HK1237372A1

HK1237372A1 - Methods of detecting trichomonas vaginalis

Info

Publication number: HK1237372A1
Application number: HK17110969.7A
Authority: HK
Inventors: 詹姆斯‧王; 素提‧阿卢古帕利; 罗莎‧余; 萨莉‧优素福
Original assignee: 西菲伊德公司
Filing date: 2014-07-15
Publication date: 2018-04-13

Description

Method for detecting trichomonas vaginalis

1. Field of the invention

Compositions and methods for detecting Trichomonas vaginalis (Trichomonas vagianalis) are provided.

2. Background of the invention

Protozoan trichomonas vaginalis causes trichomoniasis (trichomonas), a common sexually transmitted infection that can infect both men and women. There are seven hundred and forty thousand cases of trichomoniasis in the united states each year. Trichomoniasis infection may be symptomatic or asymptomatic. See, e.g., Ginocchio et al, j.clin.microbiol.2012, 50: 2601-2608. Trichomoniasis is one of a series of conditions involving vaginal discharge in women. See, for example, Centers for discease control and preference (CDC). CDC away sheet: trichomonas.2010. www.cdc. gov/std/trichomonas/STDFact-trichomonas. htm. Symptoms in women may include genital itching, burning, redness, or pain, odor, malaise urination, or a thin clear, white, yellow, or green discharge. See above. In males, trichomoniasis can cause urethritis (NGU) which is not a gonococcal bacterium. Symptoms in men may include internal itching or burning of the penis, burning after ejaculation or urination, or penile discharge. See, e.g., Workowski et al, Centers for disease Control and prediction. Sexually transmitted disease treatment guidelines, 2010.MMWR 2010; 59 (RR-12): 1 to 110; targets for Disease Control and prediction, biosafety in microbiology and biomedicalla laboratories, w, 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, methods are provided for detecting the presence or absence of Trichomonas Vaginalis (TV) in a sample from a subject. In some embodiments, methods of determining whether a subject has a Trichomonas Vaginalis (TV) infection are provided. In some embodiments, the method comprises detecting the presence or absence of a TV40S ribosomal protein (TV40Srp) gene or RNA in a sample of the subject.

In some embodiments, the subject has not previously been treated for TV infection. In some embodiments, the subject was previously treated for TV infection. In some embodiments, the previous treatment comprises more than one dose of metronidazole or tinidazole. In some embodiments, the subject does not have any symptoms of TV infection. In some embodiments, the subject has more than one symptom of TV infection. In some embodiments, the subject has one or more symptoms selected from 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; discomfort with urination; and dilute clear, white, yellow, or green effluent. In some embodiments, the subject is pregnant. In some embodiments, the subject is male and has one or more symptoms selected from: itching and/or burning within the penis; cauterizing after ejaculation and/or urination; and penile waste.

In some embodiments, the method comprises detecting an endogenous control. In some embodiments, the endogenous control is a sample sufficiency 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, beta actin, and beta globin.

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

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

In some embodiments, the TV40S ribosomal protein (TV40Srp) gene comprises SEQ id no: 4. In some embodiments, the method comprises contacting nucleic acids from the sample with a first primer pair for detecting a TV40S ribosomal protein (TV40Srp) gene or RNA. In some embodiments, the method comprises contacting nucleic acids from the sample with a second primer pair for detecting an endogenous control. In some embodiments, the method comprises contacting nucleic acids from the 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 primer that hybridizes to SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100%. In some embodiments, the polypeptide of SEQ ID NO: 4 or its complement, each of the first and second primers independently comprises 0, 1, or 2 mismatches. 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 SEQ ID NO: 1 and SEQ ID NO: 2. In some embodiments, the first primer pair produces an amplicon that is 50 to 500 nucleotides in length, 50 to 400 nucleotides in length, 50 to 300 nucleotides in length, 50 to 200 nucleotides in length, 50 to 150 nucleotides in length, 100 to 300 nucleotides in length, 100 to 200 nucleotides in length, or 100 to 150 nucleotides in length.

In some embodiments, the method comprises forming a Tv40Srp amplicon. In some embodiments, the method comprises contacting the Tv40Srp amplicon with a first probe capable of selectively hybridizing to the Tv40Srp amplicon. In some embodiments, the first probe comprises a detectable label. In some embodiments, the first probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the first probe comprises a sequence identical to SEQ ID NO: 4 or SEQ ID NO: 5, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100% identical or complementary sequence. In some embodiments, the polypeptide of SEQ ID NO: 4 or the complement thereof or to SEQ ID NO: 5 or its complement, the first probe comprises 0, 1, or 2 mismatches. 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 comprises forming an endogenous control amplicon and/or an exogenous control amplicon. In some embodiments, the method comprises contacting the endogenous control amplicon with a second probe capable of selectively hybridizing to the endogenous control amplicon and/or contacting the exogenous control amplicon with a third probe capable of selectively hybridizing to the exogenous control amplicon. In some embodiments, the second probe and the third probe each comprise a detectable label, wherein the detectable labels may be the same or different. In some embodiments, the detectable label of the second and third probes is detectably different from the detectable label of the first probe. In some embodiments, the methods comprise detecting the Tv40Srp gene or RNA, an endogenous control, and an exogenous control in a single multiplex reaction.

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

In some embodiments, a composition is provided comprising a first primer pair for detecting a 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 sufficiency control. In some embodiments, the endogenous control is selected from HMBS, GAPDH, beta actin, and beta globin. In some embodiments, the composition comprises a third primer pair for detecting an exogenous control. In some embodiments, the exogenous control is a sample processing 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 primer that hybridizes to SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100%. In some embodiments, the polypeptide of SEQ ID NO: 4 or its complement, each of the first and second primers independently comprises 0, 1, or 2 mismatches. 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 SEQ ID NO: 1 and SEQ ID NO: 2.

In some embodiments, the composition comprises a first probe capable of selectively hybridizing to the Tv40Srp amplicon generated by the first primer pair. In some embodiments, the first probe comprises a detectable label. In some embodiments, the first probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the first probe comprises a sequence identical to SEQ ID NO: 4 or SEQ ID NO: 5, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100% identical or complementary sequence. In some embodiments, the polypeptide of SEQ ID NO: 4 or the complement thereof or to SEQ ID NO: 5 or its complement, the first probe comprises 0, 1, or 2 mismatches. 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 SEQ ID NO: 5.

In some embodiments, the composition comprises a second probe capable of selectively hybridizing to an endogenous control amplicon generated by the second primer pair. In some embodiments, the endogenous control is a sample sufficiency control. In some embodiments, the endogenous control is selected from HMBS, GAPDH, beta actin, and beta globin. In some embodiments, the composition comprises a third probe capable of selectively hybridizing to the exogenous control amplicon generated by the third primer pair. In some embodiments, the exogenous control is a sample processing control. In some embodiments, the exogenous control comprises a DNA sequence that is not expected to be present in the sample. In some embodiments, the exogenous control is 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 of a subject tested for the presence or absence of trichomonas vaginalis.

In some embodiments, a kit is provided comprising a first primer pair for detecting a 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 composition in the kit. In some embodiments, the endogenous control is a sample sufficiency control. In some embodiments, the endogenous control is selected from HMBS, GAPDH, beta actin, and beta 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 composition as the primer pair for detecting Tv40Srp and the second primer pair. In some embodiments, the exogenous control is a sample processing control. In some embodiments, the exogenous control comprises a DNA sequence that is not expected to be present in the sample. In some embodiments, the exogenous control is a bacterial gene.

