Methods for detecting Trichomonas vaginalis

Abstract

The present invention provides compositions and methods for detecting Trichomonas vaginalis.

Description

1. Field of the Invention

[0001] Compositions and methods for detecting Trichomonas vaginalis are provided.

[0002] 2. Background

[0003] The protozoan Trichomonas vaginalis causes trichomoniasis, a common sexually transmitted infection that can infect both men and women. There are 7.4 million cases of trichomoniasis in the United States each year. Trichomoniasis infection can be symptomatic or asymptomatic. See, e.g., Ginocchio et al., J. Clin. Microbiol. 2012, 50: 2601-2608. In women, trichomoniasis is one of a range of conditions that include a vaginal discharge. See, e.g., Centers for Disease Control and Prevention (CDC). CDC fact sheet: trichomoniasis.2010. www.cdc.gov / std / trichomonas / STDFact-Trichomoniasis.htm. Symptoms in women may include genital itching, burning, redness, or pain, an unpleasant odor, discomfort with urination, or a thin, clear, white, yellow, or green discharge. See ibid. In men, trichomoniasis can cause nongonococcal urethritis (NGU). Symptoms in men may include itching or burning inside the penis, burning after ejaculation or urination, or discharge from the penis. See, e.g., Workowski et al., Centers for Disease Control and Prevention. Sexually transmitted disease treatment guidelines, 2010. MMWR 2010;59(RR-12):1-110; Centers for Disease Control and Prevention. Biosafety in Microbiological and Biomedical laboratories. www.cdc.gov / biosafety / publications / .

[0004] There is a need for improved methods of detecting Trichomonas vaginalis (TV). In particular, there is a need for highly specific, accurate and sensitive urine or swab-based diagnostic tests.

[0005] 3. Overview

[0006] In some embodiments, methods are provided for detecting the presence or absence of Trichomonas vaginalis (TV) in a sample from a subject. In some embodiments, methods are provided for determining whether a subject has a Trichomonas vaginalis (TV) infection. In some embodiments, the methods comprise detecting the presence or absence of a TV 40S ribosomal protein (Tv40Srp) gene or RNA in a sample from a subject.

[0007] In some embodiments, the subject has not been previously treated for TV infection. In some embodiments, the subject has been previously treated for TV infection. In some embodiments, the previous treatment included one or more doses of metronidazole or tinidazole. In some embodiments, the subject does not have any symptoms of TV infection. In some embodiments, the subject has more than one symptom of TV infection. In some embodiments, the subject has more than one symptom selected from vaginitis, urethritis, and cervicitis. In some embodiments, the subject is female and has more than one symptom selected from the following: genital itching, burning, redness, and / or pain; genital odor; urinary discomfort; and thin clear, white, yellow, or green discharge. In some embodiments, the subject is pregnant. In some embodiments, the subject is male and has more than one symptom selected from the following: itching and / or burning inside the penis; burning after ejaculation and / or urination; and penile discharge.

[0008] In some embodiments, the method includes detecting an endogenous control. In some embodiments, the endogenous control is a sample adequacy control. In some embodiments, the endogenous control is a single copy human gene. In some embodiments, the endogenous control is selected from HMBS, GAPDH, β-actin, and β-globin.

[0009] In some embodiments, the method includes detecting an exogenous control. In some embodiments, the exogenous control is a sample treatment control. In some embodiments, the exogenous control comprises a DNA sequence that is not expected to be present in the sample. In some embodiments, the exogenous control is a bacterial gene.

[0010] In some embodiments, the method comprises PCR. In some embodiments, the method comprises quantitative PCR. In some embodiments, the PCR reaction takes less than 2 hours, less than 1 hour, or less than 30 minutes from the initial denaturation step through the final extension step.

[0011] In some embodiments, the TV 40S ribosomal protein (Tv40Srp) gene comprises the sequence of SEQ ID NO: 4. In some embodiments, the method comprises contacting the nucleic acid from the sample with a first primer pair for detecting the TV 40S ribosomal protein (Tv40Srp) gene or RNA. In some embodiments, the method comprises contacting the nucleic acid from the sample with a second primer pair for detecting an endogenous control. In some embodiments, the method comprises contacting the nucleic acid from the sample with a third primer pair for detecting an exogenous control.

[0012] In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4. In some embodiments, the first primer and the second primer each independently comprise 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its complement. In some embodiments, the first primer pair comprises a first primer consisting of 15 to 30 nucleotides and a second primer consisting of 15 to 30 nucleotides. In some embodiments, the first primer pair comprises a first primer of SEQ ID NO: 1 and a second primer of SEQ ID NO: 2. In some embodiments, the first primer pair produces an amplicon that is 50 to 500 nucleotides in length, 50 to 400 nucleotides in length, 50 to 300 nucleotides in length, 50 to 200 nucleotides in length, 50 to 150 nucleotides in length, 100 to 300 nucleotides in length, 100 to 200 nucleotides in length, or 100 to 150 nucleotides in length.

[0013] In some embodiments, the method includes forming a Tv40Srp amplicon. In some embodiments, the method includes contacting the Tv40Srp amplicon with a first probe capable of selectively hybridizing to the Tv40Srp amplicon. In some embodiments, the first probe comprises a detectable label. In some embodiments, the first probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the first probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the first probe comprises 0, 1, or 2 mismatches compared to SEQ ID NO:4 or its complement or compared to SEQ ID NO:5 or its complement. In some embodiments, the first probe consists of 15 to 30 nucleotides. In some embodiments, the first probe has the sequence of SEQ ID NO:3.

[0014] In some embodiments, described method comprises forming endogenous control amplicon and / or exogenous control amplicon.In some embodiments, described method comprises endogenous control amplicon is contacted with the second probe that can selectively hybridize with endogenous control amplicon and / or exogenous control amplicon is contacted with the 3rd probe that can selectively hybridize with exogenous control amplicon.In some embodiments, the second probe and the 3rd probe each comprise detectable label, wherein said detectable label can be identical or different.In some embodiments, the detectable label of the second and the 3rd probe is different from the detectable label of the first probe in detection.In some embodiments, described method comprises detecting Tv40Srp gene or RNA, endogenous control and exogenous control in single multiple reaction.

[0015] In some embodiments, the sample is selected from the group consisting of a urine sample, an endocervical swab sample, a vaginal swab sample, and a urethral swab sample.

[0016] In some embodiments, a composition is provided comprising a first primer pair for detecting the Trichomonas vaginalis 40S ribosomal protein (Tv40Srp) gene or RNA. In some embodiments, the composition comprises a second primer pair for detecting an endogenous control. In some embodiments, the endogenous control is a sample adequacy control. In some embodiments, the endogenous control is selected from HMBS, GAPDH, β-actin, and β-globin. In some embodiments, the composition comprises a third primer pair for detecting an exogenous control. In some embodiments, the exogenous control is a sample treatment control. In some embodiments, the exogenous control is a bacterial gene.

[0017] In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4. In some embodiments, the first primer and the second primer each independently comprise 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its complement. In some embodiments, the first primer pair comprises a first primer consisting of 15 to 30 nucleotides and a second primer consisting of 15 to 30 nucleotides. In some embodiments, the first primer pair comprises a first primer of SEQ ID NO: 1 and a second primer of SEQ ID NO:2.

[0018] In some embodiments, the composition comprises a first probe capable of selectively hybridizing to the Tv40Srp amplicon produced by the first primer pair. In some embodiments, the first probe comprises a detectable label. In some embodiments, the first probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the first probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO:4 or SEQ ID NO:5. In some embodiments, the first probe comprises 0, 1, or 2 mismatches compared to SEQ ID NO:4 or its complement or compared to SEQ ID NO:5 or its complement. In some embodiments, the first probe consists of 15 to 30 nucleotides. In some embodiments, the first probe has the sequence of SEQ ID NO:3. In some embodiments, the Tv40Srp amplicon has the sequence of SEQ ID NO:5.

[0019] In some embodiments, the composition comprises a second probe capable of selectively hybridizing to an endogenous control amplicon produced by the second primer pair. In some embodiments, the endogenous control is a sample adequacy control. In some embodiments, the endogenous control is selected from HMBS, GAPDH, beta actin, and beta globin. In some embodiments, the composition comprises a third probe capable of selectively hybridizing to an exogenous control amplicon produced by a third primer pair. In some embodiments, the exogenous control is a sample treatment control. In some embodiments, the exogenous control comprises a DNA sequence that is not expected to be present in the sample. In some embodiments, the exogenous control is bacterial DNA.

[0020] In some embodiments, the composition is a lyophilized composition. In some embodiments, the composition is in solution. In some embodiments, the composition comprises nucleic acid from a sample of a subject being tested for the presence or absence of Trichomonas vaginalis.

[0021] In some embodiments, a test kit is provided, the test kit comprising a first primer pair for detecting the Trichomonas vaginalis 40S ribosomal protein (Tv40Srp) gene or RNA. In some embodiments, the test kit comprises a second primer pair for detecting an endogenous control, wherein the primer pair for detecting Tv40Srp and the second primer pair are in the same or different compositions in the test kit. In some embodiments, the endogenous control is a sample adequacy control. In some embodiments, the endogenous control is selected from HMBS, GAPDH, β-actin and β-globin. In some embodiments, the test kit comprises a third primer pair for detecting an exogenous control, wherein the third primer pair and the primer pair for detecting Tv40Srp and the second primer pair are in the same or different compositions. In some embodiments, the exogenous control is a sample processing control. In some embodiments, the exogenous control comprises a DNA sequence that is not expected to be present in the sample. In some embodiments, the exogenous control is a bacterial gene.

[0022] In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4. In some embodiments, the first primer and the second primer each independently comprise 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its complement. In some embodiments, the first primer pair comprises a first primer consisting of 15 to 30 nucleotides and a second primer consisting of 15 to 30 nucleotides. In some embodiments, the first primer pair comprises a first primer of SEQ ID NO: 1 and a second primer of SEQ ID NO:2.

[0023] In some embodiments, the kit comprises a first probe capable of selectively hybridizing to the Tv40Srp amplicon produced by a first primer pair, wherein the first probe and one or more primer pairs are in the same or different compositions. In some embodiments, the first probe comprises a detectable label. In some embodiments, the first probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the first probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4 or SEQ ID NO: 5. In some embodiments, the first probe comprises 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its complement or compared to SEQ ID NO: 5 or its complement. In some embodiments, the first probe consists of 15 to 30 nucleotides. In some embodiments, the first probe has the sequence of SEQ ID NO: 3. In some embodiments, the Tv40Srp amplicon has the sequence of SEQ ID NO:5.

[0024] In some embodiments, the kit comprises a second probe capable of selectively hybridizing to an endogenous control amplicon produced by a second primer pair, wherein the second probe is in the same or different composition as one or more of the primer pairs. In some embodiments, the kit comprises a third probe capable of selectively hybridizing to an exogenous control amplicon produced by a third primer pair, wherein the third probe is in the same or different composition as one or more of the primer pairs.

[0025] In some embodiments, the kit comprises dNTPs and / or a thermostable polymerase.In some embodiments, the kit comprises more than one lyophilized composition.

[0026] In some embodiments, a primer is provided, wherein the primer consists of the sequence of SEQ ID NO: 1, wherein the primer comprises at least one modified nucleotide. In some embodiments, a primer is provided, wherein the primer consists of the sequence of SEQ ID NO: 2, wherein the primer comprises at least one modified nucleotide. In some embodiments, a probe is provided, wherein the probe consists of the sequence of SEQ ID NO: 3, wherein the probe comprises at least one modified nucleotide and / or a detectable label. In some embodiments, the probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the probe is a fluorescence resonance energy transfer (FRET) probe. In some embodiments, the probe comprises at least one modified nucleotide.

[0027] In some embodiments, a composition is provided, wherein the composition comprises a first primer consisting of a sequence of SEQ ID NO: 2 and a second primer consisting of a sequence of SEQ ID NO: 3, wherein the first primer and the second primer each comprise at least one modified nucleotide. In some embodiments, the composition comprises a probe consisting of a sequence of SEQ ID NO: 3, wherein the probe comprises at least one modified nucleotide and / or a detectable label. In some embodiments, the probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the probe is a fluorescence resonance energy transfer (FRET) probe. In some embodiments, the probe comprises at least one modified nucleotide. In some embodiments, the composition is a lyophilized composition. In some embodiments, the composition is in solution. In some embodiments, the composition comprises nucleic acid from a sample of a subject.

[0028] Further embodiments and details of the invention are described below.

[0029] 4. Detailed description

[0030] 4.1 Definition

[0031] To aid in understanding the present invention, a number of terms and expressions are defined below:

[0032] As used herein, the terms "detect," "detecting," or "detection" may describe the general act of finding or understanding or the specific observation of a detection-labeled composition.

[0033] As used herein, the term "detectably distinct" refers to a set of labels (eg, dyes) that can be simultaneously detected and distinguished.

