HK1237372B - Methods of detecting trichomonas vaginalis - Google Patents

Description

Methods for detecting Trichomonas vaginalis

1. Field of Invention

Compositions and methods for detecting Trichomonas vaginalis are provided.

2. Background

Trichomoniasis, caused by the protozoan Trichomonas vaginalis, is a common sexually transmitted infection that can infect both men and women. There are 7.4 million cases of trichomoniasis annually in the United States. Trichomoniasis infection can be symptomatic or asymptomatic. See, for example, Giocchio et al., J. Clin. Microbiol. 2012, 50: 2601-2608. In women, trichomoniasis is one of a group of conditions involving vaginal discharge. See, for example, Centers for Disease Control and Prevention (CDC). CDC fact sheet: trichomononiasis. 2010. www.cdc.gov/std/trichomonas/STDFact-Trichomoniasis.htm. Symptoms in women may include genital itching, burning, redness, or pain, odor, urinary discomfort, or 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 penile discharge. See, for example, 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 Biomedicallaboratories. www.cdc.gov/biosafety/publications/.

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.

3. Overview

In some embodiments, a method is provided for detecting the presence or absence of Trichomonas vaginalis (TV) in a sample from a subject. In some embodiments, a method is provided for determining whether a subject has a Trichomonas vaginalis (TV) infection. In some embodiments, the method includes detecting the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene or RNA in a sample from a subject.

In some embodiments, the subject has not previously been treated for a TV infection. In some embodiments, the subject has previously been treated for a TV infection. In some embodiments, the prior treatment included one or more doses of metronidazole or tinidazole. In some embodiments, the subject does not have any symptoms of a TV infection. In some embodiments, the subject has one or more symptoms of a TV infection. In some embodiments, the subject has one or more symptoms of vaginitis, urethritis, and cervicitis. In some embodiments, the subject is female and has one or more symptoms selected from: 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 one or more symptoms selected from: penile itching and/or burning; burning after ejaculation and/or urination; and penile discharge.

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 of a human gene. In some embodiments, the endogenous control is selected from HMBS, GAPDH, β-actin, and β-globin.

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 contains a DNA sequence that is not expected to be present in the sample. In some embodiments, the exogenous control is a bacterial gene.

In some embodiments, the method includes PCR. In some embodiments, the method includes 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.

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

In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence of 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 of 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 contain 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 the first primer of SEQ ID NO: 1 and the second primer of SEQ ID NO: 2. In some embodiments, the first primer pair produces an amplicon of 50 to 500 nucleotides, 50 to 400 nucleotides, 50 to 300 nucleotides, 50 to 200 nucleotides, 50 to 150 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, or 100 to 150 nucleotides.

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 with the Tv40Srp amplicon. In some embodiments, the first probe contains a detectable label. In some embodiments, the first probe contains a fluorescent dye and a quencher molecule. In some embodiments, the first probe contains 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 contains 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 method includes forming an endogenous control amplicon and/or an exogenous control amplicon. In some embodiments, the method includes contacting the endogenous control amplicon with a second probe capable of selectively hybridizing with the endogenous control amplicon and/or contacting the exogenous control amplicon with a third probe capable of selectively hybridizing with the exogenous control amplicon. In some embodiments, the second and third probes each contain a detectable marker, wherein the detectable marker may be the same or different. In some embodiments, the detectable markers of the second and third probes are detectably different from the detectable marker of the first probe. In some embodiments, the method includes detecting the Tv40Srp gene or RNA, the endogenous control, and the exogenous control in a single multiplex reaction.

In some implementations, the sample is selected from urine samples, cervical swab samples, vaginal swab samples, and urethral swab samples.

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.

In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence of 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 of 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 contain 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 implementations, the first primer pair comprises the first primer of SEQ ID NO: 1 and the second primer of SEQ ID NO: 2.

In some embodiments, the composition comprises a first probe capable of selectively hybridizing with a Tv40Srp amplicon generated by a 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.

In some embodiments, the composition comprises a second probe capable of selectively hybridizing with an endogenous control amplicon generated by a 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, β-actin, and β-globin. In some embodiments, the composition comprises a third probe capable of selectively hybridizing with an exogenous control amplicon generated 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.

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

In some embodiments, a kit is provided comprising a first primer pair for detecting the Trichomonas vaginalis 40S ribosomal protein (Tv40Srp) gene or RNA. In some embodiments, the 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 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 kit comprises a third primer pair for detecting an exogenous control, wherein the third primer pair is in the same or different compositions as the primer pair for detecting Tv40Srp and the second 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 a bacterial gene.

In some embodiments, the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence of 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 of 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 contain 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 implementations, the first primer pair comprises the first primer of SEQ ID NO: 1 and the second primer of SEQ ID NO: 2.

In some embodiments, the kit comprises a first probe capable of selectively hybridizing with Tv40Srp amplicon generated by a first primer pair, wherein the first probe is in the same or different compositions with one or more of the primer pairs. 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 implementations, the Tv40Srp amplicon has the sequence SEQ ID NO: 5.

In some embodiments, the kit includes a second probe capable of selectively hybridizing with endogenous control amplicon generated by a second primer pair, wherein the second probe is in the same or different compositions with one or more primer pairs. In some embodiments, the kit includes a third probe capable of selectively hybridizing with exogenous control amplicon generated by a third primer pair, wherein the third probe is in the same or different compositions with one or more primer pairs.

In some embodiments, the kit comprises dNTPs and/or a heat-stable polymerase. In some embodiments, the kit comprises more than one lyophilized composition.

In some embodiments, a primer is provided, wherein the primer comprises the sequence of SEQ ID NO: 1, wherein the primer contains at least one modified nucleotide. In some embodiments, a primer is provided, wherein the primer comprises the sequence of SEQ ID NO: 2, wherein the primer contains at least one modified nucleotide. In some embodiments, a probe is provided, wherein the probe comprises the sequence of SEQ ID NO: 3, wherein the probe contains 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 contains at least one modified nucleotide.

In some embodiments, a composition is provided comprising a first primer consisting of the sequence of SEQ ID NO: 2 and a second primer consisting of the sequence of SEQ ID NO: 3, wherein each of the first and second primers comprises at least one modified nucleotide. In some embodiments, the composition comprises a probe consisting 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. In some embodiments, the composition is a lyophilized composition. In some embodiments, the composition is in solution. In some embodiments, the composition comprises nucleic acids from a sample from a subject.

Further embodiments and details of the present invention are described below.

4. Detailed Explanation

4.1 Definition

To aid in understanding this invention, several terms and phrases are defined below:

As used herein, the terms “detect,” “detecting,” or “detection” can describe the general behavior of discovering or understanding or specifically observing a composition that detects a marker.

As used in this article, the term "dissimilar in detection" refers to a set of markers (such as dyes) that can be detected and distinguished simultaneously.

As used herein, the terms "patient" and "subject" may be used interchangeably to refer to humans. In some embodiments, the methods described herein may be used on samples from non-human animals.

"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, odor, urinary discomfort, or clear, white, yellow, or green discharge. Symptoms in men include, but are not limited to, penile itching or burning, burning after ejaculation or urination, or penile discharge.

As used herein, the terms “oligonucleotide,” “polynucleotide,” “nucleic acid molecule,” etc., refer to molecules containing nucleic acids, including but not limited to DNA or RNA. This term encompasses sequences including, but not limited to, any known base analogues of DNA and RNA, such as, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridylcytosine, 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, and 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, N-uracil-5-oxyacetate, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

As used herein, the term "oligonucleotide" refers to a single-stranded polynucleotide having fewer than 500 nucleotides. In some embodiments, oligonucleotides are 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to 75, or 12 to 50 nucleotides in length. Oligonucleotides can be indicated by their length; for example, a 24-residue oligonucleotide can be referred to as a "24-mer (24-mer)".

As used herein, the term “complementary” to the target gene (or its target region) is used, and the percentage of “complementarity” between the probe sequence and the target gene sequence is the percentage of “identity” with the target gene sequence or its reverse complement. In determining the degree of “complementarity” between the probe (or its region) used in the compositions described herein and the target gene (as disclosed herein), the degree of “complementarity” is expressed as the percentage identity between the probe sequence (or its region) and its best-aligned target gene sequence or its reverse complement. The percentage is calculated by counting the number of identical aligned bases between the two sequences, dividing by the total number of consecutive nucleotides in the probe, and multiplying by 100. When the term “complementary” is used, 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.