In some embodiments, the kit comprises a first probe capable of selectively hybridizing to a Tv40Srp amplicon generated by a first primer pair, wherein the first probe is in the same or different composition as more than one 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 identical to SEQ ID NO: 4 or SEQ ID NO: 5, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100% identical or complementary sequence. In some embodiments, the polypeptide binds to seq id NO: 4 or the complement thereof or to SEQ ID NO: 5 or its complement, the first probe comprises 0, 1, or 2 mismatches. 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 SEQ ID NO: 5.

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

In some embodiments, the kit comprises dntps and/or a thermostable polymerase. In some embodiments, the kit comprises more than one lyophilized composition.

In some embodiments, a primer is provided, wherein the primer consists of SEQ ID NO: 1, wherein the primer comprises at least one modified nucleotide. In some embodiments, a primer is provided, wherein the primer consists of SEQ ID NO: 2, wherein the primer comprises at least one modified nucleotide. In some embodiments, a probe is provided, wherein the probe consists of 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, a composition is provided, wherein the composition comprises a polypeptide consisting of SEQ ID NO: 2 and a first primer consisting of the sequence of SEQ ID NO: 3, wherein the first primer and the second primer each comprise at least one modified nucleotide. In some embodiments, the composition comprises a polypeptide consisting 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 of the subject.

Further embodiments and details of the invention are described below.

4. Detailed description of the invention

4.1 definition

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

as used herein, the terms "detect", "detecting" or "detection" may describe the general behavior of a composition in which a detection marker is found or understood or specifically observed.

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

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

"Trichomonas vaginalis" refers to protozoa responsible for trichomoniasis, a common sexually transmitted infection that can infect both males and females. 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 of limonene. Symptoms in men include, but are not limited to, itching or burning within the penis, burning after ejaculation or urination, or penile discharge.

As used herein, the terms "oligonucleotide," "polynucleotide," "nucleic acid molecule," and the like, refer to nucleic acid-containing molecules, including, but not limited to, DNA or RNA. The term encompasses sequences including base analogs of any known DNA and RNA, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, meptyl cytosine, pseudoisocytosine, 5- (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseuduracyl, 1-methylguanine, 1-methylinosine, 2, 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, n6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β -D-mannosylqueosine, 5' -methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 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 less than 500 nucleotides. In some embodiments, the oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to 75, or 12 to 50 nucleotides in length. The oligonucleotide may be indicated in terms of its length, for example a 24 residue oligonucleotide may be referred to as a "24-mer (24-mer)".

As used herein, the term "complementary" to a target gene (or target region thereof), and the percentage of "complementarity" of a probe sequence to a target gene sequence is the percentage of "identity" to the target gene sequence or to the reverse complement of the target gene sequence. In determining the degree of "complementarity" between a probe (or region thereof) and a target gene (such as those disclosed herein) used in the compositions described herein, the degree of "complementarity" is expressed as the percent identity between the probe sequence (or region thereof) and the target gene sequence or the reverse complement of the target gene sequence with which it is optimally aligned. The percentage is calculated by counting the number of identical alignment bases between 2 sequences, dividing by the total number of consecutive nucleotides in the probe, and multiplying by 100. When the term "complementary" is used, the subject oligonucleotide is at least 90% complementary to the target molecule, unless otherwise indicated. In some embodiments, the subject 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 comprising a region complementary to a sequence of at least 8 contiguous 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, a primer or probe comprises a region that is complementary to a sequence of at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 contiguous nucleotides of a target molecule. When the primer or probe comprises a region that is "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%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target molecule.

The term "nucleic acid amplification" includes any method by which at least a portion of at least one target nucleic acid is replicated (typically in a template-dependent manner), including, but not limited to, a wide range of techniques for amplifying nucleic acid sequences (linearly or exponentially). Exemplary methods of performing the amplification step include Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), Ligase Detection Reaction (LDR), multiple ligation dependent probe amplification (MLPA), ligation followed by Q-replicase amplification, primer extension, Strand Displacement Amplification (SDA), hyperbranched strand displacement amplification, Multiple Displacement Amplification (MDA), Nucleic Acid Strand Based Amplification (NASBA), two-step multiplex amplification, Rolling Circle Amplification (RCA), and the like, including various versions and combinations thereof, such as, but not limited to, OLA/PCR, PCR/OLA, LDR/PCR, PCR/LDR, LCR/PCR, PCR/LCR (also known as combinatorial chain reaction — CCR), digital amplification, and the like. A description of such techniques may be found in other sources, Ausbel et al; PCR Primer: a Laboratory Manual, Diffenbach, eds., Cold Spring Harbor Press (1995); the Electronic Protocol Book, Chang Bioscience (2002); msuih et al, J.Clin.Micro.34: 501-07 (1996); the nucleic Protocols Handbook, R.Rapley, eds, Humana Press, Totowa, N.J. (2002); abramson et al, Curr Opin Biotechnol.1993 Feb; 4(1): 41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No. 6,605,451 to Barany et al, PCT publication No. WO 97/31256; wenz et al, PCT publication No. WO 01/92579; day et al, Genomics, 29 (1): 152-162(1995), Ehrlich et al, Science 252: 1643-50 (1991); innis et al, PCR Protocols: a Guide to methods and Applications, Academic Press (1990); favis et al, Nature Biotechnology 18: 561-64 (2000); and Rabenau et al, Infection 28: 97-102 (2000); belgrader, Barany, and Lubin, Development of a multiple ligation detection reaction DNA Typing Assay, six International Symposium on HumanIdentification, 1995 (available at the world Wide Web site: promega. com/geneticipproc/ussymp 6 proc/blerad. html); LCR Kit Instruction Manual, Cat. #200520, Rev. #050002, Stratagene, 2002; barany, proc.natl.acad.sci.usa 88: 188-93 (1991); bi and Sambrook, nucleic acids res.25: 2924-2951 (1997); zirvi et al, nuclear.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, nuclear.acid res.20: 1691-96 (1992); polstra et al, BMC inf.dis.2: 18- (2002); lage et al, Genome Res.2003, month 2; 13(2): 294-307, and Landegren et al, Science 241: 1077-80(1988), Demidov, V., Expert Rev Mol diagn.2002Nov.; 2(6): 542-8, Cook et al, J Microbiol methods, 5 months 2003; 53(2): 165-74, Schweitzer et al, Curropin Biotechnol.2001, month 2; 12(1): 21-7, U.S. patent No. 5,830,711, U.S. patent No. 6,027,889, U.S. patent No. 5,686,243, PCT publication No. WO0056927A3, and PCT publication No. WO9803673a 1.

In some embodiments, the amplification comprises at least one cycle of sequential steps in: 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 strands. The cycle may or may not be repeated. Amplification may include thermal cycling or may be performed under isothermal conditions.