[0034] As used herein, the terms "patient" and "subject" are used interchangeably to refer to a human. In some embodiments, the methods described herein can be used on samples from non-human animals.

[0035] "Trichomonas vaginalis" refers to the protozoan that causes trichomoniasis, a common sexually transmitted infection that can infect both men and women. Trichomoniasis can be symptomatic or asymptomatic. Symptoms of trichomoniasis include, but are not limited to, vaginitis, urethritis, and cervicitis. Symptoms in women include, but are not limited to, genital itching, burning, redness, or pain, an unpleasant odor, discomfort with urination, or a clear, white, yellow, or green discharge. Symptoms in men include, but are not limited to, itching or burning inside the penis, burning after ejaculation or urination, or discharge from the penis.

[0036] As used herein, the terms "oligonucleotide," "polynucleotide," "nucleic acid molecule," and the like refer to nucleic acid molecules, including, but not limited to, DNA or RNA. The term encompasses sequences comprising any known base analogs of DNA and RNA, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-Methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosine, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, methyl uracil-5-oxyacetate, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, methyl N-uracil-5-oxyacetate, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0037] As used herein, the term "oligonucleotide" refers to a single-stranded polynucleotide having less than 500 nucleotides. In some embodiments, an oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to 75, or 12 to 50 nucleotides in length. An oligonucleotide can be indicated by its length, for example, a 24-residue oligonucleotide can be referred to as a "24-mer (24 aggressiveness)".

[0038] As used herein, the term is "complementary" to the target gene (or its target region), and the "complementarity" percentage of the probe sequence to the target gene sequence is the "identity" percentage to the target gene sequence or to the reverse complement of the target gene sequence. In the determination of the "complementarity" degree between the probe (or its region) and the target gene (such as those disclosed herein) used in the compositions described herein, the degree of "complementarity" is expressed as the percentage identity between the probe sequence (or its region) and the target gene sequence or the reverse complement of the target gene sequence that is optimally aligned with it. Percentage is calculated by counting the number of identical alignment bases between the two sequences, divided by the total number of continuous nucleotides in the probe, and multiplied by 100. When using the term "complementary", the test oligonucleotide is at least 90% complementary to the target molecule, unless otherwise stated. In some embodiments, the test oligonucleotide is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule.

[0039] As used herein, "primer" or "probe" refers to an oligonucleotide comprising a region complementary to a sequence of at least 8 consecutive nucleotides of a target nucleic acid molecule, such as a DNA (e.g., a target gene) or mRNA (or a DNA reverse transcribed from an mRNA). In some embodiments, a primer or probe comprises a region complementary to a sequence of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of a target molecule. When a primer or probe comprises a region that is "complementary to at least x consecutive nucleotides of a target molecule," the primer or probe is at least 95% complementary to at least x consecutive nucleotides of the target molecule. In some embodiments, the primer or probe is at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule.

[0040] The term "nucleic acid amplification" includes any method by which at least a portion of at least one target nucleic acid is replicated (typically in a template-dependent manner), including but not limited to a wide range of techniques for amplifying nucleic acid sequences (linearly or exponentially). Exemplary methods for performing the amplification step include polymerase chain reaction (PCR), ligase chain reaction (LCR), ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLPA), ligation followed by Q-replicase amplification, primer extension, strand displacement amplification (SDA), hyperbranched strand displacement amplification, multiple displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), and the like, including various versions and combinations thereof, such as, but not limited to, OLA / PCR, PCR / OLA, LDR / PCR, PCR / PCR / LDR, PCR / LDR, LCR / PCR, PCR / LCR (also known as combined chain reaction—CCR), digital amplification, and the like. Descriptions of such techniques can be found in, among other sources, Ausbel et al.; PCR Primer: A Laboratory Manual, Diffenbach, ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book, Chang Bioscience (2002); Msuih et al., J. Clin. Micro. 34:501-07 (1996); The Nucleic Acid Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, NJ (2002); Abramson et al., Curr Opin Biotechnol. 1993 Feb.; 4(1):41-7, U.S. Patent No. 6,027,998; U.S. Patent No. 6,605,451, Barany et al., PCT Publication No. WO 97 / 31256; Wenz et al., PCT Publication No. WO 01 / 92579; Day et al., Genomics, 29(1):152-162 (1995), Ehrlich et al., Science 252:1643-50 (1991); Innis et al., PCR Protocols: A Guide to methods and Applications, Academic Press (1990); Favis et al., Nature Biotechnology 18:561-64 (2000); and Rabenau et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium on Human Identification, 1995 (available at the World Wide Web at promega.com / geneticidproc / ussymp6proc / blegrad.html); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93 (1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al., Nucl. Acid Res. 27:e40i-viii (1999); Dean et al., Proc Natl Acad Sci USA 99:5261-66 (2002); Barany and Gelfand, Gene 109:1-11 (1991); Walker et al., Nucl. Acid Res. 20:1691-96 (1992); Polstra et al., BMC Inf. Dis. 2:18- (2002); Lage et al., Genome Res. 2003 Feb;13(2):294-307, and Landegren et al., Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol Diagn. 2002 Nov.; 2(6): 542-8., Cook et al., J Microbiol Methods. 2003 May; 53(2): 165-74, Schweitzer et al., Curr Opin Biotechnol. 2001 Feb; 12(1): 21-7, U.S. Patent No. 5,830,711, U.S. Patent No. 6,027,889, U.S. Patent No. 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.

[0041] In some embodiments, amplification comprises at least one cycle of the following sequential steps: annealing at least one primer to a complementary or sequentially complementary sequence in at least one target nucleic acid; synthesizing at least one strand of nucleotides in a template-dependent manner using a polymerase; and denaturing the newly formed nucleic acid duplex to separate the strands. The cycles may or may not be repeated. Amplification may include thermal cycling or may be performed under isothermal conditions.

[0042] Unless otherwise indicated, the term "hybridization" as used herein means "specific hybridization," which is the preferential binding, duplexing, or hybridization of nucleic acid molecules to a particular nucleotide sequence, in some embodiments, under stringent conditions. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target sequence and will hybridize to a lesser extent, or not at all, to other sequences. In the context of nucleic acid hybridization (e.g., in array, Southern blot, or Northern blot hybridization), "stringent hybridization" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. An extensive guide to nucleic acid hybridization is found, for example, in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology--hybridization with Nucleic Acid Probes Part 1, Chapter 2, "Overview of principles of hyridization and the straegy of nucleic acid probes assays," Elsevier, NY ("Tijssen"). Typically, highly stringent hybridization and wash conditions for filter paper hybridization are selected to be about 5°C below the thermal melting point of the specific sequence at a defined ionic strength and pH. m The temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are chosen to be equal to the T for a specific probe. mThe dependence of hybridization stringency on buffer composition, temperature, and probe length is known to those skilled in the art (see, e.g., Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd ed.) Vols. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY).

[0043] As used herein, " sample " includes urine samples (including samples derived from urine samples), swabs of the cervix, and vaginal swabs collected by patients, as well as other types of human samples. In some embodiments, the urine sample is a "first catch" urine sample, which is a sample collected when the subject first begins to urinate. As used herein, urine samples include, but are not limited to, whole urine, a sample comprising cells from a urine sample, a sample comprising a cell pellet obtained by centrifuging a urine sample, a sample comprising cells separated by filtering a urine sample, etc. In some embodiments, the urine sample includes a buffer, such as a preservative. In some embodiments, the sample is a human sample other than a urine sample, such as an endocervical swab or a vaginal swab, including vaginal swabs collected by patients, and urethral swabs. In some embodiments, the swab sample includes a buffer, such as a preservative.

[0044] As used herein, an "endogenous control" refers to a portion naturally present in a sample for detection. In some embodiments, the endogenous control is a "sample adequacy control" (SAC), which can be used to determine whether there is enough sample for the assay, or whether the sample contains enough biological material, such as cells. In some embodiments, the SAC is a single-copy human gene. In some embodiments, an endogenous control, such as a SAC, is selected that can be detected in the same manner as the target gene, and, in some embodiments, detected simultaneously with the target gene.

[0045] As used herein, "exogenous control" refers to a part added to a sample or assay, such as a "sample processing control" (SPC). In some embodiments, an exogenous control includes an assay reagent. An exogenous control is typically selected to be expected not to be present in a sample for detection, or to be present in a sample at very low levels so that the amount of the part naturally present in the sample is undetectable or can be detected at a level far lower than the amount added to the sample as an exogenous control. In some embodiments, an exogenous control comprises a nucleotide sequence that is expected not to be present in the sample type for detecting the target gene. In some embodiments, an exogenous control comprises a nucleotide sequence that is known to be absent in the species from which the sample is collected. In some embodiments, an exogenous control comprises a nucleotide sequence from a species different from that from which the sample is collected. In some embodiments, an exogenous control comprises a nucleotide sequence that is known not to be present in any species. In some embodiments, an exogenous control that can be detected in the same manner as detecting the target gene is selected, and, in some embodiments, detected simultaneously with the target gene. In some embodiments, an exogenous control is bacterial DNA. In some embodiments, bacteria are species that are not expected to be found in the test sample type.

[0046] In the sequences herein, "U" and "T" are used interchangeably, whereby the two letters represent a uracil or a thymine at that position. From the context and / or intended use, one skilled in the art will understand whether a uracil or a thymine is intended and / or should be used at that position in the sequence. For example, one skilled in the art will understand that natural RNA molecules typically include uracil, while natural DNA molecules typically include thymine. Thus, where an RNA sequence includes a "T," one skilled in the art will understand that the position in the natural RNA is likely to be a uracil.

[0047] In the present disclosure, "a sequence selected from..." includes "a sequence selected from..." and "more than one sequence selected from..." Therefore, when "a sequence selected from..." is used, it is understood that one or more than one of the listed sequences can be selected.

[0048] 4.2 Detection of Trichomonas vaginalis

[0049] The present inventors have developed an assay for detecting Trichomonas vaginalis (TV). In some embodiments, the assay comprises detecting the TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, the assay comprises detecting RNA transcribed from the TV 40S ribosomal protein (Tv40Srp) gene. This assay relies on the polymerase chain reaction (PCR) and can be performed in a sequential automated manner using commercially available nucleic acid amplification systems. Exemplary non-limiting nucleic acid amplification systems that can be used to perform the methods of the invention include system, Infinity system, and Smartcycler system (Cepheid, Sunnyvale, CA). This assay uses automated systems, e.g., The system can be completed in 3 hours, and in some embodiments, in 2 hours.

[0050] 4.2.1 General approach

[0051] Compositions and methods for detecting Trichomonas vaginalis (TV) are provided. In some embodiments, the method comprises detecting the TV 40S ribosomal protein (Tv40Srp) gene.

[0052] In some embodiments, the method of detecting Trichomonas vaginalis (TV) in a subject comprises detecting the presence of the TV 40S ribosomal protein (Tv40Srp) gene in a sample from the subject. In some embodiments, the sample is selected from a urine sample, a swab of the cervix, and a vaginal swab. In some embodiments, the urine sample is a first-time urine sample.

[0053] In some embodiments, the method for detecting TV further comprises detecting at least one endogenous control, such as a sample adequacy control (SAC). In some embodiments, the method for detecting TV further comprises detecting at least one exogenous control, such as a sample processing control (SPC). In some embodiments, the method for detecting TV further comprises detecting at least one endogenous control and at least one exogenous control.

[0054] In some embodiments, the method for detecting TV comprises detecting the TV 40S ribosomal protein (Tv40Srp) gene in the sample. In some embodiments, the method for detecting TV further comprises detecting a sample adequacy control (SAC), such as a single copy human gene. In some embodiments, the method for detecting TV further comprises detecting a sample treatment control (SPC), such as exogenously added bacterial DNA. In some embodiments, the method for detecting TV further comprises detecting SAC and SPC.

[0055] In this disclosure, for convenience, the term "target gene" is used to refer to the TV 40S ribosomal protein (Tv40Srp) gene, and also refers to exogenous and / or endogenous controls. Therefore, it is understood that when discussing a target gene, the discussion is specifically intended to include the TV 40S ribosomal protein (Tv40Srp) gene, one or more endogenous controls (e.g., SAC), and one or more exogenous controls (e.g., SPC).

[0056] In some embodiments, the presence of the TV 40S ribosomal protein (Tv40Srp) gene is detected in a urine sample. In some embodiments, the target gene is detected in a urine sample to which a buffer (e.g., a preservative) has been added. In some embodiments, the buffer is added to the urine sample at a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 buffer:urine. In some embodiments, the presence of the TV 40S ribosomal protein (Tv40Srp) gene is detected in an endocervical swab sample or a vaginal swab sample. In some embodiments, the vaginal swab is a patient-collected vaginal swab. In some embodiments, the target gene is detected in an endocervical swab sample or a vaginal swab sample placed in a buffer (e.g., a preservative). In some embodiments, the swab is placed in 1 mL, 2 mL, or 2.5 mL of buffer.