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

The term "nucleic acid amplification" includes any method by which at least a portion of a 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-replicaase amplification, primer extension, strand substitution amplification (SDA), hyperbranched strand substitution amplification, multiplex substitution amplification (MDA), nucleic acid chain-based amplification (NASBA), two-step multiplex amplification, rolling circle amplification (RCA), etc., 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, etc. Descriptions of such techniques can be found in 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, N.J. (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; Wen z 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 promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit Instruction Manual, Cat.#200520, Rev.#050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:18 8-93 (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., N ucl.Acid Res.20:1691-96 (1992); Polstra et al., BMC Inf.Dis.2:18- (2002); Lage et al., Genome Res. Feb. 2003;13(2):294-307; and Landegren et al., Science 241:1077-80 (1988); Demidov, V., Expert Rev Mol Diagn. 2002 Nov.;2(6):54 2-8. Cook et al., J Microbiol Methods. May 2003; 53(2): 165-74, Schweitzer et al., Curr Opin Biotechnol. February 2001; 12(1): 21-7, found in U.S. Patent Nos. 5,830,711, 6,027,889, 5,686,243, PCT Publication No. WO0056927A3, and PCT Publication No. WO9803673A1.

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.

Unless otherwise stated, the term “hybridization” as used herein refers to “specific hybridization,” which is the preferential binding, double-stranding, or hybridization of a nucleic acid molecule to a specific 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, with less or no hybridization to other sequences. In the case of nucleic acid hybridization (e.g., in array, DNA blotting, or RNA blotting hybridization), “stringent hybridization” and “stringent hybridization washing conditions” are sequence-dependent and vary under different environmental parameters. Extensive guidance on nucleic acid hybridization can be 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 washing conditions for filter paper hybridization are chosen to be approximately 5°C, below the pyrolysis point of the specific sequence at defined ionic strengths and pH. T _{_m} is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe (within defined ionic strengths and pH). Very stringent conditions are chosen to be equal to T _{_m} for a specific probe. The dependence of hybridization stringency on buffer composition, temperature, and probe length is known to those skilled in the art (see, for example, Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd ed.) Vols. 1–3, ColdSpring Harbor Laboratory, ColdSpring Harbor Press, NY).

As used herein, "sample" includes urine samples (including samples derived from urine samples), cervical swabs, and patient-collected vaginal swabs, 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, samples containing cells derived from the urine sample, samples containing cell pellets separated by centrifugation of the urine sample, samples containing cells separated by filtering the urine sample, etc. In some embodiments, the urine sample contains a buffer solution, such as a preservative. In some embodiments, the sample is a human sample other than a urine sample, such as an internal cervical swab or vaginal swab, including patient-collected vaginal swabs and urethral swabs. In some embodiments, the swab sample contains a buffer solution, such as a preservative.

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

As used herein, "exogenous control" refers to a portion added to a sample or assay, such as a "sample processing control" (SPC). In some embodiments, the exogenous control includes the assay reagent. Exogenous controls are typically selected to be either not expected to be present in the sample for detection, or present in the sample at very low levels such that the amount of that portion naturally present in the sample is undetectable or detectable at levels much lower than the amount added to the sample as an exogenous control. In some embodiments, the exogenous control comprises a nucleotide sequence not expected to be present in the sample type used to detect the target gene. In some embodiments, the exogenous control comprises a nucleotide sequence known not to be present in the species from which the sample was collected. In some embodiments, the exogenous control comprises a nucleotide sequence from a species different from the subject from which the sample was collected. In some embodiments, the exogenous control comprises a nucleotide sequence known not to be present in any species. In some embodiments, the exogenous control is selected as one that can be detected in the same manner as the target gene, and in some embodiments, it is detected simultaneously with the target gene. In some embodiments, the exogenous control is bacterial DNA. In some embodiments, the bacteria are a species not expected to be found in the test sample type.

In the sequences described herein, “U” and “T” may be used interchangeably, with the two letters representing uracil or thymine at that position. From the context and/or intended use, those skilled in the art will understand whether uracil or thymine is intended and/or should be used at that position in the sequence. For example, those skilled in the art will understand that natural RNA molecules typically include uracil, while natural DNA molecules typically include thymine. Therefore, in the case of an RNA sequence containing “T”, those skilled in the art will understand that the position in natural RNA is likely uracil.

In this disclosure, "selected from..." includes "selected from one sequence" and "selected from more than one sequence". Therefore, when using "selected from...", it should be understood that one or more of the listed sequences can be selected.

4.2 Detection of Trichomonas vaginalis

The inventors have developed an assay for detecting Trichomonas vaginalis (TV). In some embodiments, the assay includes detecting the TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, the assay includes detecting RNA transcribed from the TV 40S ribosomal protein (Tv40Srp) gene. This assay relies on 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 present invention include the Infinity system and the Smartcycler system (Cepheid, Sunnyvale, CA). This assay uses an automated system, for example, that can be completed within 3 hours, and in some embodiments, within 2 hours.

4.2.1 General Method

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

In some embodiments, methods for detecting Trichomonas vaginalis (TV) in a subject include detecting the TV 40S ribosomal protein (Tv40Srp) gene in a sample from the subject. In some embodiments, the sample is selected from urine samples, cervical swabs, and vaginal swabs. In some embodiments, the urine sample is a first-time urine sample.

In some embodiments, the method for detecting TV also includes detecting at least one endogenous control, such as a sample adequacy control (SAC). In some embodiments, the method for detecting TV also includes detecting at least one exogenous control, such as a sample processing control (SPC). In some embodiments, the method for detecting TV also includes detecting at least one endogenous control and at least one exogenous control.

In some embodiments, methods for detecting TV include detecting the TV 40S ribosomal protein (Tv40Srp) gene in a sample. In some embodiments, methods for detecting TV also include detecting a sample adequacy control (SAC), such as a single copy of the human gene. In some embodiments, methods for detecting TV also include detecting a sample treatment control (SPC), such as exogenously added bacterial DNA. In some embodiments, methods for detecting TV also include detecting both SAC and SPC.

In this disclosure, for convenience, the term "target gene" is used to refer to the TV 40S ribosomal protein (Tv40Srp) gene, and also to exogenous and/or endogenous controls. Therefore, it is to be understood that when discussing the 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).

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 solution (such as a preservative) has been added. In some embodiments, a buffer solution 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 intracervical 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 intracervical swab sample or a vaginal swab sample placed in a buffer solution (such as a preservative). In some embodiments, the swab is placed in 1 mL, 2 mL, or 2.5 mL of buffer solution.

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, the detection is performed quantitatively. In other embodiments, the detection is performed qualitatively. In some embodiments, detecting the target gene involves forming a complex of polynucleotides and nucleic acids comprising a target gene, a DNA amplicon of the target gene, and a complement of the target gene. In some embodiments, detecting the target gene includes PCR. In some embodiments, detecting the target gene includes quantitative PCR or real-time PCR. In some embodiments, a sample adequacy control (SAC) and/or a sample processing control (SPC) are detected using the same assay as the target gene. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is detected, TV is considered 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 undetectable only if SPC and SAC are also detected in the assay.

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

In some embodiments, this assay can be used as part of a subject's routine and/or preventative healthcare. That is, in some embodiments, this assay can be used to test an individual for TV infection, regardless of whether the individual displays symptoms of TV infection or has a history of TV infection. In some embodiments, this assay is used to detect TV infection in pregnant and/or attempting pregnancy subjects. In some cases, pregnant women with TV are more likely to experience preterm birth and/or have low birth weight infants (less than 5.5 pounds).

In some embodiments, the sample to be tested is a urine sample (such as a first urine sample) or a sample derived from urine. In some embodiments, a buffer solution (such as a preservative) is added to the urine sample. In some embodiments, the buffer solution is added to the urine sample within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after sample collection.

In some embodiments, the sample to be tested is a cervical swab or a vaginal swab. In some embodiments, the swab is placed in a buffer solution. In some embodiments, the swab is placed in the buffer solution immediately. In some embodiments, the swab is placed in the buffer solution within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, or 8 hours after sample collection.

In some embodiments, urine samples of 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 are used in this method. In some embodiments, urine samples of 0.1 ml to 1 ml are used in this method.

In some implementations, the sample to be tested is another bodily fluid, such as blood, sputum, mucus, saliva, vaginal or penile discharge, semen, etc.

In some embodiments, the clinical sample to be tested is fresh (i.e., unfrozen). In other embodiments, the sample is a frozen sample. In some embodiments, the sample is a tissue sample, such as a formalin-fixed paraffin-embedded sample. In some embodiments, the sample is a liquid cytology sample.