Unless otherwise indicated, the term "hybridize" is used herein to mean "specific hybridization," which is the preferential binding, formation of a duplex, or hybridization of a nucleic acid molecule to a particular nucleotide sequence, in some embodiments, under stringent conditions. The term "stringent conditions" refers to conditions under which a probe will preferentially hybridize to its target sequence, but to a lesser extent to other sequences, or not at all. In the case of nucleic acid hybridization (e.g., in array, southern blot, or northern blot hybridization), the "stringent hybridization" and "stringent hybridization wash conditions" are sequence-dependent and differ under different environmental parameters. A broad guide to Nucleic Acid hybridization is found, for example, in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-hybridization with Nucleic Acid Probes section I, Chapter 2, "Overview of principal hybridization and the strategy of Nucleic Acid Probes, and" Elsevier, NY ("Tijssen"). Typically, highly stringent hybridization and wash conditions for filter paper hybridization are selected to be about 5 ℃ below the thermal melting point of the specific sequence at a defined ionic strength and pH. T is_mIs the temperature (at defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T of a particular probe_m. Dependence of hybridization stringency on buffer composition, temperature and probe lengthKnown to those skilled in the art (see, e.g., Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (3rd ed.) Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY).

As used herein, "sample" includes urine samples (including samples derived from urine samples), swabs of the cervix, and patient-collected vaginal swabs, as well as other types of human samples. In some embodiments, the urine sample is a "first time" urine sample, which is the sample taken when the subject first begins to urinate. As used herein, a urine sample includes, but is not limited to, whole urine, a sample comprising cells from a urine sample, a sample comprising a cell pellet isolated by centrifugation of a urine sample, a sample comprising cells isolated by filtration of a urine sample, and the like. In some embodiments, the urine sample comprises a buffer, such as a preservative. In some embodiments, the sample is a human sample other than a urine sample, such as an endocervical swab or a vaginal swab, including patient-collected vaginal swabs, and urethral swabs. In some embodiments, the swab sample comprises a buffer, such as a preservative.

As used herein, "endogenous control" refers to the portion that is naturally present in the sample 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, an endogenous control, such as SAC, is selected, which can be detected in the same manner as the target gene, and, in some embodiments, is detected simultaneously with the target gene.

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

In the sequences herein, "U" and "T" are used interchangeably, such that two letters represent uracil or thymine at that position. From the context and/or intended use, one 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, one skilled in the art will appreciate that a natural RNA molecule typically includes uracil, while a natural DNA molecule typically includes thymine. Thus, where the RNA sequence includes a "T," one of skill in the art would understand that a position in the native RNA may be uracil.

In the present disclosure, "a sequence selected from. Thus, when "a sequence selected from.. is used," it is to be understood that one, or more than one, of the listed sequences may be selected.

4.2 detection of Trichomonas vaginalis

The present inventors developed an assay to detect Trichomonas Vaginalis (TV). In some embodimentsIn one embodiment, the assay comprises detecting the TV40S ribosomal protein (TV40Srp) gene. In some embodiments, the assaying comprises detecting RNA transcribed from the TV40S ribosomal protein (TV40Srp) gene. The assay relies on the Polymerase Chain Reaction (PCR) and can be performed in a sequential automated fashion using commercially available nucleic acid amplification systems. An exemplary non-limiting nucleic acid amplification system that can be used to perform the methods of the invention includesThe system is provided with a plurality of sensors,the Infinity system, and the Smartcycler system (Cepheid, Sunnyvale, Calif.). The present assay uses an automated system, for example,the system, may be completed in 3 hours, and in some embodiments, in 2 hours.

4.2.1 general procedure

Compositions and methods for detecting Trichomonas Vaginalis (TV) are provided. In some embodiments, the method comprises detecting a TV40S ribosomal protein (TV40Srp) gene.

In some embodiments, a method of detecting Trichomonas Vaginalis (TV) in a subject comprises detecting the presence of the TV40S ribosomal protein (Tv40Srp) gene in a sample from the subject. In some embodiments, the sample is selected from a urine sample, a cervical swab, and a vaginal swab. In some embodiments, the urine sample is a first time urine sample.

In some embodiments, the method of detecting TV further comprises detecting at least one endogenous control, such as a Sample Adequacy Control (SAC). In some embodiments, the method of detecting TV further comprises detecting at least one exogenous control, such as a Sample Processing Control (SPC). In some embodiments, the method of detecting TV further comprises detecting at least one endogenous control and at least one exogenous control.

In some embodiments, a method of detecting TV comprises detecting a TV40S ribosomal protein (TV40Srp) gene in a sample. In some embodiments, the method of detecting TV further comprises detecting a Sample Adequacy Control (SAC), such as a single copy of a human gene. In some embodiments, the method of detecting TV further comprises detecting a Sample Processing Control (SPC), such as exogenously added bacterial DNA. In some embodiments, the method of detecting TV further comprises detecting SAC and SPC.

In the present disclosure, the term "target gene" is used for convenience to denote the TV40S ribosomal protein (TV40Srp) gene, and also to denote exogenous and/or endogenous controls. Thus, it is to be understood that when discussing in relation to a target gene, the discussion is specifically intended to include the TV40S 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 TV40S ribosomal protein (TV40Srp) gene is detected in a urine sample. In some embodiments, the target gene is detected in a urine sample to which a buffer (e.g., a preservative) is added. In some embodiments, the buffer is added to the urine sample at a ratio of 1: 1, 1: 2, 1: 3, 1: 4, 1: 5, 1: 6, 1: 7, 1: 8, 1: 9, or 1: 10 buffer to urine. In some embodiments, the presence of the TV40S ribosomal protein (TV40Srp) gene is detected in an endocervical swab sample or a vaginal swab sample. In some embodiments, the vaginal swab is a patient-collected vaginal swab. In some embodiments, the target gene is detected in an endocervical swab sample or a vaginal swab sample placed in a buffer (e.g., a preservative). In some embodiments, the swab is placed in 1mL, 2mL, 2.5mL of buffer.

In some embodiments, detection of the TV40S 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 comprises forming a complex of a polynucleotide and a nucleic acid 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 comprises PCR. In some embodiments, detecting the target gene comprises quantitative PCR or real-time PCR. In some embodiments, the Sample Adequacy Control (SAC) and/or the Sample Processing Control (SPC) are detected in the same assay as the target gene. In some embodiments, TV is considered detected if the TV40S ribosomal protein (TV40Srp) gene is detected, even if SPC and/or SAC is not detected in the assay. In some embodiments, TV is considered not detected only when SPC and SAC are also detected in the assay if the TV40S ribosomal protein (TV40Srp) gene is not detected.

In some embodiments, the presence of the TV40S ribosomal protein (TV40Srp) gene is measured in samples collected from a subject more than once to monitor treatment of TV infection in the subject. Treatment includes, but is not limited to, single or multiple doses of metronidazole or tinidazole. In some embodiments, subjects with a history of TV infection are monitored for TV relapse by detecting the presence or absence of the TV40S ribosomal protein (TV40Srp) gene at regular or semi-regular intervals. In some such embodiments, the patient is monitored by detecting the presence or absence of the TV40S ribosomal protein (TV40Srp) gene at least once a month, at least once every two months, at least once every three months, at least once every four months, at least once every five months, at least once every six months, at least once every september, at least once a year, or at least once every two years.