[0057] In some embodiments, detection of the TV 40S ribosomal protein (Tv40Srp) gene in a sample from a subject indicates the presence of Trichomonas vaginalis in the subject. In some embodiments, detection is performed quantitatively. In other embodiments, detection is performed qualitatively. In some embodiments, detecting the target gene comprises forming a complex comprising a polynucleotide and a nucleic acid selected from the target gene, a DNA amplicon of the target gene, and a complement of the target gene. In some embodiments, detecting the target gene comprises PCR. In some embodiments, detecting the target gene comprises quantitative PCR or real-time PCR. In some embodiments, a sample adequacy control (SAC) and / or a sample treatment control (SPC) are detected in the same assay as the target gene. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is detected, TV is considered to be detected, even if SPC and / or SAC are not detected in the assay. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is not detected, TV is considered to be undetected only when SPC and SAC are also detected in the assay.

[0058] In some embodiments, the presence of the TV 40S ribosomal protein (Tv40Srp) gene is measured in samples collected from the subject more than once to monitor the treatment of TV infection in the subject. Treatment includes, but is not limited to, single or multiple doses of metronidazole or tinidazole. In some embodiments, by detecting the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene at regular or semi-regular intervals, the recurrence of TV in a subject with a history of TV infection is monitored. In some such embodiments, the patient is monitored by detecting the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene at least once a month, at least once every two months, at least once every three months, at least once every four months, at least once every five months, at least once every six months, at least once every nine months, at least once a year, or at least once every two years.

[0059] In some embodiments, the assay can be used as part of a subject's routine and / or preventive health care. That is, in some embodiments, the assay can be used to test individuals for TV infection, regardless of whether the individual displays symptoms of TV infection or has a history of TV infection. In some embodiments, the assay is used to detect TV infection in subjects who are pregnant and / or attempting pregnancy. In some cases, pregnant women with TV are more likely to experience premature birth and / or have low birth weight babies (less than 5.5 pounds).

[0060] In some embodiments, the sample to be tested is a urine sample (e.g., a first urine sample), or is derived from a urine sample. In some embodiments, a buffer (e.g., a preservative) is added to the urine sample. In some embodiments, the buffer is added to the urine sample within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours of collecting the sample.

[0061] In some embodiments, the sample to be tested is an endocervical swab sample or a vaginal swab sample. In some embodiments, the swab is placed in a buffer. In some embodiments, the swab is immediately placed in a buffer. In some embodiments, in some embodiments, the swab is placed in a buffer within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours of collecting the sample.

[0062] In some embodiments, less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, less than 1 ml, or less than 0.75 ml of urine is used in the method. In some embodiments, 0.1 ml to 1 ml of urine is used in the method.

[0063] In some embodiments, the sample to be tested is another body fluid, such as blood, sputum, mucus, saliva, vaginal or penile discharge, semen, etc.

[0064] In some embodiments, the clinical sample to be tested is fresh (i.e., not frozen). In other embodiments, the sample is a frozen specimen. In some embodiments, the sample is a tissue sample, such as a formalin-fixed wax-embedded sample. In some embodiments, the sample is a liquid cytology sample.

[0065] In some embodiments, the sample to be tested is obtained from an individual with one or more symptoms of TV infection. Non-limiting exemplary symptoms of TV infection include vaginitis, urethritis, and cervicitis; in women: genital itching, burning, redness, or pain, an unpleasant odor, urinary discomfort, and a clear, white, yellow, or green discharge; and in men: itching or burning inside the penis, burning after ejaculation or urination, and penile discharge. In some embodiments, the sample to be tested is obtained from an individual previously diagnosed with TV infection. In some such embodiments, the individual is monitored for recurrence of TV infection.

[0066] In some embodiments, the methods described herein can be used for routine screening of healthy individuals who do not have risk factors. In some embodiments, the methods described herein are used, for example, to screen asymptomatic individuals in routine or preventive healthcare. In some embodiments, the methods described herein are used to screen women who are pregnant or attempting to become pregnant.

[0067] In some embodiments, the methods described herein can be used to assess the effectiveness of a treatment for a TV infection in a patient.

[0068] In some embodiments, the use of the TV 40S ribosomal protein (Tv40Srp) gene for detecting TV infection is provided. In some embodiments, the use of the TV 40S ribosomal protein (Tv40Srp) gene for monitoring recurrence of TV infection is provided.

[0069] In any of the embodiments described herein, the TV 40S ribosomal protein (Tv40Srp) gene can be detected in the same assay reaction as a sample processing control (SPC) and / or a sample adequacy control (SAC).

[0070] In some embodiments, a method for helping to detect TV infection in a subject is provided. The method includes detecting the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene in a sample from a subject. In some embodiments, information about the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene in a sample from a subject is communicated to a medical practitioner. As used herein, "medical practitioner" refers to an individual or entity that diagnoses and / or treats a patient, such as a hospital, clinic, doctor's office, doctor, nurse, or any of the aforementioned entities or individual's institutions. In some embodiments, detecting the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene is performed in a laboratory that receives a sample from a medical practitioner or a medical practitioner's institution. The laboratory detects by any method, including those described herein, and subsequently communicates the result to the medical practitioner. When provided to a medical practitioner by any means, the result as used herein is "communicated." In some embodiments, the communication can be oral or written, can be by telephone, in person, email or other courier, or can be communicated by storing the information directly in, for example, a database accessible to the medical practitioner, including databases not controlled by the medical practitioner. In some embodiments, the information is maintained in electronic form. In some embodiments, the information can be stored in a memory or other computer-readable medium, such as RAM, ROM, EEPROM, flash memory, computer chip, digital video disc (DVD), compact disks (CDs), hard disk drive (HDD), magnetic tape, etc.

[0071] In some embodiments, a method for detecting TV is provided. In some embodiments, a method for diagnosing TV infection is provided. In some embodiments, the method includes obtaining a sample from a subject and providing the sample to a laboratory for detecting the TV 40S ribosomal protein (Tv40Srp) gene in the sample. In some embodiments, the method also includes receiving information from the laboratory indicating the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene in the sample. As used herein, a "laboratory" is any device that detects a target gene in a sample by any method (including the methods described herein) and communicates the results to a medical practitioner. In some embodiments, the laboratory is under the control of a medical practitioner. In some embodiments, the laboratory is not under the control of a medical practitioner.

[0072] When a laboratory communicates the results of detecting the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene to a medical practitioner, in some embodiments, the laboratory indicates whether the TV 40S ribosomal protein (Tv40Srp) gene was detected in the sample. In some embodiments, the laboratory indicates whether the sample contains Trichomonas vaginalis (TV) by indicating, for example, "TV positive" or "TV negative" or "TV present" or "TV absent."

[0073] As used herein, when a method relates to detecting TV, determining the presence of TV, monitoring TV, and / or diagnosing TV infection, the method includes activities in which the steps of the method are performed, but the result for the presence of TV is negative. That is, detecting, determining, monitoring, and diagnosing TV or TV infection include instances in which the method is performed that result in either a positive or negative result.

[0074] In some embodiments, at least one endogenous control (eg, SAC) and / or at least one exogenous control (eg, SPC) are detected simultaneously with the TV 40S ribosomal protein (Tv40Srp) gene in a single reaction.

[0075] 4.2.2 Exemplary Controls

[0076] In some embodiments, the assay described herein includes detecting the TV 40S ribosomal protein (Tv40Srp) gene and at least one endogenous control. In some embodiments, the endogenous control is a sample adequacy control (SAC). In some such embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is not detected in the sample and the SAC is not detected in the sample, the assay result is considered to be "invalid" because the sample may not be sufficient. Without intending to be limited by any particular theory, an insufficient sample may be too dilute, contain too little cell material, contain assay inhibitors, etc. In some embodiments, failure to detect SAC can indicate that the assay reaction has failed. In some embodiments, the endogenous control (such as SAC) is a single-copy human gene. Non-limiting exemplary SACs include human hydroxymethyl-bilane synthase (HMBS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, β2-microglobin, cyclooxygenase 1, hypoxanthine phosphoribosyltransferase, porphobilinogen deaminase, and transferrin receptor.

[0077] In some embodiments, the assays described herein include detecting the TV 40S ribosomal protein (Tv40Srp) gene and at least one exogenous control. In some embodiments, the exogenous control is a sample processing control (SPC). In some such embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is not detected in the sample and the SPC is not detected in the sample, the assay result is considered to be "invalid" because there may be errors in the sample processing, including but not limited to, assay failure. Non-limiting exemplary errors in sample processing include insufficient sample processing, the presence of assay inhibitors, damaged reagents, and the like. In some embodiments, an exogenous control (such as an SPC) is added to the sample. In some embodiments, an exogenous control (such as an SPC) is added during the assay, such as using more than one buffer or reagent. In some embodiments, when using When the system is in use, SPC is included in In some embodiments, an exogenous control (e.g., an SPC) is a DNA sequence that is not expected to be present in the sample being assayed. Non-limiting exemplary SPCs include bacterial genes that are not expected to be present in the sample being assayed.

[0078] In some embodiments, when detecting the TV 40S ribosomal protein (Tv40Srp) gene in a sample, an endogenous control and / or an exogenous control are detected simultaneously, such as in the same assay. In some embodiments, the assay comprises reagents for simultaneously detecting the TV 40S ribosomal protein (Tv40Srp) gene, an exogenous control, and an endogenous control in the same assay reaction. In some such embodiments, for example, the assay reaction comprises a primer set for amplifying the TV 40S ribosomal protein (Tv40Srp) gene, a primer set for amplifying the endogenous control, and a primer set for amplifying the exogenous control, and a labeled probe (such as, for example, probe).

[0079] 4.2.3 Exemplary Sample Preparation

[0080] 4.2.3.1 Exemplary Buffers

[0081] In some embodiments, a buffer is added to the urine sample. In some embodiments, the buffer is added (e.g., empty) within one hour, two hours, three hours, or six hours of the time the urine sample is collected. In some embodiments, the buffer is added to the urine sample within one hour, two hours, three hours, or six hours before the sample is analyzed by the methods described herein.

[0082] In some embodiments, the swab sample is placed in a buffer. In some embodiments, the swab sample is placed in a buffer within one hour, two hours, three hours, or six hours of the time the swab sample is collected. In some embodiments, the swab sample is placed in a buffer within one hour, two hours, three hours, or six hours before the sample is analyzed by the methods described herein.

[0083] Non-limiting exemplary commercial buffers include PreservCyt (Hologic, Bedford, MA), SurePath (BD, Franklin Lakes, NJ), and CyMol (Copan Diagnostics, Murrietta, CA).

[0084] 4.2.3.2 Exemplary DNA Preparation

[0085] Sample DNA can be prepared by any appropriate method. In some embodiments, by contacting the sample with lysis buffer and DNA is bound to a DNA binding substrate, such as glass or silica substrates to prepare target DNA. The binding substrate can have any suitable form, such as particles, porous solids or film forms. For example, support can include hydroxycellulose, glass fiber, cellulose, nitrocellulose, zirconium hydroxide, titanium oxide (IV), silicon dioxide, zirconium silicate or silicon dioxide particles (for example, referring to U.S. Patent number 5,234,809). Many such DNA binding substrates are known in the art.

[0086] In some embodiments, DNA is detected in a lysate without first isolating or separating the DNA. In some embodiments, the sample is subjected to a lysis step to release the DNA. Non-limiting exemplary lysis methods include ultrasound (e.g., 2-15 seconds, 8-18 μm, at 36 kHz); chemical lysis, e.g., using detergents; and various commercially available lysis reagents. In some embodiments, DNA is detected in a sample in which the DNA is separated or separated from at least some other cellular components.

[0087] When the methods discussed herein indicate detection of a target gene, the detection can be performed on the complement of the target gene (instead of, or in addition to, the target gene sequence shown herein). In some embodiments, when detecting the complement of a target gene, a polynucleotide complementary to the complement of the target gene is used for detection. In some embodiments, the polynucleotide used for detection comprises at least a portion that is at least 90%, at least 95%, or 100% identical in sequence to the target gene, although it may comprise modified nucleotides.

[0088] 4.2.4 Exemplary Analysis Methods

[0089] As described above, a method for detecting Trichomonas vaginalis is provided. The method comprises detecting the presence of a TV 40S ribosomal protein (Tv40Srp) gene in a sample from a subject. In some embodiments, the method further comprises detecting at least one endogenous control (such as SAC) and / or at least one exogenous control (such as SPC). In some embodiments, detecting the TV 40S ribosomal protein (Tv40Srp) gene indicates the presence of TV, even if the endogenous control and / or exogenous control are not detected in the assay. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is not detected, only when a control is detected, it is considered that the result is negative for TV. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is not detected, only when an endogenous control and an exogenous control are detected, it is considered that the result is negative for TV.