In some embodiments, the samples to be tested are obtained from individuals exhibiting more than one symptom of TV infection. Non-limiting exemplary symptoms of TV infection include vaginitis, urethritis, and cervicitis; in women: genital itching, burning, redness, or pain, odor, urinary discomfort, and clear, white, yellow, or green discharge; and in men: penile itching or burning, burning after ejaculation or urination, and penile discharge. In some embodiments, the samples to be tested are obtained from individuals previously diagnosed with TV infection. In some such embodiments, individuals are monitored for recurrence of TV infection.

In some embodiments, the methods described herein can be used for routine screening of healthy individuals without risk factors. In some embodiments, the methods described herein are used, for example, to screen asymptomatic individuals in routine or preventative healthcare. In some embodiments, the methods described herein are used to screen women who are pregnant or attempting to become pregnant.

In some implementations, the methods described herein can be used to evaluate the effectiveness of treatment for TV infection in patients.

In some implementations, the TV 40S ribosomal protein (Tv40Srp) gene is provided for the detection of TV infection. In some implementations, the TV 40S ribosomal protein (Tv40Srp) gene is provided for the monitoring of TV infection recurrence.

In any of the embodiments described herein, the TV 40S ribosomal protein (Tv40Srp) gene can be detected in the same assay reaction as the Sample Processing Control (SPC) and/or Sample Adequacy Control (SAC).

In some embodiments, methods are provided to aid in the detection of TV infection in subjects. The methods include detecting the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene in samples from the subject. In some embodiments, information regarding the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene in samples from the subject is communicated to a medical practitioner. As used herein, "medical practitioner" refers to an individual or entity that diagnoses and/or treats patients, such as a hospital, clinic, physician's office, physician, nurse, or institution of any of the aforementioned entities or individuals. In some embodiments, the detection of the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene is performed in a laboratory that receives the subject's sample from a medical practitioner or the medical practitioner's institution. The laboratory performs the detection by any method, including those described herein, and subsequently communicates the results to the medical practitioner. When provided to a medical practitioner in any manner, the results are "communicated" as used herein. In some embodiments, the communication may be oral or written, and may be conducted by telephone, personal contact, email, or other messengers, or by storing the information directly in, for example, a database accessible to medical practitioners, including databases not controlled by medical practitioners. In some embodiments, the information is maintained in electronic form. In some embodiments, the information may be stored in memory or other computer-readable media, such as RAM, ROM, EEPROM, flash memory, computer chips, digital video optical discs (DVDs), compressed disks (CDs), hard disk drives (HDDs), magnetic tape, etc.

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, "laboratory" means any device that detects the target gene in a sample by any method (including those 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.

When a laboratory communicates the results of testing for the presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene to medical practitioners, 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”.

As used herein, when a method involves 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 includes examples of performing methods that result in positive or negative results.

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

4.2.2 Exemplary Comparison

In some embodiments, the assay described herein includes detection of 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 SAC is also not detected in the sample, the assay result is considered "invalid" because the sample may be insufficient. Without being limited by any particular theory, an insufficient sample may be too dilute, contain too little cellular material, contain assay inhibitors, etc. In some embodiments, failure to detect SAC may indicate a failed assay response. In some embodiments, the endogenous control (such as SAC) is a single copy of a human gene. Non-limiting exemplary SACs include human hydroxymethyl-bilirubin synthase (HMBS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, β2-microglobin, cyclooxygenase 1, hypoxanthine phosphoribosyltransferase, bile pigment deaminase, and transferrin receptor.

In some embodiments, the assay described herein includes detection of 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 also not detected in the sample, the assay result is considered "invalid" because errors may have occurred in the sample processing, including but not limited to assay failure. Non-limiting exemplary errors in sample processing include inadequate sample processing, the presence of assay inhibitors, damaged reagents, etc. In some embodiments, an exogenous control (such as an SPC) is added to the sample. In some embodiments, the exogenous control (such as an SPC) is added during the assay, such as using more than one buffer or reagent. In some embodiments, the SPC is included in a cartridge when the system is to be used. In some embodiments, the exogenous control (such as 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.

In some embodiments, when detecting the TV 40S ribosomal protein (Tv40Srp) gene in a sample, an endogenous control and/or an exogenous control are simultaneously detected, as in the same assay. In some embodiments, the assay includes 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 includes 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, as well as a labeled probe (e.g., a probe) for detecting the amplification product.

4.2.3 Exemplary Sample Preparation

4.2.3.1 Exemplary Buffer Solution

In some embodiments, a buffer solution is added to the urine sample. In some embodiments, the buffer solution is added (e.g., hollow) within one hour, two hours, three hours, or six hours after the urine sample is collected. In some embodiments, the buffer solution 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.

In some embodiments, the swab sample is placed in a buffer solution. In some embodiments, the swab sample is placed in the buffer solution within one hour, two hours, three hours, or six hours after the swab sample is collected. In some embodiments, the swab sample is placed in the buffer solution within one hour, two hours, three hours, or six hours before the sample is analyzed by the method described herein.

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

4.2.3.2 Exemplary DNA Preparation

Sample DNA can be prepared by any suitable method. In some embodiments, target DNA is prepared by contacting the sample with a lysis buffer and binding the DNA to a DNA-binding substrate, such as a glass or silica substrate. The binding substrate can be in any suitable form, such as granules, porous solids, or membranes. For example, the support can comprise hydroxycellulose, glass fiber, cellulose, nitrocellulose, zirconium hydroxide, titanium dioxide (IV), silica, zirconium silicate, or silica particles (see, for example, U.S. Patent No. 5,234,809). Many such DNA-binding substrates are known in the art.

In some embodiments, DNA is detected in the 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 sonication (e.g., 2-15 seconds, 8-18 μm, at 36 kHz); chemical lysis, e.g., using a detergent; and various commercially available lysis reagents. In some embodiments, DNA is detected in a sample in which DNA has been isolated or separated from at least some other cellular components.

When the methods discussed herein indicate the detection of a target gene, the detection may be performed on a complement of the target gene (instead of the target gene sequence shown herein, or any other target gene sequence shown herein). In some embodiments, when detecting a complement of the 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 of the sequence that is at least 90%, at least 95%, or 100% identical to the target gene, although it may contain modified nucleotides.

4.2.4 Exemplary Analysis Method

As described above, a method for detecting Trichomonas vaginalis is provided. The method includes detecting the presence of the TV 40S ribosomal protein (Tv40Srp) gene in a sample from a subject. In some embodiments, the method further includes detecting at least one endogenous control (e.g., SAC) and/or at least one exogenous control (e.g., SPC). In some embodiments, detection of the TV 40S ribosomal protein (Tv40Srp) gene indicates the presence of TV even if the endogenous and/or exogenous controls are not detected in the assay. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is not detected, the result is considered negative for TV only if the control is detected. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is not detected, the result is considered negative for TV only if both the endogenous and exogenous controls are detected.

Any analytical step that allows for the specific detection of target genes can be used in the methods provided herein. Exemplary non-limiting analytical steps 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.

In some implementations, methods for detecting target genes, such as the TV 40S ribosomal protein (Tv40Srp) gene, include amplifying the gene and/or its complement. The amplification can be performed 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 T7 polymerase, such as that provided by the SenseAmp Plus ^™ Kit available at Implen, Germany.

When amplifying a target gene, in some embodiments, an 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 associated with the target gene in a sense or antisense orientation. In some embodiments, the amplicon of the target gene is detected instead of the target gene itself. Therefore, when the methods discussed herein indicate that the target gene is detected, the detection can be performed on the amplicon of the target gene (instead of the target gene itself, or in addition to the target gene itself). In some embodiments, when detecting the amplicon of the target gene instead of the target gene, a polynucleotide complementary to the complement of the target gene is used for detection. In some embodiments, when detecting the amplicon of the target gene instead of the target gene, a polynucleotide complementary to the target gene is used for detection. Furthermore, in some embodiments, multiple 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.

In some embodiments, methods for detecting the TV 40S ribosomal protein (Tv40Srp) gene include PCR, as described below. In some embodiments, detection of more than one target gene includes real-time monitoring of the PCR reaction, which can be performed by any method. These methods include, but are not limited to, the use of molecular beacons or Scorpion probes (i.e., energy transfer (ET) probes, such as FRET probes) and the use of complementary dyes such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc.