In some embodiments, the subject assays can be used as part of routine and/or preventive health care for a subject. That is, in some embodiments, the present assay can be used to test an individual for TV infection, whether the individual shows symptoms of TV infection or has a history of TV infection. In some embodiments, the present assays are used to detect TV infection in a pregnant and/or a subject attempting to become pregnant. 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 (e.g., a first urine sample), or is derived from a urine sample. In some embodiments, a buffer (e.g., a preservative) is added to the urine sample. In some embodiments, the buffer is added to the urine sample within 1 hour, within 2 hours, within 3 hours, within 4 hours, within 5 hours, within 6 hours, within 7 hours, or within 8 hours of collecting the sample.

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

In some embodiments, less than 5ml, less than 4ml, less than 3ml, less than 2ml, less than 1ml, or less than 0.75ml of urine is used in the present methods. In some embodiments, 0.1ml to 1ml of urine is used in the present methods.

In some embodiments, the sample to be tested is another bodily fluid, such as blood, sputum, mucus, saliva, vaginal or penile feces, semen, and the like.

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

In some embodiments, the sample to be tested is obtained from an individual who has 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 of limonene; and in males: itching or burning in the penis, burning after ejaculation or urination, and penile discharge. In some embodiments, the sample to be tested is obtained from an individual previously diagnosed with TV infection. In some such embodiments, the individual is monitored for recurrence of TV infection.

In some embodiments, the methods described herein can be used to routinely screen healthy individuals for those who do not have risk factors. In some embodiments, the methods described herein are used, for example, to screen asymptomatic individuals for routine or preventive care. In some embodiments, the methods described herein are used to screen for females who are pregnant or attempting to become pregnant.

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

In some embodiments, use of the TV40S ribosomal protein (TV40Srp) gene for detecting TV infection is provided. In some embodiments, use of the TV40S ribosomal protein (TV40Srp) gene is provided for monitoring TV infection relapse.

In any of the embodiments described herein, the TV40S ribosomal protein (TV40Srp) gene can be detected in the same assay reaction as the sample treatment control (SPC) and/or the Sample Adequacy Control (SAC).

In some embodiments, methods are provided that facilitate the detection of TV infection in a subject. The method comprises detecting the presence or absence of a TV40S ribosomal protein (TV40Srp) gene in a sample from the subject. In some embodiments, information regarding the presence or absence of the TV40S ribosomal protein (TV40Srp) gene in a sample from a subject is communicated to a medical practitioner. As used herein, a "medical practitioner" refers to an individual or entity, such as a hospital, clinic, doctor's office, doctor, nurse, or any of the aforementioned entities or individuals, that diagnoses and/or treats a patient. In some embodiments, detecting the presence or absence of the TV40S ribosomal protein (TV40Srp) gene is performed in a laboratory that receives a sample of the subject from a medical practitioner or a facility of the medical practitioner. The laboratory tests by any method, including those described herein, and then communicates the results to a medical practitioner. The results as used herein are "communicated" when provided to a medical practitioner by any means. In some embodiments, the communication may be oral or written, may be by telephone, personal, email or other credit, or may be by direct storage of information in, for example, a database accessible to the medical practitioner, including databases not controlled by the medical practitioner. In some embodiments, the information is maintained in electronic form. In some embodiments, the information may be stored in memory or other computer readable medium, such as RAM, ROM, EEPROM, flash memory, computer chips, Digital Video Disks (DVDs), Compact Disks (CDs), Hard Disk Drives (HDDs), magnetic tape, or the like.

In some embodiments, methods of detecting TV are provided. In some embodiments, methods of diagnosing TV infection are provided. In some embodiments, the method comprises obtaining a sample from a subject and providing the sample to a laboratory for detection of the TV40S ribosomal protein (TV40Srp) gene in the sample. In some embodiments, the method further comprises receiving a communication from a laboratory indicating the presence or absence of a TV40S ribosomal protein (TV40Srp) gene in the sample. A "laboratory," as used herein, is any device that detects a target gene in a sample by any method, including the methods described herein, and communicates the results to a medical practitioner. In some embodiments, the laboratory is not under the control of the medical practitioner.

When the laboratory communicates to a medical practitioner the result of detecting the presence or absence of the TV40S ribosomal protein (TV40Srp) gene, in some embodiments, the laboratory indicates whether the TV40S ribosomal protein (TV40Srp) gene was detected in the sample. In some embodiments, the laboratory indicates whether the sample comprises Trichomonas Vaginalis (TV) by indicating, for example, "TV positive" or "TV negative" or "TV present" or "TV absent" or the like.

As used herein, when a method involves detecting TV, determining the presence of TV, monitoring TV, and/or diagnosing TV infection, the method includes an activity in which the steps of the method are performed, but the result is negative for the presence of TV. That is, detecting, determining, monitoring and diagnosing TV or TV infection includes examples of methods that result in positive or negative results.

In some embodiments, at least one endogenous control (e.g., SAC) and/or at least one exogenous control (e.g., SPC) are detected simultaneously with the TV40S ribosomal protein (TV40Srp) gene in a single reaction.

4.2.2 exemplary controls

In some embodiments, the assays described herein comprise detecting a TV40S 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 TV40S ribosomal protein (TV40Srp) gene is not detected in the sample, and SAC is not detected in the sample either, the assay result is considered "invalid" because the sample may not be sufficient. Without intending to be bound by any particular theory, an insufficient sample may be too dilute, contain too little cellular material, contain assay inhibitors, and the like. In some embodiments, failure to detect SAC may indicate failure of the assay reaction. In some embodiments, the endogenous control (e.g., SAC) is a single copy of the human gene. Non-limiting exemplary SACs include human hydroxymethyl-cholane synthase (HMBS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β actin, β 2-microglobulin, cyclooxygenase 1, hypoxanthine phosphoribosyl transferase, porphobilinogen deaminase, and transferrin receptor.

In some embodiments, the assays described herein comprise detecting a TV40S 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 TV40S ribosomal protein (TV40Srp) gene is not detected in the sample, and in the sampleSPC is also not detected and the assay result is considered "invalid" because there may be errors in 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, compromised reagents, and the like. In some embodiments, an exogenous control (such as SPC) is added to the sample. In some embodiments, an exogenous control (e.g., SPC) is added during the assay run, e.g., using one or more buffers or reagents. In some embodiments, when to be usedWhen systematic, SPC is included inIn the cartridge. In some embodiments, the exogenous control (e.g., SPC) is a DNA sequence that is not expected to be present in the assayed sample. Non-limiting exemplary SPCs include bacterial genes that are not expected to be present in the sample assayed.

In some embodiments, the endogenous control and/or the exogenous control are detected simultaneously when detecting the TV40S ribosomal protein (TV40Srp) gene in a sample, such as in the same assay. In some embodiments, the assay comprises reagents that simultaneously detect the TV40S ribosomal protein (TV40Srp) gene, an exogenous control, and an endogenous control in the same assay reaction. In some such embodiments, for example, the assay reaction comprises a primer set that amplifies the TV40S ribosomal protein (TV40Srp) gene, a primer set that amplifies an endogenous component control, and a primer set that amplifies an exogenous component control, and a labeled probe that detects the amplification product (such as, for example,a probe).

4.2.3 exemplary sample preparation

4.2.3.1 exemplary buffers

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

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

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

4.2.3.2 exemplary DNA preparation

Sample DNA may be prepared by any suitable method. In some embodiments, the 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 may be in any suitable form, such as a particulate, porous solid or membrane form. For example, the support may comprise hydroxycellulose, glass fibre, cellulose, nitrocellulose, zirconium hydroxide, titanium (IV) oxide, silica, zirconium silicate, or silica particles (see, e.g., U.S. Pat. No. 5,234,809). Many such DNA binding substrates are known in the art.