[0090] Any analytical procedure that allows for specific detection of a target gene can be used in the methods provided herein. Exemplary non-limiting analytical procedures include, but are not limited to, nucleic acid amplification methods, PCR methods, isothermal amplification methods, and other analytical detection methods known to those skilled in the art.

[0091] In some embodiments, a method for detecting a target gene, such as the TV 40S ribosomal protein (Tv40Srp) gene, comprises amplifying the gene and / or its complement. The amplification can be accomplished by any method. Exemplary methods include, but are not limited to, isothermal amplification, real-time PCR, endpoint PCR, and amplification from a T7 promoter annealed to DNA using a T7 polymerase, such as the SenseAmp Plus polymerase available from Implen, Germany. TM Kit provided.

[0092] When amplifying the target gene, in some embodiments, the amplicon of the target gene is formed. The amplicon can be single-stranded or double-stranded. In some embodiments, when the amplicon is single-stranded, the sequence of the amplicon is relevant to the target gene with sense or antisense orientation. In some embodiments, the amplicon of the target gene is detected rather than the target gene itself. Therefore, when the method discussed herein shows that the target gene is detected, this detection can be carried out on the amplicon of the target gene (rather than the target gene itself, or in addition to the target gene itself). In some embodiments, when detecting the amplicon of the target gene rather than the target gene, the polynucleotide complementary to the complement of the target gene is used for detection. In some embodiments, when detecting the amplicon of the target gene rather than the target gene, the polynucleotide complementary to the complement of the target gene is used for detection. In some embodiments, a plurality of polynucleotides can be used for detection, and some polynucleotides can be complementary to the target gene and some polynucleotides can be complementary to the complement of the target gene.

[0093] In some embodiments, the method for detecting the TV 40S ribosomal protein (Tv40Srp) gene comprises PCR, as described below. In some embodiments, detecting more than one target gene comprises real-time monitoring of PCR reactions, which can be accomplished by any method. Such methods include, but are not limited to, using Molecular beacons, or Scorpion probes (ie, energy transfer (ET) probes, such as FRET probes) and the use of supplementary dyes, such as SYBR Green, EvaGreen, Thiazole Orange, YO-PRO, TO-PRO, etc.

[0094] Non-limiting exemplary conditions for amplifying the target gene are as follows. Exemplary cycles include initial denaturation at 90°C to 100°C for 30 seconds to 5 minutes, followed by denaturation at 90°C to 100°C for 1 to 10 seconds, followed by annealing and amplification at 60°C to 75°C for 10 to 30 seconds. Further exemplary cycles include up to 40 cycles at 95°C for 1 minute, followed by 5 seconds at 92.5°C and 20 seconds at 68°C. In some embodiments, the cyclic denaturation step is omitted for the first cycle following the initial denaturation step. In some embodiments, Taq polymerase is used for amplification. In some embodiments, the cycles are performed at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, or at least 45 times. In some embodiments, Taq with a hot start function is used. In some embodiments, the amplification reaction is performed at In some embodiments, the amplification of the TV 40S ribosomal protein (Tv40Srp) gene, the endogenous control, and the exogenous control occurs in the same reaction. In some embodiments, the detection of the TV 40S ribosomal protein (Tv40Srp) gene occurs in less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes from the initial denaturation to the final extension.

[0095] In some embodiments, the detection of the target gene includes forming a complex comprising a polynucleotide complementary to the target gene or its complement and a nucleic acid selected from the target gene, the DNA amplicon of the target gene and the complement of the target gene. Therefore, in some embodiments, the polynucleotide forms a complex with the target gene. In some embodiments, the polynucleotide forms a complex with the complement of the target gene. In some embodiments, the polynucleotide forms a complex with the DNA amplicon of the target gene. When the double-stranded DNA amplicon is part of the complex, as used herein, the complex can include one or two chains of the DNA amplicon. Therefore, in some embodiments, the complex only includes one chain of the DNA amplicon. In some embodiments, the complex is a triplex and includes two chains of the polynucleotide and the DNA amplicon. In some embodiments, the complex is formed by hybridization between the polynucleotide and the target gene, the complement of the target gene, or the DNA amplicon of the target gene. In some embodiments, the polynucleotide is a primer or a probe.

[0096] In some embodiments, the method includes detecting a complex. In some embodiments, the complex does not necessarily need to be associated when detecting. That is, in some embodiments, a complex is formed, then the complex is dissociated or disrupted in some manner, and the components from the complex are detected. An example of such a system is In some embodiments, when the polynucleotide is a primer, detection of the complex can include amplification of the target gene, the complement of the target gene, or a DNA amplicon of the target gene.

[0097] In some embodiments, the analytical method used to detect at least one target gene in the methods described herein comprises real-time quantitative PCR. In some embodiments, the analytical method used to detect at least one target gene comprises using Probes. This assay uses energy transfer ("ET"), such as fluorescence resonance energy transfer ("FRET"), to detect and quantify synthesized PCR products. Typically, The probe contains a fluorescent dye molecule coupled to the 5'-end and a quencher molecule coupled to the 3'-end, so that the dye and quencher are in close proximity, allowing the quencher to suppress the fluorescent signal of the dye via FRET. When the probe binds to the chimeric amplicon template, the 5'-nuclease of the polymerase cleaves the probe, uncoupling the dye and quencher, thereby detecting the dye signal (e.g., fluorescence). The signal (e.g., fluorescence) increases with each PCR cycle in proportion to the amount of cleaved probe.

[0098] In some embodiments, if any signal is generated from the TaqMan probe during the PCR cycles, the target gene is considered to be detected. For example, in some embodiments, if the PCR comprises 40 cycles, the target gene is considered to be present and detected if a signal is generated at any cycle during amplification. In some embodiments, if no signal is generated at the end of the PCR cycles, the target gene is considered to be absent and undetected.

[0099] In some embodiments, the quantitative determination of the real-time PCR results is completed by constructing a standard curve from nucleic acids of known concentrations and then inferring the quantitative information of the target gene of unknown concentration. In some embodiments, the nucleic acid used to generate the standard curve is DNA (e.g., an endogenous control, or an exogenous control). In some embodiments, the nucleic acid used to generate the standard curve is purified double-stranded plasmid DNA or single-stranded DNA generated in vitro.

[0100] In some embodiments, in order for an assay to demonstrate the absence of TV in a sample, the Ct value for an endogenous control (e.g., SAC) and / or an exogenous control (e.g., SPC) must be within a predetermined valid range. That is, in some embodiments, the absence of TV cannot be confirmed unless the control is detected, indicating that the assay was successful. The Ct value is inversely proportional to the amount of nucleic acid target in the sample.

[0101] In some embodiments, a threshold Ct (or "cutoff Ct") value (below which detection is considered) for a target gene (including an endogenous control and / or an exogenous control) is previously determined. In some embodiments, substantially the same assay conditions and system (e.g., ) determines the threshold value Ct based on which the sample will be tested.

[0102] Apart from In addition to the PCR assay, other real-time PCR chemistries used to detect and quantify PCR products in the methods provided herein include, but are not limited to, molecular beacons, Scorpion probes, and supplementary dyes such as SYBR Green, EvaGreen, Thiazole Orange, YO-PRO, TO-PRO, etc., which are discussed below.

[0103] In various embodiments, in a single multiplex reaction, real-time PCR detection is used to detect the TV 40S ribosomal protein (Tv40Srp) gene, an endogenous control, and an exogenous control. In some multiplex embodiments, multiple probes are used, such as Probes (each specific for a different target). In some embodiments, each target gene-specific probe is spectrally distinguishable from the other probes used in the same multiplex reaction.

[0104] Real-time PCR is performed using any PCR instrument available in the art. Typically, instruments for real-time PCR data collection and analysis comprise a thermal cycler, optics for fluorescence excitation and emission collection, and optionally a computer and data acquisition and analysis software.

[0105] In some embodiments, detection and / or quantification of real-time PCR products is accomplished using dyes that bind to double-stranded DNA products, such as SYBR Green, EvaGreen, Thiazole Orange, YO-PRO, TO-PRO, etc. In some embodiments, the analytical method used in the methods described herein is (DNA-mediated annealing, selection, extension, and ligation) assay. In some embodiments, the analytical method used to detect and quantify the target gene in the methods described herein is a bead-based flow cytometric assay. See Lu J. et al. (2005) Nature 435:834-838, which is incorporated herein by reference in its entirety. An example of a bead-based flow cytometric assay is the Luminex, Inc. Technology. See www.luminexcorp.com / technology / index.html. In some embodiments, the analytical method for detecting and quantifying the level of at least one target gene in the methods described herein is by gel electrophoresis and detection with a labeled probe (e.g., a probe labeled with a radioactive or chemiluminescent label), such as by Northern blotting. In some embodiments, exemplary probes contain one or more affinity-enhanced nucleotide analogs as discussed below, such as locked nucleic acid ("LNA") analogs that contain a bicyclic sugar moiety instead of a deoxyribose or ribose sugar. See, e.g., Varallyay, E. et al. (2008) Nature Protocols 3(2): 190-196, which is incorporated herein by reference in its entirety. In some embodiments, detection and quantification of one or more target genes is accomplished using a microfluidic device and single molecule detection.

[0106] Optionally, the sample DNA is modified prior to hybridization. The target DNA / probe duplex is then passed through a channel in a microfluidic device containing a detector that records the unique signals of the three labels. In this way, individual molecules are detected and counted by their unique signals. See Fuchs et al., U.S. Patent Nos. 7,402,422 and 7,351,538 to US Genomics, Inc., each of which is incorporated herein by reference in its entirety.

[0107] 4.2.5 Exemplary Automation and Systems

[0108] In some embodiments, gene expression is detected using an automated sample processing and / or analysis platform. In some embodiments, a commercially available automated analysis platform is utilized. For example, in some embodiments, a commercially available automated analysis platform is utilized. system (Cepheid, Sunnyvale, CA).

[0109] The present invention is illustrated using the GeneXpert system. Exemplary sample preparation and analysis methods are described below. However, the present invention is not limited to a particular detection method or analysis platform. Those skilled in the art will appreciate that any number of platforms and methods may be utilized.

[0110] Utilizing a proprietary, single-use cartridge, sample extraction, amplification, and detection can all be performed within this proprietary "lab-in-a-cartridge" (see, e.g., U.S. Patents 5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185; each of which is incorporated herein by reference in its entirety).

[0111] The cartridge components include, but are not limited to, processing chambers containing reagents, filters, and capture technology for extracting, purifying, and amplifying target nucleic acids. Valves enable fluid transfer between chambers and contain nucleic acid cleavage and filtration components. Optical windows enable real-time optical detection. Reaction tubes enable very rapid thermal cycling.

[0112] In some embodiments, The system includes multiple modules for scalability. Each module includes multiple cartridges along with sample handling and analysis components.

[0113] After the sample is added to the cartridge, it is contacted with a lysis buffer and the released DNA binds to a DNA-binding substrate such as silica or glass. The sample supernatant is then removed and the DNA is eluted in an elution buffer such as Tris / EDTA buffer. The eluate can then be processed in the cartridge to detect the target gene as described herein. In some embodiments, the eluate is used to reconstitute at least some of the PCR reagents, which are present in the cartridge as lyophilized particles.

[0114] In some embodiments, PCR is used to amplify and analyze the presence of the target gene. In some embodiments, PCR uses a Taq polymerase with a hot start function, such as AptaTaq (Roche). In some embodiments, the initial denaturation is at 90°C to 100°C for 30 seconds to 5 minutes; the cycling denaturation temperature is 90°C to 100°C for 1 to 10 seconds; the cycling annealing and amplification temperature is 60°C to 75°C for 10 to 30 seconds; and up to 50 cycles are performed.

[0115] In some embodiments, a double-denaturation method is used to amplify low copy number targets. In some embodiments, the double-denaturation method includes a first denaturation step followed by the addition of primers and / or probes for detecting the target gene. All or most of the DNA-containing samples (such as DNA eluates) are then denatured for a second time, after which, in some cases, a portion of the sample is aliquoted for circulation and detection of the target gene. Without intending to be limited by any particular theory, the double denaturation scheme can increase the probability that the low copy number target gene (or its complement) will be present in the aliquot selected for circulation and detection because the second denaturation effectively doubles the number of targets (i.e., it divides the target and its complement into two separate templates), after which the aliquot is selected for circulation. In some embodiments, the first denaturation step comprises heating to a temperature of 90°C to 100°C for a total time of 30 seconds to 5 minutes. In some embodiments, the second denaturation step comprises heating to a temperature of 90°C to 100°C for a total time of 5 seconds to 3 minutes. In some embodiments, the first denaturation step and / or the second denaturation step are performed by separately heating aliquots of the sample. In some embodiments, each aliquot can be heated the above number of times. As a non-limiting example, a first denaturation step for a DNA-containing sample (such as a DNA eluate) can include heating at least one, at least two, at least three, or at least four aliquots of the sample (sequentially or simultaneously) separately to a temperature of 90°C to 100°C for 60 seconds each. As a non-limiting example, a second denaturation step for a DNA-containing sample (such as a DNA eluate) containing an enzyme, primer, and probe can include heating at least one, at least two, at least three, or at least four aliquots of the eluate (sequentially or simultaneously) separately to a temperature of 90°C to 100°C for 5 seconds each. In some embodiments, the aliquot is the entire DNA-containing sample (such as a DNA eluate). In some embodiments, the aliquot is less than the entire DNA-containing sample (such as a DNA eluate).