Non-limiting exemplary conditions for amplifying the target gene are as follows. An exemplary cycle includes an 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. A further exemplary cycle includes 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 denaturation cycle 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, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 times. In some embodiments, Taq polymerase with hot-start capability is used. In some embodiments, the amplification reaction occurs in a tube, and the amplification of the TV 40S ribosomal protein (Tv40Srp) gene, endogenous control, and exogenous control occurs in the same reaction. In some embodiments, the detection of the TV 40S ribosomal protein (Tv40Srp) gene, from the onset of denaturation to the final extension, occurs in less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes.

In some embodiments, detection of a target gene involves forming a complex comprising a polynucleotide complementary to the target gene or its complement, and a nucleic acid selected from the target gene, a DNA amplicon of the target gene, and a complement of the target gene. Thus, in some embodiments, the polynucleotide forms a complex with the target gene. In some embodiments, the polynucleotide forms a complex with a complement of the target gene. In some embodiments, the polynucleotide forms a complex with a DNA amplicon of the target gene. When a double-stranded DNA amplicon is part of the complex, as used herein, the complex may contain one or both strands of the DNA amplicon. Thus, in some embodiments, the complex contains only one strand of the DNA amplicon. In some embodiments, the complex is a triplet and contains both strands of the polynucleotide and the DNA amplicon. In some embodiments, the complex is formed by hybridization between the polynucleotide and the target gene, a complement of the target gene, or a DNA amplicon of the target gene. In some embodiments, the polynucleotide is a primer or probe.

In some embodiments, the method includes detecting the complex. In some embodiments, the complex does not necessarily need to be associated during detection. That is, in some embodiments, the complex is formed, then the complex is dissociated or destroyed in some way, and the components from the complex are detected. An example of this system is assay. In some embodiments, when the polynucleotide is the primer, the detection of the complex may include amplification of the target gene, a complement of the target gene, or a DNA amplicon of the target gene.

In some embodiments, the analytical method for detecting at least one target gene in the methods described herein includes real-time quantitative PCR. In some embodiments, the analytical method for detecting at least one target gene includes the use of a probe. The assay uses energy transfer (“ET”), such as fluorescence resonance energy transfer (“FRET”), to detect and quantify the synthesized PCR product. Typically, the probe comprises a fluorescent dye molecule coupled to the 5’-end and a quencher molecule coupled to the 3’-end, such that the dye and quencher are in close proximity, allowing the quencher to inhibit the fluorescence signal of the dye via FRET. When the polymerase replicates the probe-bound chimeric amplicon template, the polymerase’s 5’-nuclease cleaves the probe, uncoupling the dye and quencher, thereby detecting the dye signal (e.g., fluorescence). The signal (e.g., fluorescence) increases proportionally to the amount of probe cleaved per PCR cycle.

In some implementations, the target gene is considered detected if any signal is generated from the TaqMan probe during a PCR cycle. For example, in some implementations, if the PCR consists of 40 cycles, the target gene is considered present and detected if a signal is generated in any cycle during amplification. In some implementations, the target gene is considered absent and undetectable if no signal is generated at the end of the PCR cycle.

In some embodiments, quantification of real-time PCR results is accomplished by constructing a standard curve from known concentrations of nucleic acids and subsequently extrapolating quantitative information about unknown concentrations of target genes. 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.

In some implementations, in order to determine whether TV is present in the sample, the Ct values for endogenous controls (such as SAC) and/or exogenous controls (such as SPC) must be within a predetermined valid range. That is, in some implementations, the absence of TV cannot be confirmed unless a control is detected, indicating that the assay is successful. The Ct value is inversely proportional to the amount of nucleic acid target in the sample.

In some implementations, a threshold Ct (or “cutoff value Ct”) value (below which is considered detected) for the target gene (including endogenous and/or exogenous controls) is determined in advance. In some implementations, the threshold Ct is determined using substantially the same assay conditions and systems (e.g.), and the sample is tested based on this.

In addition to assays, other real-time PCR chemistry used in the methods provided herein for the detection and quantification of PCR products includes, but is 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.

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

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

In some embodiments, the detection and/or quantification of real-time PCR products are performed 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 a (DNA-mediated annealing, selection, extension, and ligation) assay. In some embodiments, the analytical method used to detect and quantify the target genes in the methods described herein is a bead-based flow cytometry assay. See Lu J. et al. (2005) Nature 435:834-838, which is incorporated herein by reference in its entirety. An example of bead-based flow cytometry assays is the technology of Luminex, Inc. See www.luminexcorp.com/technology/index.html. In some embodiments, the analytical method used to detect and quantify the level of at least one target gene in the methods described herein is by gel electrophoresis and detection with labeled probes (e.g., probes labeled with radioactive or chemiluminescent labels), such as by RNA blotting. In some embodiments, exemplary probes contain one or more affinity-enhancing nucleotide analogs, such as locked nucleic acid (“LNA”) analogs, discussed below, which contain a bicyclic sugar moiety instead of deoxyribose or ribose. See, for example, Várallyay, E. et al. (2008) Nature Protocols 3(2): 190-196, which is incorporated herein by reference in its entirety. In some embodiments, the detection and quantification of one or more target genes are accomplished using microfluidic devices and single-molecule detection.

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

4.2.5 Exemplary Automation and Systems

In some implementations, automated sample processing and/or analysis platforms are used to detect gene expression. In some implementations, commercially available automated analysis platforms are utilized. For example, in some implementations, systems (Cepheid, Sunnyvale, CA) are used.

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 analytical platform. Those skilled in the art will understand that any number of platforms and methods can be used.

Using proprietary, single-purpose cartridges, sample extraction, amplification, and detection can all be performed within this proprietary "laboratory in a cartridge" (see, for example, 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).

The components of the tube include, but are not limited to, a processing chamber containing reagents, a filter, and capture technology for extracting, purifying, and amplifying target nucleic acids. Valves allow for fluid transfer between chambers and include nucleic acid lysis and filtration components. Optical windows enable real-time optical detection. Reaction tubes enable very rapid thermal cycling.

In some implementations, the system includes multiple modules for scalability. Each module includes multiple cylinders, along with sample processing and analysis components.

After the sample is added to the cartridge, it comes into contact with 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 eluent can then be processed in the cartridge to detect the target gene as described herein. In some embodiments, the eluent is used to reconstruct at least some PCR reagents, which are present in the cartridge as lyophilized particles.

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

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. Then, all or most of the DNA-containing sample (e.g., DNA eluate) is denatured a second time, after which, in some cases, a portion of the sample is aliquoted for cycling and detection of the target gene. Without being limited by any particular theory, the double-denaturation protocol can increase the probability that a low copy number target gene (or its complement) will be present in the aliquot selected for cycling and detection because the second denaturation effectively doubles the number of targets (i.e., it separates the target and its complement into two separate templates), after which the aliquot is selected for cycling. 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 and/or second denaturation steps are performed by separately heating aliquots of the sample. In some embodiments, each aliquot may be heated the aforementioned number of times. As a non-limiting example, a first denaturing step for a DNA-containing sample (such as DNA eluent) may 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 denaturing step for a DNA-containing sample (such as DNA eluent) containing enzymes, primers, and probes may include heating at least one, at least two, at least three, or at least four aliquots of the eluent (sequentially or simultaneously) separately to a temperature of 90°C to 100°C for 5 seconds each. In some embodiments, the aliquots are the entire DNA-containing sample (such as DNA eluent). In some embodiments, the aliquots are less than the entire DNA-containing sample (such as DNA eluent).

In some implementations, the following procedure is used to detect target genes in DNA-containing samples, such as DNA eluates: One or more aliquots of the DNA-containing sample are separately heated 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 separately heated 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 using the following two steps: (1) 94°C for 5 seconds, (2) 66°C for 30 seconds.

This invention is not limited to specific primer and/or probe sequences. Exemplary amplification primers and detection probes are described in the examples.

In some implementations, in-line centrifugation is used, for example, with samples having low cell content. The sample (with or without buffer added) is centrifuged and the supernatant is removed. The precipitate is then resuspended in a smaller volume of supernatant or buffer. The resuspended precipitate is then analyzed as described herein.

4.2.6 Exemplary Data Analysis

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

In some embodiments, computer-based analysis programs are used to translate the raw data generated by the assays into predictive data for clinicians. Clinicians can access the predictive data using any suitable method. Therefore, in some embodiments, the invention provides a further benefit: clinicians who may not be trained in genetics or molecular biology do not need to understand the raw data. The data is provided directly to the clinician in its most useful form. The clinician can then immediately utilize the information to optimize the care of the patient.