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

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

4.2.4 exemplary analytical methods

As described above, methods for detecting trichomonas vaginalis are provided. The method comprises detecting the presence of a TV40S ribosomal protein (TV40Srp) gene in a sample from the subject. In some embodiments, the methods further comprise detecting at least one endogenous control (such as SAC) and/or at least one exogenous control (such as SPC). In some embodiments, detection of the TV40S ribosomal protein (TV40Srp) gene indicates the presence of TV, even if the endogenous control and/or the exogenous control are not detected in the assay. In some embodiments, if the TV40S ribosomal protein (TV40Srp) gene is not detected, the result is considered negative for TV only when the control is detected. In some embodiments, if the TV40S ribosomal protein (TV40Srp) gene is not detected, the result is considered negative for TV only when the endogenous control and the exogenous control are detected.

Any assay step that allows for specific detection of a target gene 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 of skill in the art.

In some embodiments, methods of detecting a target gene, such as the TV40S ribosomal protein (Tv40Srp) geneA method comprising amplifying a gene and/or its complement. The amplification may be accomplished by any method. Exemplary methods include, but are not limited to, isothermal amplification, real-time PCR, end-point PCR, and amplification from a T7 promoter annealed to DNA using T7 polymerase, such as SenseAmp Plus available from Implen, Germany^TMSupplied by Kit.

When the target gene is amplified, in some embodiments, an amplicon of the target gene is formed. The amplicon may be single stranded or double stranded. In some embodiments, when the amplicon is single stranded, the sequence of the amplicon is related to the target gene in a sense or antisense orientation. In some embodiments, the amplicon of the target gene is detected rather than the target gene itself. Thus, when the methods discussed herein indicate the detection of a target gene, the detection can be performed on the amplicon of the target gene (rather than, or in addition to, the target gene itself). In some embodiments, when detecting an amplicon of a 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 an amplicon of a target gene instead of the target gene, a polynucleotide complementary to the target gene is used for detection. Furthermore, in some embodiments, multiple polynucleotides may be used for detection, and some polynucleotides may be complementary to a target gene and some polynucleotides may be complementary to the complement of the target gene.

In some embodiments, the method of detecting the TV40S ribosomal protein (TV40Srp) gene comprises PCR, as described below. In some embodiments, detecting more than one target gene includes monitoring a PCR reaction in real time, which can be accomplished by any method. The method includes, but is not limited to, usingMolecular beacons, or Scorpion probes (i.e., Energy Transfer (ET) probes such as FRET probes) and the use of supplemental dyes such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, and the like.

Non-limiting exemplary methods for amplifying target genesThe conditions were as follows. Exemplary cycles include a cycle of initial denaturation at 90 ℃ to 100 ℃ for 30 seconds to 5 minutes, followed by denaturation at 90 ℃ to 100 ℃ for 1 to 10 seconds, followed by annealing and amplification at 60 ℃ to 75 ℃ for 10 to 30 seconds. A further exemplary cycle includes up to 40 cycles of 1 minute at 95 ℃, followed by 5 seconds at 92.5 ℃, and 20 seconds at 68 ℃. In some embodiments, for the first cycle following the initial denaturation step, the cyclic denaturation step is omitted. In some embodiments, Taq polymerase is used for amplification. In some embodiments, the cycling is performed at least 10 times, at least 15 times, at least 20 times, at least 25 times, at least 30 times, at least 35 times, at least 40 times, or at least 45 times. In some embodiments, Taq with hot start functionality is used. In some embodiments, the amplification reaction is inBarrel and amplification of TV40S ribosomal protein (TV40Srp) gene, endogenous control and exogenous control occurred in the same reaction. In some embodiments, the detection of the TV40S ribosomal protein (TV40Srp) gene occurs in less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes from initial denaturation to final extension.

In some embodiments, detection of the target gene comprises forming a complex comprising a polynucleotide complementary to the target gene or its complement and a nucleic acid selected from the group consisting of the target gene, a DNA amplicon of the target gene, and the 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 the 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 a complex, as used herein, the complex may comprise one or both strands of the DNA amplicon. Thus, in some embodiments, the complex comprises only one strand of the DNA amplicon. In some embodiments, the complex is a triplex and comprises the polynucleotide and both strands of the DNA amplicon. In some embodiments, the complex is formed by hybridization between the polynucleotide and the target gene, the complement of the target gene, or the DNA amplicon of the target gene. In some embodiments, the polynucleotide is a primer or a probe.

In some embodiments, the method comprises detecting the complex. In some embodiments, the complexes do not have to be correlated at the time of detection. That is, in some embodiments, a complex is formed, then the complex is dissociated or disrupted in some manner, and components from the complex are detected. An example of such a system isAnd (4) measuring. In some embodiments, when the polynucleotide is a primer, detection of the complex may include amplification of the target gene, the complement of the target gene, or a DNA amplicon of the target gene.

In some embodiments, the assay for detecting at least one target gene in the methods described herein comprises real-time quantitative PCR. In some embodiments, the assay for detecting at least one target gene comprises the use ofAnd (3) a probe. The assay uses energy transfer ("ET"), such as fluorescence resonance energy transfer ("FRET"), to detect and quantify the synthesized PCR products. In general,the probe comprises a fluorescent dye molecule coupled to the 5 '-terminus and a quencher molecule coupled to the 3' -terminus, such that the dye and quencher are in close proximity, allowing the quencher to suppress the fluorescent signal of the dye via FRET. When the polymerase replicatesWhen the probe binds to the chimeric amplicon template, the 5' -nuclease of the polymerase cleaves the probe, uncouples the dye and the quencher, and a dye signal (e.g., fluorescence) is detected. The signal (e.g., fluorescence) increases with each PCR cycle in proportion to the amount of cleaved probe.

In some embodiments, a target gene is considered to be detected if any signal is generated from the TaqMan probe during a PCR cycle. For example, in some embodiments, if the PCR comprises 40 cycles, the target gene is considered to be present and detected if a signal is generated at any cycle during amplification. In some embodiments, if no signal is generated at the end of a PCR cycle, the target gene is considered absent and not detected.

In some embodiments, the quantification of the real-time PCR assay results is accomplished by constructing a standard curve from a known concentration of nucleic acid and then inferring quantitative information for the unknown concentration of the target gene. 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 embodiments, the Ct values for an endogenous control (e.g., SAC) and/or an exogenous control (e.g., SPC) must be within a predetermined effective range in order to be determined to indicate that TV is not present in the sample. That is, in some embodiments, the absence of TV cannot be confirmed unless a control is detected, indicating that the assay was successful. The Ct value is inversely proportional to the amount of nucleic acid target in the sample.

In some embodiments, a threshold Ct (or "cutoff Ct") value (below which detection is deemed to be detected) for a target gene (including endogenous controls and/or exogenous controls) is determined previously. In some embodiments, substantially the same assay conditions and systems are used (e.g., as in) The threshold Ct is determined, based on which the sample will be tested.

Except thatIn addition to assays, thereof for detecting and quantifying PCR products in the methods provided hereinHis real-time PCR chemistry includes, but is not limited TO, molecular beacons, Scorpion probes and supplemental dyes, such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, and the like, which are discussed below.