[0116] In some embodiments, the following protocol is used to detect a target gene in a DNA-containing sample, such as a DNA eluate: one or more aliquots of the DNA-containing sample are heated separately to 95°C for 60 seconds each. An enzyme, primers, and probe are added to the DNA-containing sample and one or more aliquots are heated separately to 95°C for 5 seconds each. At least one aliquot of the DNA-containing sample containing the enzyme, primers, and probe is then heated to 94°C for 60 seconds. The aliquots are then cycled 45 times with the following two steps: (1) 94°C for 5 seconds, and (2) 66°C for 30 seconds.

[0117] The present invention is not limited to specific primer and / or probe sequences. Exemplary amplification primers and detection probes are described in the Examples.

[0118] In some embodiments, offline centrifugation is used, for example, with samples having low cell content. The sample (with or without buffer added) is centrifuged and the supernatant removed. The pellet is then resuspended in a smaller volume of supernatant or buffer. The resuspended pellet is then analyzed as described herein.

[0119] 4.2.6 Exemplary Data Analysis

[0120] In some embodiments, if the Ct value for the TV 40S ribosomal protein (Tv40Srp) gene is below a certain threshold, the presence of TV is detected. In some embodiments, the effective range of Ct values ​​is 9 to 39.9 Ct. In some such embodiments, if no amplification above background is observed from TV-specific primers after 40 cycles, the sample is considered negative for TV.

[0121] In some embodiments, computer-based analysis programs are used to translate the raw data generated by the detection assay into clinician's predicted value data. Clinicians can use any suitable means to obtain predicted data. Therefore, in some embodiments, the present invention provides further benefits, i.e., clinicians who may not be trained in genetics or molecular biology do not need to understand the raw data. Data are directly provided to clinicians in their most useful form. Clinicians can then immediately utilize information to optimize the care of the experimenter.

[0122] The present invention contemplates any method of receiving, processing and transmitting information to a laboratory for measuring, information providing, medical individuality and experimenter and receiving, processing and transmitting information from a laboratory for measuring, information providing, medical individuality and experimenter. For example, in some embodiments of the present invention, sample (for example, biopsy or serum or urine sample) is obtained from the experimenter and performs feature analysis (profiling) service (for example, clinical laboratory at medical facility, genome analysis business etc.), which is located in any part of the world (for example, different from the country where the experimenter lives or the country where the information is finally used) to produce raw data. In the case where sample comprises tissue or other biological samples, the experimenter can visit a medical center to obtain a sample and send it to an analysis center, or the experimenter can collect a sample (for example, a urine sample) and directly send it to an analysis center. In the case where the sample comprises the biological information determined before, information can be directly sent to analysis services (for example, the information card containing information can be scanned by a computer and data will be transferred to the computer of an analysis center using an electronic communication system) by the experimenter. Once received by analysis services, sample is processed and produces feature analysis results (profile) (i.e., expression data), specific to the diagnosis or prognosis information required for the experimenter.

[0123] The analysis data will then be prepared in a format suitable for interpretation by a treatment clinician. For example, rather than providing raw expression data, the format prepared can represent a diagnosis or risk assessment (e.g., the presence of TV) to the experimenter, suggesting or not suggesting a specific treatment option. The data can be presented to the clinician by any suitable method. For example, in some embodiments, analysis services produce a report that can be printed to a clinician (e.g., at a care point) or presented to the clinician on a computer monitor.

[0124] In some embodiments, first analyze information at the care point or at a regional facility. The raw data is then sent to a central processing facility for further analysis and / or the raw data is converted into information for a clinician or patient. The central processing facility provides the privacy of data analysis (all data are stored in the central facility with a unified security protocol), the advantages of speed and uniformity. The central processing facility can then control the fate of the data after treating the subject. For example, using an electronic communication system, the central facility can provide data to a clinician, subject, or researcher.

[0125] In some embodiments, the subject can directly access the data using an electronic communication system. The subject can then choose to further intervene or consult based on the results. In some embodiments, the data is used for research purposes. For example, the data can be used to further optimize the inclusion or exclusion of markers that are useful indicators of a particular condition or stage of a disease or as companion diagnostics to determine a course of treatment.

[0126] 4.2.7 Exemplary Polynucleotides

[0127] In some embodiments, polynucleotides are provided. In some embodiments, synthetic polynucleotides are provided. As used herein, synthetic polynucleotides refer to polynucleotides synthesized in vitro chemically or enzymatically. Chemical synthesis of polynucleotides includes, but is not limited to, synthesis using a polynucleotide synthesizer such as OligoPilot (GE Healthcare), ABI3900 DNA synthesizer (Applied Biosystems). Enzymatic synthesis includes, but is not limited to, generating polynucleotides by enzymatic amplification, for example, PCR. Polynucleotides may comprise one or more nucleotide analogs (i.e., modified nucleotides) discussed herein.

[0128] In some embodiments, polynucleotides are provided that comprise a region that is at least 90%, at least 95%, or 100% identical to, or at least 90%, at least 95%, or 100% complementary to, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of a TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, polynucleotides are provided that comprise a region that is at least 90%, at least 95%, or 100% identical to, or complementary to, 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides of a TV 40S ribosomal protein (Tv40Srp) gene. Non-limiting exemplary polynucleotides are shown in Table 1.

[0129] In various embodiments, the polynucleotide comprises fewer than 500, fewer than 300, fewer than 200, fewer than 150, fewer than 100, fewer than 75, fewer than 50, fewer than 40, or fewer than 30 nucleotides. In various embodiments, the polynucleotide is between 6 and 200, between 8 and 200, between 8 and 150, between 8 and 100, between 8 and 75, between 8 and 50, between 8 and 40, between 8 and 30, between 15 and 100, between 15 and 75, between 15 and 50, between 15 and 40, or between 15 and 30 nucleotides in length.

[0130] In some embodiments, polynucleotide is a primer. In some embodiments, primer is labeled with a detectable portion. In some embodiments, primer is not labeled. Primer as used herein is a polynucleotide that can selectively hybridize with a target gene or with an amplicon (collectively referred to as " template ") amplified from a target gene, and, in the presence of a template, a polymerase and suitable buffer and reagent can extend to form a primer extension product.

[0131] In some embodiments, polynucleotide is a probe. In some embodiments, the probe is labeled with a detectable moiety. Detectable moieties as used herein include direct detectable moieties, such as fluorescent dyes, and indirect detectable moieties, such as in conjunction with a pair member. In some embodiments, when the detectable moiety is in conjunction with a pair member, the probe can be detected by incubating the probe with a detectable label that is incorporated into a second pair member. In some embodiments, the probe is unlabeled, such as when the probe is a capture probe, for example, on a microarray or on a bead. In some embodiments, the probe is non-extendable, for example, extended by a polymerase. In other embodiments, the probe is extendable.

[0132] In some embodiments, the polynucleotide is a FRET probe labeled with a fluorescent dye (donor) at the 5'-end and a quencher (acceptor) at the 3'-end. The quencher is a chemical group that absorbs (i.e., suppresses) fluorescence emission from the dye when the groups are in close proximity (i.e., attached to the same probe). Thus, in some embodiments, the emission spectrum of the dye should overlap to a considerable extent with the absorption spectrum of the quencher. In other embodiments, the dye and quencher are not at the ends of the FRET probe.

[0133] 4.2.7.1 Exemplary Polynucleotide Modifications

[0134] In some embodiments, the methods for detecting at least one target gene described herein utilize one or more modified polynucleotides, such as polynucleotides comprising one or more affinity-enhanced nucleotide analogs. Modified polynucleotides for use in the methods described herein include primers for reverse transcription, PCR amplification primers, and probes. In some embodiments, the incorporation of affinity-enhancing nucleotides increases the binding affinity and specificity of the polynucleotide for its target nucleic acid compared to a polynucleotide containing only deoxyribonucleotides, and allows the use of shorter polynucleotides or shorter regions of complementarity between the polynucleotide and the target nucleic acid.

[0135] In some embodiments, affinity-enhancing nucleotide analogs include more than one base-modified, sugar-modified, and / or backbone-modified nucleotide.

[0136] In some embodiments, modified bases for affinity-enhancing nucleotide analogs include 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthine, and hypoxanthine.

[0137] In some embodiments, affinity-enhancing nucleotide analogs include nucleotides with modified sugars such as 2'-substituted sugars, such as 2'-O-alkyl-ribose sugars, 2'-amino-deoxyribose sugars, 2'-fluoro-deoxyribose sugars, 2'-fluoro-arabinose sugars, and 2'-O-methoxyethyl-ribose (2'MOE) sugars. In some embodiments, the modified sugar is arabinose sugar, or d-arabinosyl-hexitol sugar.

[0138] In some embodiments, affinity-enhancing nucleotide analogs include backbone modifications such as the use of peptide nucleic acids (PNA; e.g., oligomers comprising nucleobases linked together by an amino acid backbone). Other backbone modifications include phosphorothioate linkages, phosphodiester-modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, methyl phosphonates, alkyl phosphonates, phosphates, alkyl phosphonothioates, phosphoramidates, carbamates, carbonates, phosphotriesters, acetamidates, carboxymethyl esters, methylphosphothioate, phosphorodithioate, p-ethoxy, and combinations thereof.

[0139] In some embodiments, a polynucleotide includes at least one affinity-enhancing nucleotide analog with a modified base, at least one nucleotide (which may be the same nucleotide) with a modified sugar, and / or at least one non-naturally occurring internucleotide linkage.

[0140] In some embodiments, the affinity-enhancing nucleotide analogs contain locked nucleic acid ("LNA") sugars that are bicyclic sugars. In some embodiments, the polynucleotides used in the methods described herein comprise one or more nucleotides having LNA sugars. In some embodiments, the polynucleotides contain one or more regions consisting of nucleotides having LNA sugars. In other embodiments, the polynucleotides contain nucleotides having LNA sugars interspersed with deoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm. Des. 14(11): 1138-1142.

[0141] 4.2.7.2 Exemplary Primers

[0142] In some embodiments, primers are provided. In some embodiments, the primers are at least 90%, at least 95%, or 100% identical to, or at least 90%, at least 95%, or 100% complementary to, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of a TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, primers are provided that comprise a region that is at least 90%, at least 95%, or 100% identical to, or complementary to, 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides of a TV 40S ribosomal protein (Tv40Srp) gene. Non-limiting exemplary primers are shown in Table 1. In some embodiments, the primers may also include portions or regions that are not identical or complementary to the target gene. In some embodiments, the regions of the primers that are at least 90%, at least 95%, or 100% identical or complementary to the target gene are contiguous, such that any regions of the primers that are not identical or complementary to the target gene do not disrupt the regions of identity or complementarity.

[0143] In some embodiments, the primer comprises a portion that is at least 90%, at least 95%, or 100% identical to a region of the target gene. In some such embodiments, the primer comprising a region that is at least 90%, at least 95%, or 100% identical to a region of the target gene is capable of selectively hybridizing to an amplicon produced by amplification of the target gene. In some embodiments, the primer is complementary to a sufficient portion of the amplicon so that it selectively hybridizes to the amplicon under the specific assay conditions used.

[0144] As used herein, "selective hybridization" means that a polynucleotide, such as a primer or probe, will hybridize to a specific nucleic acid in a sample with an affinity at least 5 times greater than that of another nucleic acid present in the same sample having a different nucleotide sequence in the hybridization region. Exemplary hybridization conditions are discussed herein, for example, in the context of a reverse transcription reaction or a PCR amplification reaction. In some embodiments, a polynucleotide will hybridize to a specific nucleic acid in a sample with an affinity at least 10 times greater than that of another nucleic acid present in the same sample having a different nucleotide sequence in the hybridization region.

[0145] In some embodiments, the primer comprises a detectable moiety.

[0146] In some embodiments, primer pairs are provided.Design such primer pairs to amplify target gene, such as TV 40S ribosomal protein (Tv40Srp) gene, or endogenous control such as sample adequacy control (SAC), or exogenous control such as a part for sample treatment control (SPC).In some embodiments, primer pairs are designed to produce an amplicon of 50 to 1500 nucleotide length, 50 to 1000 nucleotide length, 50 to 750 nucleotide length, 50 to 500 nucleotide length, 50 to 400 nucleotide length, 50 to 300 nucleotide length, 50 to 200 nucleotide length, 50 to 150 nucleotide length, 100 to 300 nucleotide length, 100 to 200 nucleotide length, or 100 to 150 nucleotide length.Non-restrictive exemplary primer pairs are presented in Table 1.