This invention contemplates any method capable of receiving, processing, and transmitting information to and from laboratories performing assays, providing information, medical individuals, and subjects. For example, in some embodiments of the invention, samples (e.g., biopsy, serum, or urine samples) are obtained from subjects and profiling services (e.g., clinical laboratories at medical facilities, genomic analysis services, etc.) are performed, located anywhere in the world (e.g., different from the country of residence of the subject or the country where the information is ultimately used) to generate raw data. In cases where the sample contains tissue or other biological samples, the subject may visit a medical center to obtain the sample and send it to the analysis center, or the subject may collect the sample themselves (e.g., urine samples) and send it directly to the analysis center. In cases where the sample contains previously identified biological information, the information may be sent directly from the subject to the analysis service (e.g., by scanning an information card containing the information with a computer and transmitting the data to a computer at the analysis center using an electronic communication system). Once received by the analysis service, the sample is processed and profiling results (i.e., expression data) are generated, specific to the diagnostic or prognostic information required by the subject.

The analyzed data is then prepared in a format suitable for interpretation by the treating clinician. For example, instead of providing raw expression data, the prepared format may represent a diagnosis or risk assessment of the subject (e.g., presence of TV), suggesting or discouraging a particular treatment option. The data can be presented to the clinician using any suitable method. For example, in some implementations, the analysis service generates reports that can be printed for the clinician (e.g., at the point of care) or displayed to the clinician on a computer monitor.

In some implementations, information is first analyzed at the point of care or at a regional facility. The raw data is then sent to a central processing facility for further analysis and/or to transform the raw data into information for clinicians or patients. Central processing facilities offer advantages in data analysis, including privacy (all data is stored in the central facility under a uniform security protocol), speed, and uniformity. The central processing facility can then control the fate of the data after the subjects have been treated. For example, using electronic communication systems, the central facility can make the data available to clinicians, subjects, or researchers.

In some implementations, subjects are able to access data directly using electronic communication systems. Subjects may choose to undergo further intervention or consultation based on the results. In some implementations, the data is used for research purposes. For example, the data may be used to further optimize the inclusion or exclusion of useful indicators as specific conditions or stages of the disease, or as biomarkers for companion diagnoses, to determine the treatment course.

4.2.7 Exemplary Polynucleotides

In some embodiments, a polynucleotide is provided. In some embodiments, a synthetic polynucleotide is provided. As used herein, a synthetic polynucleotide refers to a polynucleotide synthesized in vitro by chemical or enzymatic methods. Chemical synthesis of polynucleotides includes, but is not limited to, synthesis using a polynucleotide synthesizer such as the OligoPilot (GE Healthcare), the ABI 3900 DNA synthesizer (Applied Biosystems), etc. Enzymatic synthesis includes, but is not limited to, generating polynucleotides by enzymatic amplification, for example, PCR. Polynucleotides may contain more than one nucleotide analogue (i.e., modified nucleotide) discussed herein.

In some embodiments, a polynucleotide is provided comprising a region comprising 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 that are at least 90%, at least 95%, or 100% identical, or at least 90%, at least 95%, or 100% complementary to the TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, a polynucleotide is provided comprising a region comprising 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides that are at least 90%, at least 95%, or 100% identical, or complementary to the TV 40S ribosomal protein (Tv40Srp) gene. Non-limiting exemplary polynucleotides are shown in Table 1.

In various embodiments, the polynucleotide comprises less than 500, less than 300, less than 200, less than 150, less than 100, less than 75, less than 50, less than 40, or less than 30 nucleotides. In various embodiments, the polynucleotide has a length of nucleotides 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.

In some embodiments, the polynucleotide is the primer. In some embodiments, the primer is labeled with a detectable portion. In some embodiments, the primer is unlabeled. As used herein, the primer is a polynucleotide capable of selectively hybridizing with a target gene or with an amplicon amplified from a target gene (collectively, the “template”), and, in the presence of the template, polymerase and suitable buffers and reagents can be extended to form a primer extension product.

In some embodiments, the polynucleotide is the probe. In some embodiments, the probe is labeled with a detectable moiety. Detectable moieties, as used herein, include directly detectable moieties, such as fluorescent dyes, and indirectly detectable moieties, such as binding pair members. In some embodiments, when the detectable moieties are members of a binding pair, the probe can be detected by incubating the probe with a detectable label bound to a second member of the binding pair. In some embodiments, the probe is unlabeled, such as when the probe is a capture probe, for example, on a microarray or beads. In some embodiments, the probe is non-extendable, for example, extended by a polymerase. In other embodiments, the probe is extendable.

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, wherein the quencher is a chemical group that absorbs (i.e., inhibits) fluorescence emission from the dye when the groups are close together (i.e., attached to the same probe). Therefore, in some embodiments, the emission spectrum of the dye should overlap considerably with the absorption spectrum of the quencher. In other embodiments, the dye and quencher are not at the end of the FRET probe.

4.2.7.1 Exemplary Polynucleotide Modifications

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

In some implementations, affinity-enhancing nucleotide analogs include nucleotides with one or more base modifications, sugar modifications, and/or backbone modifications.

In some embodiments, the bases used for modifying 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.

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

In some embodiments, affinity-enhancing nucleotide analogs include backbone modifications such as the use of peptide nucleic acids (PNAs; for example, oligomers comprising nucleobases linked together by an amino acid backbone). Other backbone modifications include thiophosphonothioyl linkages, phosphodiester-modified nucleic acids, combinations of phosphodiester and thiophosphonothioyl nucleic acids, methyl phosphonate, alkyl phosphonate, phosphate esters, alkyl thiophosphonates, aminophosphate esters, carbamates, carbonates, triphosphate esters, acetamide esters, carboxymethyl esters, methyl thiophosphonothioyl, dithiophosphate esters, p-ethoxy, and combinations thereof.

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

In some embodiments, the affinity-enhancing nucleotide analog contains locked nucleic acid (“LNA”) sugars, which are bicyclic sugars. In some embodiments, the polynucleotide used in the methods described herein comprises more than one nucleotide having an LNA sugar. In some embodiments, the polynucleotide contains more than one region consisting of nucleotides having an LNA sugar. In other embodiments, the polynucleotide contains nucleotides having LNA sugars dispersed with deoxyribonucleotides. See, for example, Frieden, M. et al. (2008) Curr. Pharm. Des. 14(11): 1138-1142.

4.2.7.2 Exemplary Primers

In some embodiments, primers are provided. In some embodiments, the primers are at least 90%, at least 95%, or 100% identical, complementary, or 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 TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, primers are provided comprising a region that is at least 90%, at least 95%, or 100% identical, or complementary, to at least 90%, at least 95%, or 100% identical, or complementary to at least 90%, at least 95%, or 100% identical, or complementary to at least 6 to 100, 8 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides of the TV 40S ribosomal protein (Tv40Srp) gene. Non-limiting exemplary primers are shown in Table 1. In some embodiments, primers may also contain portions or regions that are not identical to or complementary to the target gene. In some embodiments, regions of primers that are at least 90%, at least 95%, or 100% identical to or complementary to the target gene are continuous, such that any region of primers that are not identical to or complementary to the target gene does not disrupt the identical or complementary region.

In some embodiments, the primers contain a portion that is at least 90%, at least 95%, or 100% identical to the region of the target gene. In some such embodiments, primers containing a region that is at least 90%, at least 95%, or 100% identical to the region of the target gene are capable of selectively hybridizing with amplicones generated by amplification of the target gene. In some embodiments, the primers are complementary to a sufficient portion of the amplicon so that they selectively hybridize with the amplicon under the specific assay conditions used.

As used herein, “selective hybridization” means that a polynucleotide, such as a primer or probe, will hybridize with 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 but having a different nucleotide sequence in the hybridization region. Exemplary hybridization conditions are discussed herein, for example, in the case of reverse transcription or PCR amplification reactions. In some embodiments, the polynucleotide will hybridize with 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 but having a different nucleotide sequence in the hybridization region.

In some implementations, the primers contain a detectable portion.

In some embodiments, primer pairs are provided. Such primer pairs are designed to amplify a target gene, such as the TV 40S ribosomal protein (Tv40Srp) gene, or as an endogenous control such as a sample adequacy control (SAC), or as part of an exogenous control such as a sample processing control (SPC). In some embodiments, the primer pairs are designed to produce amplicons of lengths of 50 to 1500 nucleotides, 50 to 1000 nucleotides, 50 to 750 nucleotides, 50 to 500 nucleotides, 50 to 400 nucleotides, 50 to 300 nucleotides, 50 to 200 nucleotides, 50 to 150 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, or 100 to 150 nucleotides. Non-limiting exemplary primer pairs are shown in Table 1.