In various embodiments, the TV40S ribosomal protein (TV40Srp) gene, endogenous controls, and exogenous controls are detected using real-time PCR detection in a single multiplex reaction. In some multiplex embodiments, multiple probes are used, e.g.Probes (each specific for a different target). In some embodiments, each target gene-specific probe is spectrally distinguishable from other probes used in the same multiplex reaction.

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

In some embodiments, detection and/or quantification of real-time PCR products is accomplished using dyes that bind double-stranded DNA products, such as SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, and the like. In some embodiments, the assay method for the methods described herein is(DNA-mediated annealing, selection, extension and ligation) assay. In some embodiments, the assay methods for detecting and quantifying the target gene in the methods described herein are bead-based flow cytometry assays. See Lu j et al (2005) Nature 435: 834-838, which is incorporated herein by reference in its entirety. An example of a bead-based flow cytometry assay is Luminex, IncProvided is a technique. Html, see www.luminexcorp.com/technology/index. In some embodiments, for testingThe assay to measure and quantify the level of at least one target gene in the methods described herein is by gel electrophoresis and detection with a labeled probe (e.g., a probe labeled with a radioactive or chemiluminescent label), such as by northern blot. In some embodiments, exemplary probes contain one or more affinity-enhanced nucleotide analogs as discussed below, such as locked nucleic acid ("LNA") analogs, which contain a bicyclic sugar moiety instead of a deoxyribose or ribose sugar. See, e.g., V rallyyay, e. et al (2008) Nature Protocols 3 (2): 190-196, which are herein incorporated by reference in their entirety. In some embodiments, the detection and quantification of more than one target gene is accomplished using a microfluidic device and single molecule detection.

Optionally, the sample DNA is modified prior to hybridization. The target DNA/probe duplex is then passed through a channel in a microfluidic device that contains a detector that records the unique signals of the 3 labels. 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 of which is incorporated herein by reference in its entirety.

4.2.5 exemplary Automation and System

In some embodiments, gene expression is detected using an automated sample processing and/or analysis platform. In some embodiments, a commercially available automated analysis platform is utilized. For example, in some embodiments, use is made ofSystem (Cepheid, Sunnyvale, Calif.).

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

Utilizing an own, single-use cartridge. Sample extraction, amplification and detection can all be performed in this own "lab-in-a-cartridge" (see, e.g., U.S. Pat. Nos. 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).

Components of the cartridge include, but are not limited to, a processing chamber containing reagents, filters, and capture technologies for extracting, purifying, and amplifying target nucleic acids. The valve enables fluid transfer between the chambers and contains the nucleic acid lysis and filtration components. The optical window enables real-time optical detection. The reaction tube enables very rapid thermal cycling.

In some embodiments of the present invention, the substrate is,the system includes a plurality of modules for scalability. Each module includes a plurality of cartridges, along with sample processing and analysis components.

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

In some embodiments, PCR is used to amplify and analyze the presence of a target gene. In some embodiments, PCR uses Taq polymerase with hot start functionality, such as aptaq (Roche). In some embodiments, the initial denaturation is at 90 ℃ to 100 ℃ for 30 seconds to 5 minutes; the cyclic denaturation temperature is 90 ℃ to 100 ℃ for 1 to 10 seconds; the cycle annealing and amplification temperature is 60 ℃ to 75 ℃ for 10 to 30 seconds; and up to 50 cycles.

In some embodiments, a dual-denaturation method is used to amplify low copy number targets. In some embodiments, the dual-denaturation method comprises a first denaturation step followed by the addition of primers and/or probes for the detection of the target gene. All or most of the DNA-containing sample (e.g., DNA eluate) is then denatured a second time, after which, in some cases, a portion of the sample is aliquoted for recycling and detection of the target gene. Without intending to be bound by any particular theory, the dual denaturation scheme can increase the probability that a low copy number target gene (or its complement) will be present in the aliquots selected for cycling and detection, as the second denaturation is effective to double the number of targets (i.e., it divides the target and its complement into two separate templates) before selecting the aliquots for cycling. In some embodiments, the first denaturation step comprises heating to a temperature of 90 ℃ to 100 ℃ for a total time of 30 seconds to 5 minutes. In some embodiments, the second denaturation step comprises heating to a temperature of 90 ℃ to 100 ℃ for a total time of 5 seconds to 3 minutes. In some embodiments, the first denaturation step and/or the second denaturation step is performed by separately heating aliquots of the sample. In some embodiments, each aliquot may be heated the above-described number of times. As a non-limiting example, a first denaturation step for a DNA-containing sample (e.g., a DNA eluate) can include heating at least one, at least two, at least three, or at least four aliquots of the sample (sequentially or simultaneously) separately to temperatures of 90 ℃ to 100 ℃ for 60 seconds each. As a non-limiting example, the second denaturation step for a DNA-containing sample (e.g., a DNA eluate) containing enzymes, primers, and probes can comprise heating at least one, at least two, at least three, or at least four aliquots (sequentially or simultaneously) of the eluate separately to temperatures of 90 ℃ to 100 ℃ for 5 seconds each. In some embodiments, the aliquot is the entire DNA-containing sample (e.g., DNA eluate). In some embodiments, the aliquot is less than the entire DNA-containing sample (e.g., DNA eluate).

In some embodiments, the target gene in a DNA-containing sample, such as a DNA eluate, is detected using the following protocol: more than one aliquot of the DNA-containing sample was heated separately to 95 ℃ for 60 seconds each. Enzymes and primers and probes were added to the DNA-containing sample and one or more aliquots were heated separately to 95 ℃ for 5 seconds each. At least one aliquot of the DNA-containing sample containing the enzyme, primers and probes is then heated to 94 ℃ for 60 seconds. The aliquots were then cycled 45 times in the following 2 steps: (1)94 ℃ for 5 seconds, and (2)66 ℃ for 30 seconds.

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

In some embodiments, offline centrifugation is used, e.g., with samples having low cell content. The samples (with or without buffer addition) were centrifuged and the supernatant removed. The pellet is then resuspended in a smaller volume of supernatant or buffer. The resuspended pellet was then analyzed as described herein.

4.2.6 exemplary data analysis

In some embodiments, the presence of TV is detected if the Ct value for the TV40S ribosomal protein (TV40Srp) gene is below a certain threshold. In some embodiments, an effective range of Ct values is 9 to 39.9 Ct. In some such embodiments, a sample is considered negative for TV if no amplification above background is observed from the TV-specific primers after 40 cycles.

In some embodiments, a computer-based analysis program is used to translate the raw data generated by the detection assay into clinician-predicted data. The clinician may obtain the predictive data using any suitable means. Thus, in some embodiments, the invention provides the further benefit that 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 subject.

The present invention contemplates any method capable of receiving, processing, and communicating information to and from laboratories that perform assays, information provision, medical individuals, and subjects. For example, in some embodiments of the invention, a sample (e.g., a biopsy or a serum or urine sample) is obtained from a subject and subjected to a profiling service (e.g., a clinical laboratory at a medical facility, a genomic analysis service, etc.) located in any part of the world (e.g., different from the country in which the subject resides or the country in which the information is ultimately used) to generate the raw data. Where the sample comprises a tissue or other biological sample, the subject may visit a medical center to have the sample obtained and sent to an analysis center, or the subject may collect the sample (e.g., a urine sample) by himself and send it directly to the analysis center. In the case where the sample contains previously determined biological information, the information may be sent directly from the subject to an analysis service (e.g., an information card containing the information may be scanned by a computer and the data will be transferred to a computer of an analysis center using an electronic communication system). Once received by the analysis service, the sample is processed and a profile (i.e., expression data) is generated that is specific to the diagnostic or prognostic information desired for the subject.