[0147] 4.2.7.3 Exemplary Probes

[0148] In various embodiments, the method for detecting the presence of Trichomonas vaginalis comprises hybridizing the nucleic acid of the sample with a probe. In some embodiments, the probe comprises a portion complementary to a target gene, such as the TV 40S ribosomal protein (Tv40Srp) gene, or an endogenous control such as a sample adequacy control (SAC), or an exogenous control such as a sample processing control (SPC). In some embodiments, the probe comprises a portion that is at least 90%, at least 95%, or 100% identical to a region of the target gene. In some such embodiments, a probe that is at least 90%, at least 95%, or 100% complementary to a target gene is complementary to a sufficient portion of the target gene so that it selectively hybridizes to the target gene under the specific assay conditions used. In some embodiments, the probe that is complementary to the target gene comprises a region that is at least 90%, at least 95%, or 100% complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of the target gene. Non-limiting exemplary probes are shown in Table 1. The probe that is at least 90%, at least 95%, or 100% complementary to the target gene may also include a portion or region that is not complementary to the target gene. In some embodiments, the region of the probe that is at least 90%, at least 95%, or 100% complementary to the target gene is continuous, so that any region of the probe that is not complementary to the target gene does not disrupt the complementary region.

[0149] In some embodiments, the probe comprises a portion that is at least 90%, at least 95%, or 100% identical to a region of a target gene, such as a TV 40S ribosomal protein (Tv40Srp) gene, or an endogenous control such as a sample adequacy control (SAC), or an exogenous control such as a sample processing control (SPC). In some such embodiments, a probe comprising a region that is at least 90%, at least 95%, or 100% identical to a region of a target gene is capable of selectively hybridizing to an amplicon generated by amplifying the target gene. In some embodiments, the probe is at least 90%, at least 95%, or 100% complementary to a sufficient portion of the amplicon so that it selectively hybridizes to the amplicon under the specific assay conditions used. In some embodiments, the probe that is complementary to the amplicon comprises a region that is at least 90%, at least 95%, or 100% complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of the amplicon. The probe that is at least 90%, at least 95%, or 100% complementary to the amplicon can also comprise a portion or region that is not complementary to the amplicon. In some embodiments, the region of the probe that is at least 90%, at least 95%, or 100% complementary to the amplicon is continuous, such that any region of the probe that is not complementary to the amplicon does not disrupt the region of complementarity.

[0150] In some embodiments, the method of detecting more than one target gene comprises: (a) amplifying a region of the target gene; and (b) detecting the amplified region using real-time PCR and a detection probe (which can be simultaneous with the amplification step (a)).

[0151] As described above, in some embodiments, real-time PCR detection can be performed using FRET probes, which include, but are not limited to, Probes, molecular beacon probes and Scorpion probes. In some embodiments, real-time PCR detection utilizes The probe is carried out, Probe, i.e., a linear probe that is usually covalently bound to a fluorescent dye at one end of the DNA and covalently bound to a quencher molecule elsewhere (such as at the other end of the DNA). The FRET probe comprises a sequence complementary to the region of the amplicon, so that when the FRET probe hybridizes with the amplicon, the dye fluorescence is quenched, and when the probe is digested during the amplification of the amplicon, the dye is released from the probe and a fluorescent signal is generated. In some embodiments, the amount of the target gene in the sample is proportional to the amount of fluorescence measured during the amplification.

[0152] The probe typically comprises a region of contiguous nucleotides having a sequence that is at least 90%, at least 95%, or 100% identical or complementary to a region of the target gene, such that the probe can selectively hybridize to a PCR amplicon of the region of the target gene. In some embodiments, the probe comprises a region of at least 6 contiguous nucleotides having a sequence that is completely complementary to or identical to a region of the target gene that is present in the region of the target gene. In some embodiments, the probe comprises a region of at least 8 contiguous nucleotides, at least 10 contiguous nucleotides, at least 12 contiguous nucleotides, at least 14 contiguous nucleotides, or at least 16 contiguous nucleotides that are at least 90%, at least 95%, or 100% identical or complementary to the target gene to be detected.

[0153] In some embodiments, having The region of the amplicon to which the probe sequence is at least 90%, at least 95%, or 100% complementary is at or near the middle of the amplicon molecule. In some embodiments, the 5'-end and 3'-end of the region of complementarity independently have at least 2 nucleotides of the amplicon, such as at least 3 nucleotides, such as at least 4 nucleotides, such as at least 5 nucleotides.

[0154] In some embodiments, molecular beacons can be used to detect PCR products. Like probes, molecular beacons use FRET to detect PCR products by attaching fluorescent dyes and quenchers to the ends of the probes. The molecular beacon remains intact during the PCR cycle. When the molecular beacon probe is free in solution, it forms a stem-loop structure, which allows the dye and quencher to be close enough to cause fluorescence quenching. When the molecular beacon hybridizes to the target, the stem-loop structure is eliminated, so that the dye and quencher are spatially separated and the dye fluoresces. Molecular beacons can be obtained, for example, from GeneLink TM Obtain (see www.genelink.com / newsite / products / mbintro.asp).

[0155] In some embodiments, Scorpion probe can be used as sequence-specific primer and for PCR product detection. Like molecular beacons, when not hybridized with target nucleic acid, Scorpion probe forms stem-loop structure. However, unlike molecular beacons, Scorpion probe realizes sequence-specific initiation (priming) and PCR product detection. Fluorescent dye molecule is connected to the 5'-end of Scorpion probe, and quencher is connected elsewhere, such as 3'-end. The 3' part of probe is complementary to the extension product of PCR primer, and this complementary part is connected to the 5'-end of probe by non-amplifiable part. After Scorpion primer extension, the target specific sequence of probe combines its complement in the extended amplicon, thus opens stem-loop structure and allows the dye at 5'-end to fluoresce and produce signal. Scorpion probe can obtain (referring to www.premierbiosoft.com / tech_notes / Scorpion.html) from, for example, Premier Biosoft International.

[0156] In some embodiments, labels that can be used on FRET probes include colorimetric and fluorescent dyes such as Alexa Fluor dyes, BODIPY dyes, such as BODIPY FL; Cascade Blue; Cascade Yellow; coumarins and their derivatives, such as 7-amino-4-methylcoumarin, aminocoumarins, and hydroxycoumarins; cyanine dyes, such as Cy3 and Cy5; eosin and erythrosine; fluorescein and its derivatives, such as fluorescein isothiocyanate; and macrocyclic chelates of lanthanide ions, such as QuantumDye. TM Marina Blue; Oregon Green; rhodamine dyes such as rhodamine red, tetramethylrhodamine, and rhodamine 6G; Texas Red; fluorescence energy transfer dyes such as thiazole orange-ethidium heterodimer; and TOTAB.

[0157] Specific examples of dyes include, but are not limited to, those mentioned above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor568, Alexa Fluor594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750; Amine-reactive BODIPY dyes, such as BODIPY 493 / 503, BODIPY 530 / 550, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY 630 / 650, BODIPY 650 / 655, BODIPY FL, BODIPY R6G,BODIPY TMR, and, BODIPY-TR; Cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2',4',5',7'-tetrabromosulfone fluorescein, and TET.

[0158] Examples of dye-quencher pairs (i.e., donor / acceptor pairs) include, but are not limited to, fluorescein / tetramethylrhodamine; IAEDANS / fluorescein; EDANS / dabcyl; fluorescein / fluorescein; BODIPY FL / BODIPY FL; fluorescein / QSY 7 or QSY 9 dyes. When the donor and acceptor are the same, in some embodiments, FRET can be detected by fluorescence depolarization. Alexa Fluor 555 / Alexa Fluor 594; Alexa Fluor 568 / Alexa Fluor 647; Alexa Fluor 594 / Alexa Fluor 647; ... Alexa Fluor 555 / QSY 7 or QSY 9; Alexa Fluor 568 / QSY 7 or QSY 9; Alexa Fluor 568 / QSY 21; Alexa Fluor 594 / QSY 21; and Alexa Fluor 647 / QSY 21. In some cases, the same quencher can be used for multiple dyes, for example, a broad spectrum quencher such as Iowa Quencher (Integrated DNA Technologies, Coralville, IA) or Black Hole Quencher TM (BHQ TM ; Sigma-Aldrich, St. Louis, MO).

[0159] In some embodiments, for example, in a multiplex reaction in which two or more moieties (e.g., amplicons) are detected simultaneously, each probe comprises a detectably distinct dye, such that the dyes can be distinguished when detected simultaneously in the same reaction. One skilled in the art can select a panel of detectably distinct dyes for use in a multiplex reaction.

[0160] Specific examples of fluorescently labeled ribonucleotides that can be used to prepare PCR probes for use in some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), and include Alexa Fluor 488-5-UTP, fluorescein-12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP, tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences (GE Healthcare), such as Cy3-UTP and Cy5-UTP.

[0161] Examples of fluorescently labeled deoxyribonucleotides for use in preparing PCR probes for use in the methods described herein include dinitrophenyl (DNP)-1′-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, rhodamine green-5-dUTP, Alexa Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP, Alexa Fluor 594-7-OBEA-dCTP, Alexa Fluor 647-12-OBEA-dCTP. Fluorescently labeled nucleotides are commercially available and can be purchased from, for example, Invitrogen.

[0162] In some embodiments, dyes and other moieties, such as quenchers, are introduced into the polynucleotides used in the methods described herein, such as FRET probes, by modified nucleotides. "Modified nucleotides" refer to nucleotides that have been chemically modified but still function as nucleotides. In some embodiments, the modified nucleotides have covalently linked chemical moieties, such as dyes or quenchers, and can, for example, be introduced into the polynucleotides by solid phase synthesis of polynucleotides. In other embodiments, the modified nucleotides include one or more reactive groups that can react with dyes or quenchers before, during, or after the modified nucleotides are introduced into the nucleic acid. In a specific embodiment, the modified nucleotides are amine-modified nucleotides, that is, nucleotides modified to have reactive amine groups. In some embodiments, the modified nucleotides include modified base moieties, such as uridine, adenosine, guanosine, and / or cytosine. In specific embodiments, the amine-modified nucleotide is selected from 5-(3-aminoallyl)-UTP; 8-[(4-amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP, N6-(6-amino)butyl-ATP, N4-[2,2-oxo-bis-(ethylamine)]-CTP; N6-(6-amino)hexyl-ATP; 8-[(6-amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP. In some embodiments, nucleotides with different nucleobase moieties are similarly modified, for example, 5-(3-aminoallyl)-GTP instead of 5-(3-aminoallyl)-UTP. Many amine-modified nucleotides are commercially available from, for example, Applied Biosystems, Sigma, JenaBioscience, and TriLink.

[0163] Exemplary detectable moieties also include, but are not limited to, members of a binding pair. In some such embodiments, the first member of the binding pair is linked to a polynucleotide. The second member of the binding pair is linked to a detectable label, such as a fluorescent label. When the polynucleotide linked to the first member of the binding pair is incubated with the second member of the binding pair that is linked to a detectable label, the first and second members of the binding pair associate and the polynucleotide can be detected. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens, and the like.

[0164] In some embodiments, multiple target genes are detected in a single multiplex reaction. In some such embodiments, each probe targeting a unique amplicon is spectrally distinguishable upon release from the probe, in which case each target gene is detected by a unique fluorescent signal. In some embodiments, two or more target genes are detected using the same fluorescent signal, in which case detection of the signal indicates the presence of one or both target genes.

[0165] One skilled in the art can select an appropriate detection method for the assay of choice, for example, a real-time PCR assay.The detection method of choice need not be the method described above, and can be any method.

[0166] 4.3 Exemplary Compositions and Kits

[0167] In another aspect, compositions are provided. In some embodiments, compositions are provided for use in the methods described herein.

[0168] In some embodiments, a composition comprising at least one target gene-specific primer is provided. The term "target gene-specific primer" includes primers having the following: a region of continuous nucleotides having (i) at least 90%, at least 95%, or 100% identity with a region of the target gene, or (ii) a region of continuous nucleotides having a sequence at least 90%, at least 95%, or 100% complementary to a sequence of continuous nucleotides found in the target gene. In some embodiments, a composition comprising at least one pair of target gene-specific primers is provided. The term "target gene-specific primer pair" includes primer pairs suitable for amplifying a defined target gene region. A target gene-specific primer pair typically comprises a first primer and a second primer, wherein the first primer comprises a sequence at least 90%, at least 95%, or 100% identity with a sequence in a region of the target gene, and the second primer comprises a sequence at least 90%, at least 95%, or 100% complementary to a region of the target gene. Primer pairs are generally suitable for amplifying a target gene region of 50 to 1500 nucleotides in length, 50 to 1000 nucleotides in length, 50 to 750 nucleotides in length, 50 to 500 nucleotides in length, 50 to 400 nucleotides in length, 50 to 300 nucleotides in length, 50 to 200 nucleotides in length, 50 to 150 nucleotides in length, 100 to 300 nucleotides in length, 100 to 200 nucleotides in length, or 100 to 150 nucleotides in length. Non-limiting exemplary primers and primer pairs are shown in Table 1.