4.2.7.3 Exemplary Probe

In various embodiments, methods for detecting the presence of Trichomonas vaginalis include hybridizing the nucleic acids of a sample with a probe. In some embodiments, the probe contains 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 treatment control (SPC). In some embodiments, the probe contains 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 the target gene is complementary to a sufficient portion of the target gene, thereby selectively hybridizing with the target gene under the specific assay conditions used. In some embodiments, the probe 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, which is at least 90%, at least 95%, or 100% complementary to the target gene, may also comprise portions or regions that are not complementary to the target gene. In some embodiments, the probe region that is at least 90%, at least 95%, or 100% complementary to the target gene is continuous, such that any region of the probe that is not complementary to the target gene does not disrupt the complementary region.

In some embodiments, the probe comprises a region that is at least 90%, at least 95%, or 100% identical to a region of 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 treatment 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 the target gene is capable of selectively hybridizing with 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, thereby enabling it to selectively hybridize with the amplicon under the specific assay conditions used. In some embodiments, the probe complementary to the amplicon comprises a region at least 90%, at least 95%, or 100% complementary to at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive nucleotides of the amplicon. The probe at least 90%, 95%, or 100% complementary to the amplicon may also include portions or regions that are not complementary to the amplicon. In some embodiments, the probe regions at least 90%, 95%, or 100% complementary to the amplicon are continuous, such that any region of the probe that is not complementary to the amplicon does not disrupt the complementary region.

In some implementations, the method for detecting more than one target gene includes: (a) amplifying a region of the target gene; and (b) detecting the amplified region using real-time PCR and a detection probe (which may be performed simultaneously with amplification step (a)).

As described above, in some embodiments, real-time PCR detection can be performed using FRET probes, including, but not limited to, probes, molecular beacon probes, and Scorpion probes. In some embodiments, real-time PCR detection utilizes probes, which are linear probes typically covalently bound to a fluorescent dye at one end of DNA and covalently bound to a quencher molecule elsewhere (e.g., at the other end of DNA). FRET probes contain sequences complementary to the regions of the amplicons, such that when the FRET probe hybridizes with the amplicons, the dye fluorescence is quenched, and when the probe is digested during amplicon amplification, 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 amplification.

The probe typically contains a region of consecutive nucleotides having a sequence that is at least 90%, at least 95%, or 100% identical or complementary to a region of the target gene, thereby allowing the probe to selectively hybridize with the PCR amplicon of the target gene region. In some embodiments, the probe contains a region of at least 6 consecutive nucleotides having a sequence that is completely complementary to or identical to a region of the target gene present in that region. In some embodiments, the probe contains a region of at least 8, at least 10, at least 12, at least 14, or at least 16 consecutive nucleotides that is at least 90%, at least 95%, or 100% identical or complementary to the target gene to be detected.

In some embodiments, an amplicon region having a sequence that is at least 90%, at least 95%, or 100% complementary to the probe sequence is located in or near the middle of the amplicon molecule. In some embodiments, at least two nucleotides, such as at least three nucleotides, at least four nucleotides, or at least five nucleotides, are independently present at the 5'-end and 3'-end of the complementary region.

In some implementations, molecular beacons can be used to detect PCR products. Like probes, molecular beacons are used to detect PCR products via FRET, where a probe with a fluorescent dye and a quencher attached to its end is used. Unlike probes, molecular beacons remain intact during PCR cycles. When free in solution, the molecular beacon probe forms a stem-loop structure, allowing the dye and quencher to come close enough to induce fluorescence quenching. When the molecular beacon hybridizes with a target, the stem-loop structure dissolves, spatially separating the dye and quencher, and the dye fluoresces. Molecular beacons can be obtained, for example, from GeneLink ^™ (see www.genelink.com/newsite/products/mbintro.asp).

In some implementations, Scorpion probes can be used as sequence-specific primers and for PCR product detection. Like molecular beacons, Scorpion probes form a stem-loop structure when not hybridizing with the target nucleic acid. However, unlike molecular beacons, Scorpion probes achieve sequence-specific priming and PCR product detection. A fluorescent dye molecule is attached to the 5' end of the Scorpion probe, and a quencher is attached elsewhere, such as to the 3' end. The 3' portion of the probe is complementary to the extension product of the PCR primer, and this complementary portion is attached to the 5' end of the probe via a non-amplifiable portion. Upon extension of the Scorpion primer, the target-specific sequence of the probe binds to its complementary portion within the extended amplicon, thereby opening the stem-loop structure and allowing the dye at the 5' end to fluoresce and generate a signal. Scorpion probes can be obtained, for example, from Premier Biosoft International (see www.premierbiosoft.com/tech_notes/Scorpion.html).

In some implementations, labeling that can be used on FRET probes includes 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, aminocoumarin, and hydroxycoumarin; anthocyanin dyes such as Cy3 and Cy5; eosin and erythrosine; fluorescein and its derivatives such as fluorescein isothiocyanate; macrocyclic chelates of lanthanides such as QuantumDye ^™ ; Marina Blue; Oregon Green; rhodamine dyes such as rhodamine red, tetramethylrhodamine, and rhodamine 6G; Texas Red; fluorescent energy transfer dyes such as thiazole orange-ethidium heterodimer; and TOTAB.

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, and Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, 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, BO DIPY 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.

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. In some embodiments, when the donor and acceptor are the same, FRET can be detected by fluorescence depolarization when the dye is the same. Certain specific examples of dye/quencher pairs (i.e., donor/acceptor pairs) include, but are not limited to, Alexa Fluor 350/Alexa Fluor 488; Alexa Fluor 488/Alexa Fluor 546; Alexa Fluor 488/Alexa Fluor 555; Alexa Fluor 488/Alexa Fluor 568; Alexa Fluor 488/Alexa Fluor 594; Alexa Fluor 488/Alexa Fluor 647; Alexa Fluor 546/Alexa Fluor 568; Alexa Fluor 546/Alexa Fluor 594; Alexa Fluor 546/Alexa Fluor 647; Alexa Fluor 555/Alexa Fluor 594; Alexa Fluor 555/Alexa Fluor 647; Alexa Fluor 568/Alexa Fluor 647; Alexa Fluor 594/Alexa Fluor 647; Alexa Fluor 350/QSY35; Alexa Fluor 350/dabcyl; Alexa Fluor488/QSY 35; Alexa Fluor488/dabcyl; Alexa Fluor 488/QSY 7 or QSY 9; Alexa Fluor555/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, broad-spectrum quenchers such as Iowa quencher (Integrated DNA Technologies, Coralville, IA) or Black Hole quencher ^™ (BHQ ^™ ; Sigma-Aldrich, St. Louis, MO).

In some implementations, such as in multiplex reactions where two or more components (e.g., amplicon) are detected simultaneously, each probe contains a different detectable dye, allowing for dye differentiation during simultaneous detection in the same reaction. Those skilled in the art can select a set of different detectable dyes for multiplex reactions.

Specific examples of fluorescently labeled ribonucleotides that can be used to prepare PCR probes for some embodiments of the methods described herein are available from Molecular Probes (Invitrogen), and these include Alexa Fluor488-5-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, such as Cy3-UTP and Cy5-UTP, are available from Amersham Biosciences (GE Healthcare).

Examples of fluorescently labeled deoxyribonucleotides used to prepare 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 F Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650-14-dUTP, BODIPY 650/665-14-dUTP; 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.

In some embodiments, a dye and other moieties, such as quenchers, are introduced into the polynucleotide used in the methods described herein, such as FRET probes, via modified nucleotides. "Modified nucleotide" refers to a nucleotide that has been chemically modified but still functions as a nucleotide. In some embodiments, the modified nucleotide has a covalently linked chemical moieties, such as dyes or quenchers, and can be introduced into the polynucleotide, for example, via solid-phase synthesis of the polynucleotide. In other embodiments, the modified nucleotide includes one or more reactive groups that can react with the dye or quencher before, during, or after the introduction of the modified nucleotide into the nucleic acid. In a specific embodiment, the modified nucleotide is an amine-modified nucleotide, i.e., a nucleotide modified to have a reactive amine group. In some embodiments, the modified nucleotide comprises a 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-oxy-di-(ethylamine)]-CTP; N6-(6-amino)hexyl-ATP; 8-[(6-amino)hexyl]-amino-ATP; 5-propynylamino-CTP, 5-propynylamino-UTP. In some embodiments, nucleotides with different nucleobase moieties are similarly modified, for example, 5-(3-aminoallyl)-GTP replaces 5-(3-aminoallyl)-UTP. Many amine-modified nucleotides are commercially available from, for example, Applied Biosystems, Sigma, Jena Bioscience, and TriLink.