The analytical data is then prepared in a format suitable for interpretation by the treating clinician. For example, rather than providing raw expression data, the prepared format may represent a diagnosis or risk assessment (e.g., presence of TV) for the subject, suggesting or not suggesting a particular treatment option. The data may be presented to the clinician by any suitable method. For example, in some embodiments, the analysis service generates a report that can be printed to a clinician (e.g., at a point of care) or presented to the clinician on a computer display.

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

In some embodiments, the subject is able to obtain the data directly using an electronic communication system. The subject may choose to intervene or consult further based on the results. In some embodiments, the data is for research use. For example, the data may be used to further optimize the inclusion or exclusion of markers that are useful indicators of a particular condition or stage of a disease or that are diagnostic in concert to determine a course of treatment.

4.2.7 exemplary polynucleotides

In some embodiments, a polynucleotide is provided. In some embodiments, synthetic polynucleotides are provided. As used herein, a synthetic polynucleotide refers to a polynucleotide synthesized in vitro, either chemically or enzymatically. Chemical synthesis of polynucleotides includes, but is not limited to, using a polynucleotide synthesizer such as OligoPilot (GE Healthcare), ABI3900DNA synthesizer (Applied Biosystems), and the like. Enzymatic synthesis includes, but is not limited to, the production of polynucleotides by enzymatic amplification, e.g., PCR. A polynucleotide may comprise more than one nucleotide analog (i.e., modified nucleotides) as discussed herein.

In some embodiments, a polynucleotide is provided that comprises a region that is at least 90%, at least 95%, or 100% identical, or at least 90%, at least 95%, or 100%, or 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 TV40S ribosomal protein (TV40Srp) gene. In some embodiments, a polynucleotide is provided comprising a region that is at least 90%, at least 95%, or 100% identical, or complementary, to 6 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides of the TV40S 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 is between 6 and 200, between 8 and 150, between 8 and 100, between 8 and 75, between 8 and 50, between 8 and 40, between 8 and 30, between 15 and 100, between 15 and 75, between 15 and 50, between 15 and 40, or between 15 and 30 nucleotides in length.

In some embodiments, the polynucleotide is a primer. In some embodiments, the primer is labeled with a detectable moiety. In some embodiments, the primer is not labeled. A primer as used herein is a polynucleotide capable of selectively hybridizing to a target gene or to an amplicon (collectively referred to as a "template") amplified from a target gene, and, in the presence of the template, a polymerase and suitable buffers and reagents, can be extended to form a primer extension product.

In some embodiments, the polynucleotide is a probe. In some embodiments, the probe is labeled with a detectable moiety. A detectable moiety, as used herein, includes both a directly detectable moiety, such as a fluorescent dye, and an indirectly detectable moiety, such as a binding pair member. In some embodiments, when the detectable moiety is a member of a binding pair, the probe can be detected by incubating the probe with a detectable label that binds to a second member of the binding pair. In some embodiments, the probe is unlabeled, such as when the probe is a capture probe, e.g., on a microarray or on a bead. In some embodiments, the probe is not extendible, for example, by polymerase extension. 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, which is a chemical group that absorbs (i.e., suppresses) fluorescent emission from the dye when the groups are in close proximity (i.e., attached to the same probe). Thus, in some embodiments, the emission spectrum of the dye should overlap to a substantial extent 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, the methods of detecting at least one target gene described herein utilize more than one modified polynucleotide, such as a polynucleotide comprising more than one affinity-enhanced nucleotide analog. Modified polynucleotides for use 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 a polynucleotide for its target nucleic acid and allows for the use of shorter polynucleotides or regions of shorter complementarity between the polynucleotide and the target nucleic acid, as compared to polynucleotides containing only deoxyribonucleotides.

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

In some embodiments, the modified base for the affinity enhancing nucleotide analog comprises 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-chloro-6-aminopurine, xanthine, and hypoxanthine.

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

In some embodiments, the affinity-enhancing nucleotide analogs include backbone modifications such as the use of peptide nucleic acids (PNAs; e.g., oligomers comprising nucleobases linked together by an amino acid backbone). Other backbone modifications include phosphorothioate linkages, phosphodiester modified nucleic acids, combinations of phosphodiester and phosphorothioate nucleic acids, methyl phosphonates, alkyl phosphonates, phosphates, alkyl thiophosphonates, phosphoramidates, carbamates, carbonates, phosphotriesters, acetamide esters, carboxymethylesters, methylphosphides, phosphorodithioates, p-ethoxys, and combinations thereof.

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

In some embodiments, the affinity-enhancing nucleotide analog contains a locked nucleic acid ("LNA") sugar, which is a bicyclic sugar. In some embodiments, a polynucleotide for use in a method described herein comprises more than one nucleotide with a LNA sugar. In some embodiments, the polynucleotide contains more than one region consisting of nucleotides with LNA sugars. In other embodiments, the polynucleotide contains nucleotides with LNA sugars interspersed with deoxyribonucleotides. See, e.g., Frieden, m. et al (2008) curr. pharm. des.14 (11): 1138-1142.

4.2.7.2 exemplary primers

In some embodiments, a primer is provided. In some embodiments, the primer is at least 90%, at least 95%, or 100% identical, or at least 90%, at least 95%, or 100% complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of the TV40S ribosomal protein (TV40Srp) gene. In some embodiments, a primer is provided comprising a region that is at least 90%, at least 95%, or 100% identical, or complementary, to 6 to 100, 8 to 75, 8 to 50, 8 to 40, or 8 to 30 consecutive nucleotides of the TV40S ribosomal protein (TV40Srp) gene. Non-limiting exemplary primers are shown in table 1. In some embodiments, the primer may also comprise a portion or region that is not identical or complementary to the target gene. In some embodiments, regions of the primer that are at least 90%, at least 95%, or 100% identical or complementary to the target gene are contiguous such that any region of the primer that is not identical or complementary to the target gene does not disrupt the identical or complementary regions.

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

As used herein, "selectively hybridize" means that a polynucleotide, such as a primer or probe, will hybridize to a particular nucleic acid in a sample with at least 5-fold greater affinity than to another nucleic acid present in the same sample that has a different nucleotide sequence at the region of hybridization. Exemplary hybridization conditions are discussed herein, for example, in the case of a reverse transcription reaction or a PCR amplification reaction. In some embodiments, a polynucleotide will hybridize to a particular nucleic acid in a sample with at least 10-fold greater affinity than it will hybridize to another nucleic acid having a different nucleotide sequence at the hybridization region present in the same sample.

In some embodiments, the primer comprises a detectable moiety.

