or more different detection oligonucleotides. Embodiment 63. The method according to embodiment 62, wherein the two or more different target nucleic acids are from different species. Embodiment 64. The method according to embodiment 63, wherein the different species are Candida species.

Claims

1. A set of oligonucleotides for use in amplifying a T. vaginalis target nucleic acid sequence in a sample, comprising: (a) A promoter primer comprising a nucleic acid sequence having a length of 25 to 30 consecutive nucleotides and having a target-specific sequence comprising SEQ ID NO: 42, and having a promoter sequence of T7 RNA polymerase attached to its 5'-end, and a non-promoter primer comprising a nucleic acid having a length of 25 to 30 consecutive nucleotides and having a target-specific sequence comprising SEQ ID NO: 13; (b) A promoter primer comprising a nucleic acid sequence having a length of 25 to 30 consecutive nucleotides and having a target-specific sequence comprising SEQ ID NO: 42, and having a promoter sequence of T7 RNA polymerase attached to its 5'-end, and a non-promoter primer comprising a nucleic acid having a length of 20 to 30 consecutive nucleotides and having a target-specific sequence comprising SEQ ID NO: 14; (c) A promoter primer comprising a nucleic acid sequence having a length of 23 to 30 consecutive nucleotides and having a target-specific sequence comprising SEQ ID NO: 45, and having a promoter sequence of T7 RNA polymerase attached to its 5'-end, and a non-promoter primer comprising a nucleic acid having a length of 22 to 30 consecutive nucleotides and having a target-specific sequence comprising SEQ ID NO: 15, or (d) A promoter primer comprising a nucleic acid sequence having a length of 22 to 30 consecutive nucleotides and having a target-specific sequence comprising SEQ ID NO: 47, and having a promoter sequence of T7 RNA polymerase attached to its 5'-end, and a non-promoter primer comprising a nucleic acid having a length of 22 to 30 consecutive nucleotides and having a target-specific sequence comprising SEQ ID NO: 15; The set of oligonucleotides.

2. The set of oligonucleotides according to claim 1, wherein the promoter primer comprises a target-specific sequence consisting of the nucleotide sequence of SEQ ID NO: 42, 45, or 47.

3. The set of oligonucleotides according to claim 1 or 2, wherein the promoter sequence of the T7 RNA polymerase comprises SEQ ID NO: 65 or 66.

4. The set of oligonucleotides according to claim 3, wherein the promoter primer comprises a nucleic acid sequence of SEQ ID NO: 4, 9, or 11.

5. The set of oligonucleotides according to claim 4, wherein the promoter primer comprises a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 4, 9, or 11.

6. The set of oligonucleotides according to any one of claims 1 to 5, wherein the non-promoter primer comprises a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 13, 14, or 15.

7. The set of oligonucleotides according to any one of claims 1 to 6, further comprising one or more additional oligonucleotides suitable for use in amplifying one or more additional target nucleic acids.

8. The set of oligonucleotides according to any one of claims 1 to 7, further comprising a detection oligonucleotide for detecting a T. vaginalis target nucleic acid amplification product, wherein the detection oligonucleotide has a nucleic acid base length of 13 to 30 and comprises a target-specific sequence comprising the nucleotide sequence of SEQ ID NO: 56, 57, 58, 59, 60, 61, 62, or its complement, and the detection oligonucleotide contains a fluorophore, or a fluorophore and a quencher.

9. The set of oligonucleotides according to claim 8, wherein the detection oligonucleotide comprises a target-specific sequence consisting of the nucleotide sequence of SEQ ID NO: 56, 57, 58, 59, 60, 61, or 62.

10. The set of oligonucleotides according to claim 8 or 9, wherein the detection oligonucleotide comprises a nucleic acid sequence of SEQ ID NO: 20, 21, 22, 23, 25, 26, 27, or 28.

11. The set of oligonucleotides according to claim 10, wherein the detection oligonucleotide comprises a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 20, 21, 22, 23, 25, 26, 27, or 28.

12. The set of oligonucleotides according to any one of claims 7 to 11, further comprising one or more additional detection oligonucleotides suitable for use in detecting amplification products of one or more additional target nucleic acids.