Exemplary detectable portions also include, but are not limited to, members of a binding pair. In some such embodiments, a first member of the binding pair is linked to a polynucleotide. A second member of the binding pair is linked to a detectable label, such as a fluorescent label. When a polynucleotide linked to the first member of the binding pair is incubated with the second member of the binding pair linked to the detectable label, the first and second members of the binding pair associate and the polynucleotide becomes detectable. Exemplary binding pairs include, but are not limited to, biotin and streptavidin, antibodies and antigens, etc.

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 fluorescence signal. In some embodiments, the same fluorescence signal is used to detect two or more target genes, in which case the signal detection indicates the presence of one or two target genes.

Those skilled in the art can select a suitable detection method for the chosen assay, such as real-time PCR. The selected detection method does not need to be one of the methods mentioned above and can be any method.

4.3 Exemplary Compositions and Kits

On the other hand, a composition is provided. In some embodiments, the composition is provided for use in the methods described herein.

In some embodiments, compositions comprising at least one target gene-specific primer are provided. The term "target gene-specific primer" includes a primer having a sequence having (i) a sequence that is at least 90%, at least 95%, or 100% identical to a region of the target gene, or (ii) a sequence that is at least 90%, at least 95%, or 100% complementary to a sequence of a region of consecutive nucleotides found in the target gene. In some embodiments, compositions comprising at least one pair of target gene-specific primers are provided. The term "target gene-specific primer pair" includes a primer pair suitable for amplifying a defined target gene region. A target gene-specific primer pair typically comprises a first primer and a second primer, the first primer containing a sequence that is at least 90%, at least 95%, or 100% identical to a region of the target gene, and the second primer containing a sequence that is at least 90%, at least 95%, or 100% complementary to a region of the target gene. Primer pairs are generally suitable for amplifying target gene regions of lengths of 50 to 1500 nucleotides, 50 to 1000 nucleotides, 50 to 750 nucleotides, 50 to 500 nucleotides, 50 to 400 nucleotides, 50 to 300 nucleotides, 50 to 200 nucleotides, 50 to 150 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, or 100 to 150 nucleotides. Non-limiting exemplary primers and primer pairs are shown in Table 1.

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).

In some embodiments, the composition comprises at least one target gene-specific probe. The term "target gene-specific probe" includes a probe having a sequence having (i) a sequence that is at least 90%, at least 95%, or 100% identical to a region of the target gene, or (ii) 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.

In some embodiments, the composition (including the above-described composition comprising one or more target gene-specific primers) contains one or more probes for detecting the target gene. In some embodiments, the composition contains probes for detecting endogenous controls (such as SAC) and/or probes for detecting exogenous controls (such as SPC).

In some embodiments, the composition is an aqueous composition. In some embodiments, the aqueous composition comprises a buffering 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 adapted to be restored by the addition of a fluid. The dry composition may include more than one buffering component and/or other components.

In some embodiments, the composition further includes one or more other components. These other components include, but are not limited to, salts such as NaCl, KCl, and _MgCl₂ ; polymerases, including heat-stable polymerases such as Taq; dNTPs; bovine serum albumin (BSA); reducing agents such as β-mercaptoethanol; EDTA, etc. Those skilled in the art can select appropriate composition components based on the intended use of the composition.

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 a probe, a molecular beacon, or a Scorpion probe.

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, thereby producing a unique fluorescent emission associated with a specific target gene when the probe is digested during a PCR reaction. In some embodiments, when the composition comprises multiple FRET probes, each probe is labeled with a different donor/acceptor pair, thereby each producing a unique fluorescent emission associated with a specific probe sequence and/or target gene when the probe is digested during a PCR reaction. In some embodiments, the sequence of the FRET probe is complementary to a target region of the target gene. In other embodiments, the FRET probe has a sequence containing more than one base mismatch when compared to the sequence of the best-matching target region of the target gene.

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, 9, 10, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides of the FRET probe are co-occurring in or complementary to a region of the TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, when compatible with a sequence or complement of the TV 40S ribosomal protein (Tv40Srp) gene, the FRET probe includes sequences having one, two, or three mismatches.

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

In some embodiments, more than one primer and/or probe is “linear.” A “linear” primer is a single-stranded molecule and typically does not contain, for example, a short region of at least 3, 4, or 5 consecutive nucleotides of a polynucleotide complementary to another region within the same polynucleotide, thereby forming an inner double helix. In some embodiments, primers for reverse transcription contain 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, having 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.

In some embodiments, the kit contains one or more pairs of linear primers (“forward primer” and “reverse primer”) for amplifying the target gene. Thus, in some embodiments, the first primer contains 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, having a sequence 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. Furthermore, in some embodiments, the second primer contains 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, 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 consecutive nucleotides at the second position of the target gene, thereby causing the amplicon to extend from the first position of the target gene to the second position of the target gene in a PCR reaction using the two primers.

In some embodiments, the kit contains at least two, at least three, or at least four sets of primers, each used to amplify different target genes, such as endogenous controls and/or exogenous controls.

In some embodiments, the probes and/or primers used in the compositions described herein comprise deoxyribonucleotides. In some embodiments, the probes and/or primers used in the compositions described herein comprise deoxyribonucleotides and one or more nucleotide analogs, such as LNA analogs or other stable duplex nucleotide analogs described above. In some embodiments, the probes and/or primers used in the compositions described herein comprise all nucleotide analogs. In some embodiments, the probes and/or primers comprise one or more stable duplex nucleotide analogs, such as LNA analogs, in complementary regions.

In some embodiments, the kit for the real-time PCR method described herein also includes reagents for reverse transcription and amplification reactions. In some embodiments, the kit contains 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 contains hybridization buffers optimized for probe and primer specificity.

A kit typically comprises a package containing one or more containers holding reagents, either as a separate composition or, optionally, as a mixture (where reagent compatibility is permitted). The kit may also include one or more other materials that the user may require, such as one or more buffer solutions, one or more diluents, one or more standards, and/or any other materials for sample preparation, washing, or any other steps in the assay.

The kit preferably includes instructions for use of one or more of the methods described herein. These instructions may be attached to packaging materials or included as a product information leaflet. Instructions are typically handwritten or printed materials, but are not limited to these. The invention is contemplated in any medium capable of storing these instructions and communicating them to the end user. Such media include, but are not limited to, electronic storage media (e.g., disks, magnetic tapes, magnetic cylinders, magnetic chips), optical media (e.g., CD-ROMs), etc. As used herein, the term "instructions for use" may include the website address providing the instructions.

In some embodiments, the kit may contain the reagents described above provided in more than one sample cartridge. These cartridges allow extraction, amplification, and detection to be performed in this self-contained "in-cartridge laboratory" (see, for example, 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 genome copy number levels and detecting pathogens may be provided in separate cartridges within the kit, or these reagents (suitable for multiplex detection) may be provided in a single cartridge.

In some implementations, any kit described herein may include a urine sample and/or swab container for collecting urethral swab samples, vaginal swab samples, or cervical swab samples.

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

5. Examples

5.1. Example 1: Detection of Trichomonas vaginalis

The assay was designed to detect the gene of 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 for detecting a single copy of the human gene, used as a target for sample adequacy control (SAC), were included. Primers and probes for detecting a bacterial gene, included as a target for sample treatment control (SPC), were also included.

Table 1: Primer and probe sequences

The final primer and probe compositions for multiplex assays are shown in Table 2.

Table 2: Primer and probe concentrations

F1 and F2 are different dyes for detection, which can be detected and distinguished simultaneously in multiple reactions. Each probe also contains a quencher (e.g., Q1, mentioned above).

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.

For each sample to be tested, add approximately 7 mL of the first, voided urine to 1 mL of buffer (preferably within 2 hours of sample collection). Immediately place a cervical swab collected by a physician or a vaginal swab collected by yourself (in a clinical setting) into 2.5 mL of buffer.

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

The measurement results are explained in Table 3. The effective range of Ct values for TV, SAC, and SPC targets is 9–39.9 Ct.