[0169] In some embodiments, the composition comprises at least one pair of target gene-specific primers. In some embodiments, the composition further comprises a pair of target gene-specific primers for amplifying an endogenous control (such as SAC) and / or a pair of target gene-specific primers for amplifying an exogenous control (such as SPC).

[0170] In some embodiments, the composition comprises at least one target gene-specific probe. The term "target gene-specific probe" includes probes having the following: a continuous nucleotide region having (i) at least 90%, at least 95%, or 100% identity to a region of the target gene, or (ii) a continuous nucleotide region having a sequence that is at least 90%, at least 95%, or 100% complementary to a sequence of a continuous nucleotide region found in the target gene. Non-limiting exemplary target-specific probes are shown in Table 1.

[0171] In some embodiments, the composition (including the above-mentioned composition comprising one or more target gene-specific primers) comprises one or more probes for detecting the target gene. In some embodiments, the composition comprises a probe for detecting an endogenous control (such as SAC) and / or a probe for detecting an exogenous control (such as SPC).

[0172] In some embodiments, the composition is an aqueous composition. In some embodiments, the aqueous composition comprises a buffer component, such as phosphate, tris, HEPES, etc., and / or other components, as discussed below. In some embodiments, the composition is dry, for example, lyophilized, and suitable for reconstitution by adding a fluid. The dry composition can include one or more buffer components and / or other components.

[0173] In some embodiments, the composition further comprises one or more other components. Other components include, but are not limited to, salts such as NaCl, KCl, and MgCl2; polymerases, including thermostable polymerases such as Taq; dNTPs; bovine serum albumin (BSA); reducing agents such as β-mercaptoethanol; EDTA, and the like. Those skilled in the art can select appropriate composition components based on the intended use of the composition.

[0174] In some embodiments, a composition comprising at least one polynucleotide for detecting at least one target gene is provided. In some embodiments, the polynucleotide is used as a primer for a reverse transcriptase reaction. In some embodiments, the polynucleotide is used as a primer for amplification. In some embodiments, the polynucleotide is used as a primer for PCR. In some embodiments, the polynucleotide is used as a probe for detecting at least one target gene. In some embodiments, the polynucleotide is detectably labeled. In some embodiments, the polynucleotide is a FRET probe. In some embodiments, the polynucleotide is probes, molecular beacons, or Scorpion probes.

[0175] In some embodiments, the composition comprises at least one FRET probe having a sequence that is at least 90%, at least 95%, or 100% identical, or at least 90%, at least 95%, or 100% complementary to a region of the TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, the FRET probe is labeled with a donor / acceptor pair so that when the probe is digested during the PCR reaction, it produces a unique fluorescence emission associated with a specific target gene. In some embodiments, when the composition comprises a plurality of FRET probes, each probe is labeled with a different donor / acceptor pair so that when the probe is digested during the PCR reaction, each produces a unique fluorescence emission associated with a specific probe sequence and / or target gene. In some embodiments, the sequence of the FRET probe is complementary to the target region of the target gene. In other embodiments, the FRET probe has a sequence that contains one or more base mismatches when compared to the sequence of the best matching target region of the target gene.

[0176] In some embodiments, the composition comprises a FRET probe consisting of at least 8, at least 9, at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides, wherein at least a portion of the sequence is at least 90%, at least 95%, or 100% identical to, or at least 90%, at least 95%, or 100% complementary to, a region of the TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, at least 8, at least 9, at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides of the FRET probe are identically present in a region of the TV 40S ribosomal protein (Tv40Srp) gene, or are complementary to a region of the TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, the FRET probe comprises a sequence having one, two, or three mismatches with the sequence or complement of the TV 40S ribosomal protein (Tv40Srp) gene.

[0177] In some embodiments, the kit comprises the polynucleotides discussed above. In some embodiments, the kit comprises at least one primer and / or probe discussed above. In some embodiments, the kit comprises at least one polymerase, such as a thermostable polymerase. In some embodiments, the kit comprises dNTPs. In some embodiments, the kit for the real-time PCR method described herein comprises one or more target gene-specific FRET probes and / or one or more primers for amplifying the target gene.

[0178] In some embodiments, more than one primer and / or probe is "linear". A "linear" primer refers to a polynucleotide that is a single-stranded molecule and generally does not include, for example, a short region of at least 3, 4 or 5 consecutive nucleotides that is complementary to another region within the same polynucleotide, such that the primer forms an internal duplex. In some embodiments, the primer for reverse transcription comprises a region of at least 4, such as at least 5, such as at least 6, such as at least 7 or more consecutive nucleotides at the 3'-end of the target gene that has a sequence complementary to a region of at least 4, such as at least 5, such as at least 6, such as at least 7 or more consecutive nucleotides at the 5'-end of the target gene.

[0179] In some embodiments, the kit comprises one or more linear primers (a "forward primer" and a "reverse primer") for amplifying a target gene. Thus, in some embodiments, the first primer comprises a region of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides that is at least 90%, at least 95%, or 100% identical to the sequence of a region of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides at a first position of the target gene. Moreover, in some embodiments, the second primer comprises a region of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides having a sequence that is at least 90%, at least 95%, or 100% complementary to the sequence of a region of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides at a second position of the target gene, such that a PCR reaction using the two primers results in an amplicon extending from the first position of the target gene to the second position of the target gene.

[0180] In some embodiments, the kit comprises at least two sets, at least three sets, or at least four sets of primers, each for amplifying a different target gene, such as an endogenous control and / or an exogenous control.

[0181] In some embodiments, probes and / or primers used in the compositions described herein comprise deoxyribonucleotides. In some embodiments, probes and / or primers used in the compositions described herein comprise deoxyribonucleotides and one or more nucleotide analogs, such as LNA analogs or other duplex-stabilizing nucleotide analogs described above. In some embodiments, probes and / or primers used in the compositions described herein comprise all nucleotide analogs. In some embodiments, probes and / or primers comprise one or more duplex-stabilizing nucleotide analogs, such as LNA analogs, in the region of complementarity.

[0182] In some embodiments, the kit for real-time PCR method as described herein also includes reagents for reverse transcription and amplification reaction. In some embodiments, the kit includes enzymes such as thermostable DNA polymerases, such as Taq polymerase. In some embodiments, the kit also includes deoxyribonucleotide triphosphates (dNTPs) for amplification. In other embodiments, the kit includes a buffer optimized for hybridization specific to the probe and primer.

[0183] The kit generally includes packaging with one or more containers for holding reagents, as one or more separate compositions or, optionally, as a mixture (wherein allowing the reagents to be compatible). The kit may also include other one or more materials that may be desired from the user's perspective, such as one or more buffers, one or more diluents, one or more standards, and / or any other materials for sample processing, washing, or any other steps in performing the assay.

[0184] The kit preferably includes instructions for performing one or more of the methods described herein. The instructions included in the kit may be attached to the packaging material or may be included as a package insert. The instructions are typically handwritten or printed material, but they are not limited thereto. The present invention contemplates any medium capable of storing the instructions and communicating them to the end user. Such media include, but are not limited to, electronic storage media (e.g., disks, tapes, magnetic cylinders, magnetic chips), optical media (e.g., CD ROMs), etc. As used herein, the term "instructions" may include a website address that provides instructions.

[0185] In some embodiments, the kit may contain more than one The above reagents are provided in sample cartridges. These cartridges allow extraction, amplification, and detection to be performed in a self-contained "laboratory in a cartridge" (see, e.g., U.S. Patents 5,958,349, 6,403,037, 6,440,725, 6,783,736, 6,818,185; each of which is incorporated herein by reference in its entirety). Reagents for measuring genomic copy number levels and detecting pathogens can be provided in separate cartridges within the kit, or these reagents (suitable for multiplexed detection) can be provided in a single cartridge.

[0186] In some embodiments, any kit described herein can include a urine sample and / or a swab container for collecting a urethral swab sample, a vaginal swab sample, or an endocervical swab sample.

[0187] The following examples are for illustrative purposes only and are not intended to be limiting in any way.

[0188] 5. Examples

[0189] 5.1. Example 1: Detection of Trichomonas vaginalis

[0190] The assay was designed to detect the gene for Trichomonas vaginalis (TV) 40S ribosomal protein (Tv40Srp) by PCR using the primers and probes shown in Table 1. In addition to TV-specific primers and probes, primers and probes were included to detect a single copy human gene used as a sample adequacy control (SAC) target. Primers and probes were also included to detect a bacterial gene that was included in the multiplex reaction as a sample processing control (SPC) target.

[0191] Table 1: Primer and probe sequences

[0192]

[0193] The final primer and probe compositions for the multiplex assay are shown in Table 2.

[0194] Table 2: Primer and probe concentrations

[0195]

[0196] F1 and F2 are detectably distinct dyes that can be detected and distinguished simultaneously in multiplex reactions. Each probe also contains a quencher (eg, Q1, described above).

[0197] Each reaction contained 42-58 mM KCl, 3.5-5.0 mM MgCl2, 250-350 μM dNTPs, 50 mM Tris, pH 8.6, and 0.01% sodium azide. AptaTaq (0.27-0.37 units / μl; Roche) was used for amplification.

[0198] For each sample to be tested, approximately 7 mL of first-time, freshly voided urine is added to 1 mL of buffer (preferably within 2 hours of sample collection). Physician-collected endocervical swabs or self-collected (in a clinical setting) vaginal swabs are immediately placed in 2.5 mL of buffer.

[0199] 500 μL of buffered urine or swab sample was loaded onto The sample is mixed with a lysis reagent to release the nucleic acids. After lysis, the nucleic acids released from the sample are captured on a DNA-binding substrate. The nucleic acids are eluted from the substrate and used to recover the reagents for real-time PCR (described above). The reaction cycle used is: Used in the system Cartridge, 95 °C for 1 min, followed by 40 cycles of 92.5 °C for 5 sec, and at 68 °C for 20 sec.

[0200] The assay results are explained in Table 3. The valid range of Ct values ​​for TV, SAC, and SPC targets was 9-39.9 Ct.

[0201] Table 3: Xpert TV assay results and interpretation

[0202]

[0203]

[0204]

[0205] 5.2 Example 2: Clinical Performance

[0206] The performance characteristics of the Xpert TV assay were evaluated at 13 institutions in the U.S. Due to the low prevalence of Trichomonas vaginalis and the difficulty in obtaining fresh Trichomonas vaginalis-positive specimens from male subjects, the sample population used for this study was provided using artificial male urine specimens.

[0207] Subjects included consenting asymptomatic and symptomatic, sexually active men and women who were seen in settings including, but not limited to: OB / GYN, sexually transmitted disease (STD), adolescent, public health facilities, and family planning clinics.

[0208] Study samples consisted of prospectively collected male urine, female urine, cervical swabs, and patient-collected vaginal swabs (collected in a clinical setting). An artificial male urine sample was included to supplement the male sample size.

[0209] The Xpert TV assay performance was compared to that of a transcription-mediated assay ( The Trichomonas vaginalis assay (GenProbe Hologic, San Diego, USA) was compared to an FDA-cleared in vitro quantitative nucleic acid amplification comparator assay for the detection of ribosomal RNA of Trichomonas vaginalis. Samples with discrepant results between the Xpert TV assay and the comparator assay were analyzed using bidirectional sequencing of separate replicate genomic DNA sequences. See Bandea, et al., Journal of Clinical Microbiology. 2013, 51(4): 1298-1300.

[0210] In Xpert TV assay runs using qualified samples, 97.3% (5327 / 5474) of these samples were successful on the first attempt. The remaining 147 gave ambiguous results on the first attempt (91 errors, 44 invalid results, and 12 no results). After a single retest, 119 of the 147 samples yielded valid results; 17 of the samples were ambiguous on the second attempt, and 11 samples were not retested. The overall assay success rate was 99.5% (5446 / 5474).

[0211] Results from the Xpert TV assay were compared with the comparator assay, and discrepancies were sequenced in both directions. Sensitivity and specificity by sex, sample type, and symptom status are provided in Table 4.

[0212]

[0213] 5.3 Example 3: Detection Limit

[0214] The sensitivity or limit of detection (LoD) of the Xpert TV assay was evaluated using two strains of Trichomonas vaginalis, a metronidazole-susceptible 30001 TM ), and a metronidazole-resistant ( Trichomonas vaginalis 30238 TM Both strains were tested in T. vaginalis -negative pooled male urine mixed with buffer (MU) and T. vaginalis -negative pooled vaginal swabs in buffer (VS).