13. Further comprising a target capture oligonucleotide (TCO) for use in capturing T. vaginalis target nucleic acid in a sample, (i) the TCO comprises a nucleic acid sequence having a target-specific sequence containing SEQ ID NO: 39, 40, or 41, and an immobilization capture probe binding region that binds to an immobilization capture probe, or (ii) the TCO comprises a nucleic acid sequence containing SEQ ID NO: 1, 2, or 3, the set of oligonucleotides according to any one of claims 1 to 12.

14. (i) the TCO comprises a target-specific sequence consisting of the nucleotide sequence of SEQ ID NO: 39, 40, or 41, or (ii) the TCO comprises a nucleic acid sequence consisting of the nucleotide sequence of SEQ ID NO: 1, 2, or 3, the set of oligonucleotides according to claim 13.

15. A kit or combination for detecting T. vaginalis in a sample, comprising the set of oligonucleotides according to any one of claims 1 to 14.

16. Further, the kit or combination according to claim 15, further comprising the target capture oligonucleotide (TCO) according to claim 13 or 14.

17. The kit or combination according to claim 15 or 16, wherein the promoter primer is present in a target capture mixture, the non-promoter primer is present in a first-phase amplification mixture, and the promoter primer and the detection oligonucleotide are present in a second-phase amplification mixture.

18. The target capture mixture contains one or more additional promoter primers, the first-phase amplification mixture contains one or more additional non-promoter primers, the second-phase amplification mixture contains one or more additional promoter primers and one or more detection oligonucleotides, and the one or more additional promoter primers, non-promoter primers, and detection oligonucleotides are suitable for the amplification and detection of species other than T. vaginalis. The kit or combination according to claim 17.

19. The kit or combination according to claim 18, wherein at least one of the species other than T. vaginalis is a Candida species.

20. A method for detecting T. vaginalis in a sample using the set of oligonucleotides according to claim 13 or 14, the method comprising (a)Under conditions that allow hybridization of the promoter primer to the first portion of the T. vaginalis target nucleic acid sequence, contacting the sample with the promoter primer, thereby generating a preamplification hybrid comprising the promoter primer and the target nucleic acid sequence; (b)Isolating the preamplification hybrid on a solid support by target capture, wherein target capture comprises contacting the sample with the target capture oligonucleotide (TCO), and the preamplification hybrid comprises the target nucleic acid sequence hybridized to each of the TCO and the promoter primer, isolating, and subsequently washing to remove any of the promoter primers that did not hybridize to the first portion of the target nucleic acid sequence in step (a); (c)In a first-phase isothermal transcription-related amplification reaction, amplifying at least a portion of the target nucleic acid sequence of the preamplification hybrid isolated in step (b) in a first-phase amplification reaction mixture under conditions that support its linear amplification but not its exponential amplification, thereby yielding a reaction mixture comprising a first amplification product, wherein the first-phase amplification reaction mixture comprises the non-promoter primer, wherein the first amplification product is not a template for nucleic acid synthesis during the first-phase isothermal transcription-related amplification reaction, yielding; (d)Combining the reaction mixture comprising the first amplification product with an additional promoter primer to generate a second-phase amplification reaction mixture, wherein the second-phase amplification reaction mixture further comprises the detection oligonucleotide, generating; (e)In a second phase, performing an isothermal transcription-related amplification reaction in the second-phase amplification reaction mixture to perform exponential amplification of the first amplification product, thereby synthesizing a second amplification product; (f)Detecting the synthesis of the second amplification product in the second-phase amplification reaction mixture using the detection oligonucleotide at regular time intervals; (g)Quantifying the target nucleic acid sequence in the sample using the results of step (f). A method comprising.

21. The method according to claim 20, wherein the non-promoter primer is enzymatically extended in the first-phase isothermal transcription-related amplification reaction.

22. The method according to claim 20 or 21, wherein the detection oligonucleotide in step (d) is a fluorescence-labeled sequence-specific hybridization probe comprising a nucleic acid sequence of SEQ ID NO: 20, 21, 22, 23, 25, 26, 27, or 28.

23. The method according to any one of claims 20 to 22, comprising two or more different promoter primers and two or more different non-promoter primers, wherein the two or more different promoter primers and the two or more different non-promoter primers amplify different target nucleic acids to generate two or more different amplification products.

24. The method according to claim 23, wherein the two or more different promoter primers and the two or more different non-promoter primers amplify T. vaginalis and Candida species.

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