Table 3: Xpert TV Measurement Results and Interpretation

5.2 Example 2: Clinical Performance

The performance characteristics of the Xpert TV assay were evaluated at 13 institutions in the United States. Due to the low prevalence of Trichomonas vaginalis and the difficulty in obtaining fresh Trichomonas vaginalis-positive samples from male subjects, the sample population used in this study utilized artificial male urine samples.

Subjects included informed consent-giving asymptomatic and symptomatic sexually active men and women, observed in locations including, but not limited to: OB/GYN, sexually transmitted diseases (STDs), adolescents, public health facilities, and family planning clinics.

The study sample consisted of intended collections of male and female urine, cervical swabs, and patient-collected vaginal swabs (collected in a clinical setting). Artificial male urine samples were included to supplement the male sample size.

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

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

Results from Xpert TV assays were compared with those from a comparator, and bidirectional sequencing was performed on discrepancies. Sensitivity and specificity for sex, sample type, and symptom status are provided in Table 4.

5.3 Example 3: Detection Limit

The sensitivity or limit of detection (LoD) of the Xpert TV assay was evaluated using two Trichomonas vaginalis strains: one metronidazole-susceptible (Trichomonas vaginalis 30001 ^™ ) and one metronidazole-resistant (Trichomonas vaginalis 30238 ^™ ). Both strains were tested in male urine (MU) mixed with buffer and vaginal swabs (VS) containing Trichomonas vaginalis-negative pools in buffer.

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

Table 5: Detection limits for Trichomonas vaginalis using Xpert TV

5.4 Example 4: Determination of repeatability

Eight samples of different concentrations of Trichomonas vaginalis were tested at three sites on 12 different days by two different operators (8 samples x 1 test/day x 12 days x 2 operators x 3 sites). Three batches of Xpert TV assays were used at each of the three test sites. Xpert TV assays were performed according to the Xpert TV assay procedure. The results are summarized in Table 6.

The repeatability of Xpert TV assays was also evaluated based on the fluorescence signal expressed as Ct values for each target being detected. Mean, standard deviation (SD), and coefficient of variation (CV) for measurements across locations, batches, days, operators, and for each group member are provided in Table 7.

5.5. Example 5: Analysis of Inclusivity

The analytical inclusiveness of the Xpert TV assay was evaluated by testing 17 Trichomonas vaginalis strains in triplicate at concentrations not exceeding 3x the limit of detection (3x LoD). Each strain was tested in vaginal swabs containing Trichomonas vaginalis-negative pools in buffer (VS) and in male urine mixed with buffer (MU). See Table 8. Under these study conditions, all strains reported TV detection ED results. The Xpert TV assay demonstrated 100% inclusiveness in both sample types.

Table 8: Analytical Inclusivity Table

5.6. Example 6: Analysis of Specificity

A panel of 47 organisms (including common bacteria, fungi, and viruses of the urogenital tract, as well as other protozoa closely related to Trichomonas vaginalis) were tested using the Xpert TV assay. Each bacterial or fungal strain was tested at 1 x ^10⁷ cfu/mL or higher. Strains that did not produce countable clones were diluted to 0.5 McFarland units, approximately equivalent to 1.5 x 10⁸ cfu/mL for E. coli. Viral strains were purchased as heat-inactivated stock from ZeptoMetrix Corp. and tested at 1 x ^10⁶ U/mL or ^10⁶ genomes/mL. Protozoa were cultured in growth medium, visually counted using an optical microscope, and tested at 1 x ^10⁶ cells/mL. Tests were performed in triplicate. The organisms tested and the Xpert TV assay results are listed in Table 9.

A study of the organism *Trichomonas tenax* reported its TV detection results using the Xpert TV assay. Under the conditions of this study, the analytical specificity of the Xpert TV assay was 98%.

Table 9: Analytical Specificity Table

5.7. Example 7: Interfering Substance

In non-clinical studies, Xpert TV assays were used to evaluate potentially interfering endogenous and exogenous substances that may be present in the urogenital tract and in cervical and vaginal swabs or first urine samples.

The substance was diluted separately in pooled negative vaginal swab matrix and pooled negative male urine matrix. The substance was tested in the same matrix containing Trichomonas vaginalis cells at no more than three times the detection limit for each sample type. Eight replicates of each negative and positive sample were tested using Xpert TV and compared with results obtained in a control of the same sample without the addition of potential interfering substances. The substance and test concentrations are listed in Tables 10 and 11.

Under the study conditions, no invalid results were reported in tests using substances diluted in a negative urine matrix; as expected, all tests reported no detectable TV. 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.

Under the conditions studied, no invalid results were reported in tests using diluted negative swab matrix; as expected, all test reports showed no detection of TV.

No false-negative TV results were reported in tests of substances diluted in the pooled positive swab matrix. TV was detected as expected using test reports of all substances.

Table 10: Possible interfering substances in urine samples

Table 11: Potential interfering substances in swab samples

5.8. Example 8: Carrying Contaminants

The study consisted of repeated tests of a high ( ^10⁶ cells/mL) TV-positive vaginal swab pool in a buffer sample followed immediately by a TV-negative vaginal swab pool processed within the same GeneXpert module. The study also consisted of a combination of a TV-negative vaginal swab pool in a buffer sample processed within the same GeneXpert module followed immediately by a high ( ^10⁶ cells/mL) TV-positive vaginal swab pool in the buffer sample. This test protocol was repeated 20 times on two GeneXpert modules, for a total of 82 runs, yielding 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.

5.9. Example 9: Testing Alternative Primers and Probes for TV Detection

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

Table 12: Alternative primer and probe sequences

It was found that the TV forward ALT1 cross-reacted with *Pentatric hominis* (Pth), another closely related trichomonad found in the human gut. When the TV forward ALT1 was used for assays 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. The TV forward ALT2 was not as sensitive as designed, detecting TV with a higher Ct value of 31.5. Similarly, the TV forward ALT3 was not as sensitive as designed, also detecting TV with a higher Ct value. The TV reverse ALT1 also resulted in less sensitive assays, detecting TV with a higher Ct value. Finally, the TV probe ALT1 was less sensitive and consistent than designed.

Just as each individual publication, patent, patent application or other document is individually incorporated by reference for all purposes, all publications, patents, patent applications and other documents cited in this application are incorporated herein by reference in their entirety for all purposes.

While various specific embodiments have been described and illustrated, it will be understood that changes may be made without departing from the spirit and scope of the invention.

Tables of certain sequences

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Claims (22)

1. Use of a first primer pair in the preparation of 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 the Trichomonas vaginalis 40S ribosomal protein gene or RNA in the sample, wherein an increased level of the 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, 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 use as claimed in claim 1, wherein the control is an endogenous control. 3. The use according to claim 2, wherein the endogenous control is selected from HMBS, GAPDH, β-actin and β-globin. 4. The use as claimed in claim 1, wherein the control is an exogenous control. 5. The use as claimed in claim 4, wherein the exogenous control is a bacterial gene. 6. The use according to claim 1, wherein the detection comprises PCR. 7. The use according to claim 1, wherein the detection comprises quantitative PCR. 8. The use as described in 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 a sample with the first primer pair, the first primer pair being used to detect the Trichomonas vaginalis 40S ribosomal protein gene or RNA. 10. The use of claim 9, wherein the detection comprises contacting nucleic acid from the sample with a second primer pair and/or a third primer pair, the second primer pair being used to detect an endogenous control and the third primer pair being used to detect 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 with Trichomonas vaginalis 40S ribosomal protein, wherein the sequence of the first probe is as 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 use according to claim 10, wherein the detection includes 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 with the endogenous control amplicon and/or the exogenous control amplicon is contacted with a third probe capable of selectively hybridizing with the exogenous control amplicon, wherein the second probe and the third probe each contain a detectable marker, wherein the detectable marker may be the same or different. 14. The use of claim 13, wherein the detection comprises detecting the Trichomonas vaginalis 40S ribosomal protein gene or RNA with a single multiplex reaction, an endogenous control, and an exogenous control. 15. The use according to claim 1, wherein the sample is selected from urine samples, cervical swab samples, vaginal swab samples, and urethral swab samples. 16. A composition comprising a first primer pair and/or a first probe for detecting the 40S ribosomal protein gene or RNA of Trichomonas vaginalis, 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 the 40S ribosomal protein gene or RNA of Trichomonas vaginalis, 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 for detecting Trichomonas vaginalis 40S ribosomal protein and the second primer pair 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 a different composition from 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 marker, used to detect the amplification product of the primer pair of claim 21.