[0215] The limit of detection (LoD) was assessed over three days using 20 replicates tested at a minimum of five concentrations for each strain and sample type. The LoD was estimated by logistic regression. The LoD was defined as the lowest number of cells / mL that could be distinguished from a negative sample with 95% confidence in the replicates or the lowest concentration at which 19 of 20 replicates were positive. Studies were conducted using two different batches of Xpert TV reagent, and the proposed LoD for each strain is the higher of the two determinations (Table 5). For swab samples in buffer, the limit of detection was 5 cells / mL. For urine samples in buffer, the limit of detection was 6 cells / mL. The proposed LoD was validated by analyzing at least 20 replicates diluted to the estimated LoD concentration.

[0216] Table 5: Detection limits of Trichomonas vaginalis using Xpert TV

[0217]

[0218] 5.4 Example 4: Determination of Repeatability

[0219] A set of eight samples with varying concentrations of Trichomonas vaginalis were tested on 12 different days by two different operators at each of three sites (8 samples x 1 test / day x 12 days x 2 operators x 3 sites). Three batches of the Xpert TV assay were used at each of the three test sites. The Xpert TV assay was performed according to the Xpert TV assay protocol. The results are summarized in Table 6.

[0220] The reproducibility of the Xpert TV assay was also evaluated with respect to the fluorescent signal expressed as Ct values ​​for each detected target. The mean, standard deviation (SD), and coefficient of variation (CV) between sites, between batches, between days, between operators, and within the assay for each panel member are provided in Table 7.

[0221]

[0222]

[0223] 5.5. Example 5: Analyzing Inclusiveness

[0224] The analytical inclusiveness of the Xpert TV assay was evaluated by testing 17 Trichomonas vaginalis strains in triplicate at concentrations no greater than 3x the analytical limit of detection (3x LoD). Each strain was tested in a Trichomonas vaginalis-negative pooled vaginal swab (VS) in buffer and in male urine (MU) mixed with buffer. See Table 8. Under the conditions of this study, all strains reported an ED result for TV detection. The Xpert TV assay demonstrated 100% inclusiveness in both sample types.

[0225] Table 8: Analysis inclusiveness table

[0226]

[0227] 5.6. Example 6: Analytical Specificity

[0228] The Xpert TV assay tests for a panel of 47 organisms (including bacteria, fungi, and viruses commonly found in the urogenital tract, as well as other protozoa closely related to Trichomonas). 7 Each bacterial or fungal strain was tested for cfu / mL or higher. Strains that did not produce countable colonies were diluted to 0.5 McFarland units, which is approximately equivalent to 1.5 x 108 cfu / mL for E. coli. Viral strains were purchased from ZeptoMetrix Corp. as heat-inactivated stocks and diluted to 1 x 10 6 U / mL or 10 6 Protozoa were cultured in growth medium, visually counted by light microscopy and counted as 1 x 10 6 Cells / mL tested. Tests were performed in triplicate. The tested organisms and Xpert TV assay results are listed in Table 9.

[0229] TV detection results were reported for one organism, Trichomonas tenax, using the Xpert TV assay. Under the conditions of this study, the analytical specificity of the Xpert TV assay was 98%.

[0230] Table 9: Analytical Specificity Table

[0231]

[0232]

[0233]

[0234]

[0235] 5.7. Example 7: Interfering substances

[0236] In nonclinical studies, the Xpert TV assay was used to evaluate potentially interfering endogenous and exogenous substances that may be within the urogenital tract and present in cervical and vaginal swabs or first urine samples.

[0237] The substances were diluted in a matrix of pooled negative vaginal swabs and a matrix of pooled negative male urine. The substances were tested in the same matrix spiked with Trichomonas vaginalis cells at a concentration no greater than three times the limit of detection for each sample type. Eight replicates of each set of negative and positive samples were tested using the Xpert TV assay and compared to the results obtained in a control of the same samples without the addition of potentially interfering substances. The substances and test concentrations are listed in Tables 10 and 11.

[0238] Under the conditions studied, no invalid results were reported in tests using the substance diluted in a negative urine matrix; as expected, all tests reported no detection of TV. Assay interference was observed in tests using 0.75% v / v blood and 1.8 mg / mL azithromycin diluted in a positive urine matrix. No false negative results were reported for tests using 0.5% v / v blood and 1 mg / mL azithromycin.

[0239] Under the conditions studied, no invalid results were reported in tests utilizing dilution in a pooled matrix of negative swabs; as expected, all tests reported no detection of TV.

[0240] No false negative TV results were reported in tests of substances diluted in pooled positive swab matrix. As expected, tests with all substances reported detection of TV.

[0241] Table 10: Potential interfering substances in urine samples

[0242]

[0243]

[0244] Table 11: Possible interfering substances in swab samples

[0245]

[0246]

[0247] 5.8. Example 8: Carryover

[0248] The study was conducted by the high (10 6 The study consisted of replicate testing of a TV-negative vaginal swab pool in buffer immediately followed by a high (10 cells / mL) TV-positive vaginal swab pool in buffer processed in the same GeneXpert module. 6The test protocol was repeated 20 more times on two GeneXpert modules for a total of 82 runs, resulting in 40 positive and 42 negative samples. All 40 positive samples were correctly reported as having TV detected, and all 42 negative samples were correctly reported as not having TV detected.

[0249] 5.9. Example 9: Testing of alternative primers and probes for detecting TV

[0250] To develop the TV assay described herein, four different forward primers, two different reverse primers, and two different probes for detecting the TV 40S ribosomal protein (Tv40Srp) gene were tested for sensitivity and specificity (e.g., cross-reactivity with other species) in the assay. Table 12 shows the primers and probes tested.

[0251] Table 12: Alternative primer and probe sequences

[0252]

[0253]

[0254] TV forward ALT1 was found to cross-react with Pentatrichomonas hominis (Pth), another closely related Trichomonas species found in the human intestine. When TV forward ALT1 was used in an assay using 1000 copies of TV and 500,000 copies of Pth, TV was detected with a Ct of 30.7 and Pth with a Ct of 26.3. TV forward ALT2 was not as sensitive as the final design, detecting TV with a higher Ct value of 31.5. Similarly, TV forward ALT3 was not as sensitive as the final design, also detecting TV with a higher Ct value. TV reverse ALT1 also resulted in a less sensitive assay, detecting TV with a higher Ct value. Ultimately, TV probe ALT1 was not as sensitive and consistent as the final design.

[0255] All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document were individually indicated to be incorporated by reference for all purposes.

[0256] While various specific embodiments have been illustrated and described, it will be appreciated that changes can be made without departing from the spirit and scope of the invention.

[0257] Table of some sequences

[0258] Sequence Listing <110> Sifide <120> Methods for detecting Trichomonas vaginalis <130> CEPHD-33923 / WO-1 / PRI <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 26 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 1 gtaacaacct tggagttctt cttaag 26 <210> 2 <211> 26 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 2 acatcaatct acaagacacc acttga 26 <210> 3 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 3 agtttggctg cttagcttcg ac 22 <210> 4 <211> 508 <212> DNA <213> Trichomonas vaginalis <400> 4 ggccggcctt tctgatgggt aagtctaaag cttgcggtcg tctcgctgct cgtaaactcc 60 gtcttgcaca caagtccaac ttgtgggctt ccaacgcata ccgccgttcc cttggtacat 120 caatctacaa gacaccactt gagggtacat caatggcatc tggcatcgtc gtcggcaagg 180 tcgctgtcga agccaagcag ccaaactctg ctattcgtaa agctgtccgt gttcagctta 240 agaagaactc taaggttgtc acagctttcg ttccacgcga tggttccctc cgtcttattg 300 atgataacga ccgtgttctt attgccggta tgggtcgttc tggccgttct gtcggtgacc 360 ttccaggatg ccgtttcaaa gttatcaagg tcgctggttt ctccctcctt gctctttggc 420 tcggcaagaa ggagaagccg cgcagctaaa taaatactct tgggtttacc ggtaaataaa 480 aacatatatt acgaaataca aatattat 508 <210> 5 <211> 146 <212> DNA <213> Artificial sequence <220> <223> Synthetic <400> 5 acatcaatct acaagacacc acttgaaggc acctcaatgg cctccggcat tgttgtcggc 60 aaagttgctg tcgaagctaa gcagccaaac tccgctattc gtaaagcagt tcgtgttcag 120 cttaagaaga actctaaagt tgttac 146 <210> 6 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 6 gagttcttct taagctgaac ac 22 <210> 7 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 7 gagttcttct tgagctgaac ac 22 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 8 aacaaccttg gagttcttct ta 22 <210> 9 <211> 20 <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 9 atctacaaga caccacttga 20 <210> 10 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> synthetic <400> 10 agtttggctg cttggcttcg ac 22

Claims

1. Use of a first primer pair in preparing a reagent for detecting the presence or absence of Trichomonas vaginalis in a sample from a subject, wherein the first primer pair is used to detect the presence or absence of Trichomonas vaginalis 40S ribosomal protein gene or RNA in the sample, wherein an increased level of Trichomonas vaginalis 40S ribosomal protein gene or RNA in the sample compared to a control indicates the presence of Trichomonas vaginalis in the sample, wherein the Trichomonas vaginalis 40S ribosomal protein gene comprises the sequence of SEQ ID NO: 4, and wherein the first primer pair comprises a first primer as shown in SEQ ID NO: 1 and a second primer as shown in SEQ ID NO:

2.

2. The method of claim 1, wherein the control is an endogenous control.

3. The method of claim 2, wherein the endogenous control is selected from the group consisting of HMBS, GAPDH, β-actin and β-globin.

4. The use of claim 1, wherein the control is an exogenous control.

5. The use of claim 4, wherein the exogenous control is a bacterial gene.

6. The use of claim 1, wherein the detection comprises PCR.

7. The use of claim 1, wherein the detection comprises quantitative PCR.

8. The use of claim 6 or 7, wherein the PCR reaction takes less than 2 hours from the initial denaturation step through the final extension step.

9. The use of claim 1, wherein the detection comprises contacting nucleic acid from the sample with the first primer pair, wherein the first primer pair is used to detect Trichomonas vaginalis 40S ribosomal protein gene or RNA.

10. The use of claim 9, wherein the detection comprises contacting the nucleic acid from the sample with a second primer pair for detecting an endogenous control and / or a third primer pair for detecting an exogenous control.

11. The use of claim 9, wherein the detection comprises forming a Trichomonas vaginalis 40S ribosomal protein amplicon and contacting the amplicon with a first probe capable of selectively hybridizing to Trichomonas vaginalis 40S ribosomal protein, wherein the sequence of the first probe is shown in SEQ ID NO:

3.

12. The use of claim 11, wherein the first probe comprises a fluorescent dye and a quencher molecule.

13. The method of claim 10, wherein the detection comprises forming an endogenous control amplicon and / or an exogenous control amplicon, and The endogenous control amplicon is contacted with a second probe capable of selectively hybridizing to the endogenous control amplicon and / or the exogenous control amplicon is contacted with a third probe capable of selectively hybridizing to the exogenous control amplicon, wherein the second and third probes each comprise a detectable label, wherein the detectable labels can be the same or different.

14. The use of claim 13, wherein the detection comprises detecting Trichomonas vaginalis 40S ribosomal protein gene or RNA, an endogenous control, and an exogenous control in a single multiplex reaction.

15. The use of claim 1, wherein the sample is selected from the group consisting of a urine sample, an endocervical swab sample, a vaginal swab sample, and a urethral swab sample.

16. A composition comprising a first primer pair and / or a first probe for detecting Trichomonas vaginalis 40S ribosomal protein gene or RNA, wherein the first primer pair comprises a first primer and a second primer, wherein the first primer pair comprises a first primer as shown in SEQ ID NO: 1 and a second primer as shown in SEQ ID NO: 2, and wherein the sequence of the first probe is shown in SEQ ID NO:

3.

17. A kit comprising a first primer pair and / or a first probe for detecting Trichomonas vaginalis 40S ribosomal protein gene or RNA, wherein the first primer pair comprises a first primer as shown in SEQ ID NO: 1 and a second primer as shown in SEQ ID NO: 2, and wherein the sequence of the first probe is shown in SEQ ID NO:

3.

18. The kit of claim 17, wherein the kit comprises a second primer pair for detecting an endogenous control, wherein the first primer pair and the second primer pair for detecting Trichomonas vaginalis 40S ribosomal protein are in the same or different compositions in the kit.

19. The kit of claim 18, wherein the kit comprises a third primer pair for detecting an exogenous control, wherein the third primer pair is in the same or different composition as the primer pair for detecting Trichomonas vaginalis 40S ribosomal protein and the second primer pair.

20. The kit of claim 17, wherein the first probe comprises a fluorescent dye and a quencher molecule.

21. A primer pair consisting of a first primer and a second primer, wherein the first primer consists of the sequence of SEQ ID NO: 1 and the second primer consists of the sequence of SEQ ID NO:

2.

22. A probe consisting of the sequence of SEQ ID NO: 3 and a detectable label, for detecting the amplification product of the primer pair of claim 21.

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