In some embodiments, a primer pair is provided. Such primer pairs are designed to amplify a portion of a target gene, such as the TV40S ribosomal protein (TV40Srp) gene, or an endogenous control, such as a sample sufficiency control (SAC), or an exogenous control, such as a Sample Processing Control (SPC). In some embodiments, the primer pair is designed to produce an amplicon that is 50 to 1500 nucleotides in length, 50 to 1000 nucleotides in length, 50 to 750 nucleotides in length, 50 to 500 nucleotides in length, 50 to 400 nucleotides in length, 50 to 300 nucleotides in length, 50 to 200 nucleotides in length, 50 to 150 nucleotides in length, 100 to 300 nucleotides in length, 100 to 200 nucleotides in length, or 100 to 150 nucleotides in length. Non-limiting exemplary primer pairs are shown in table 1.

4.2.7.3 exemplary Probe

In various embodiments, a method of detecting the presence of Trichomonas vaginalis comprises hybridizing nucleic acids of a sample to a probe. In some embodiments, the probe comprises a portion complementary to a target gene, such as the TV40S ribosomal protein (TV40Srp) gene, or an endogenous control, such as a sample sufficiency control (SAC), or an exogenous control, such as a Sample Processing Control (SPC). In some embodiments, the probe comprises a portion that is at least 90%, at least 95%, or 100% identical to a region of the target gene. In some such embodiments, a probe that is at least 90%, at least 95%, or 100% complementary to the target gene is complementary to a sufficient portion of the target gene such that it selectively hybridizes to the target gene under the particular 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. Probes that are 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 region of the probe that is at least 90%, at least 95%, or 100% complementary to the target gene is contiguous, 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 portion that is at least 90%, at least 95%, or 100% identical to a region of a target gene, such as a TV40S ribosomal protein (TV40Srp) gene, or an endogenous control, such as a sample sufficiency control (SAC), or an exogenous control, such as a Sample Processing Control (SPC). In some such embodiments, a probe comprising a region at least 90%, at least 95%, or 100% identical to a region of a target gene is capable of selectively hybridizing to an amplicon produced by amplification of 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 such that it selectively hybridizes to the amplicon under the particular assay conditions used. In some embodiments, a probe complementary to an amplicon comprises a region that is at least 90%, at least 95%, or 100% complementary to at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 consecutive nucleotides of the amplicon. Probes that are at least 90%, at least 95%, or 100% complementary to an amplicon may also comprise portions or regions that are not complementary to an amplicon. In some embodiments, the region of the probe that is at least 90%, at least 95%, or 100% complementary to the amplicon is contiguous, such that any region of the probe that is not complementary to the amplicon does not disrupt the complementary region.

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

As described above, in some embodiments, real-time PCR detection can be performed using FRET probes, which include, but are not limited to,probes, molecular beacon probes and Scorpion probes. In some embodiments, real-time PCR detection utilizesA probe is carried out, theProbes are linear probes that are typically covalently bound to a fluorescent dye at one end of the DNA and to a quencher molecule elsewhere (e.g., at the other end of the DNA). The FRET probe comprises a sequence complementary to a region of the amplicon, such that, when the FRET probe is hybridized to the amplicon, the dye fluorescence is quenched, and when the probe is digested during amplification of the amplicon, the dye is released from the probe and generates a fluorescent signal. 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 comprises a contiguous region of nucleotides having a sequence at least 90%, at least 95%, or 100% identical or complementary to a region of the target gene, such that the probe can selectively hybridize to a PCR amplicon of the region of the target gene. In some embodiments, the probe comprises a region of at least 6 contiguous nucleotides having a sequence that is completely complementary to or identical to a region of the target gene present in the region of the target gene. In some embodiments, the probe comprises a region that is at least 90%, at least 95%, or 100% identical or complementary to at least 8 contiguous nucleotides, at least 10 contiguous nucleotides, at least 12 contiguous nucleotides, at least 14 contiguous nucleotides, or at least 16 contiguous nucleotides of the target gene to be detected.

In some embodiments, has a chemical bond withThe amplicon region of sequences at least 90%, at least 95%, or 100% complementary to the probe sequence is at or near the middle of the amplicon molecule. In some embodiments, there are at least 2 nucleotides, such as at least 3 nucleotides, such as at least 4 nucleotides, such as at least 5 nuclei of the amplicon at the 5 '-end and the 3' -end of the region of complementarity independentlyA nucleotide.

In some embodiments, molecular beacons may be used to detect PCR products. ImageLike the probe, the molecular beacon detects a PCR product using FRET through a probe in which a fluorescent dye and a quencher are attached to the end of the probe. Is different fromProbes, molecular beacons remain intact during PCR cycles. Molecular beacon probes form a stem-loop structure when free in solution, allowing the dye and quencher to be close enough to cause fluorescence quenching. When the molecular beacon hybridizes to the target, the stem-loop structure is eliminated, so that the dye and quencher are spatially separated and the dye fluoresces. Molecular beacons may be derived, for example, from GeneLink^TMObtained (see www.genelink.com/newsite/products/mb intro. asp).

In some embodiments, the Scorpion probe can be used as a sequence-specific primer and for PCR product detection. Like molecular beacons, Scorpion probes form stem-loop structures when not hybridized to a target nucleic acid. However, unlike molecular beacons, the Scorpion probe enables sequence-specific priming (priming) and PCR product detection. The fluorescent dye molecule is attached to the 5 '-end of the Scorpion probe and the quencher is attached elsewhere, such as the 3' -end. The 3 'portion of the probe is complementary to the extension product of the PCR primer, and the complementary portion is linked 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 complement 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 are available, for example, from Premier BiosoftInternational (see www.premierbiosoft.com/tech _ sites/Scorpion. html).

In some embodiments, labels that may be used on FRET probes include colorimetry and fluorescent dyes such as Alexa Fluor dyes, BODIPY dyes such as BODIPY FL; cas (Cas)cade Blue; cascade Yellow; coumarins and derivatives thereof, such as 7-amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes such as Cy3 and Cy 5; eosin and erythrosine; fluorescein and its derivatives, such as fluorescein isothiocyanate; macrocyclic chelates of lanthanide ions, e.g. QuantumDye^TM(ii) a 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 described above and the following: alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430, Alexa Fluor488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor532, Alexa Fluor546, Alexa Fluor555, Alexa Fluor 568, Alexa Fluor594, Alexa Fluor 610, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750; amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/655, BODIPY FL, BODIPY R6G, BODIPY TMR, and, BODIPY-TR; cy3, Cy5, 6-FAM, fluorescein isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, SYPRO, TAMRA, 2 ', 4', 5 ', 7' -tetrabromosulfone fluorescein, and TET.

Examples of dye quencher pairs (i.e., donor/acceptor pairs) include, but are not limited to, fluorescein/tetra methylrhodamine; IAEDANS/fluorescein; EDANS/dabcyl; fluorescein/fluorescein; BODIPY FL/BODIPY FL; fluorescein/QSY 7 or QSY9 dyes. When the donor and acceptor are the same, FRET may be detected by fluorescence depolarization in some embodiments. Some 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; al (Al)exaFluor 488/Alexa Fluor 647; alexa Fluor546/Alexa Fluor 568; alexa Fluor546/Alexa Fluor 594; alexa Fluor546/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/QSY 35; alexa Fluor 350/dabcyl; alexa Fluor 488/QSY 35; alexa Fluor 488/dabcyl; alexa Fluor 488/QSY 7 or QSY 9; alexa Fluor 555/QSY 7 or QSY 9; alexa Fluor 568/QSY 7 or QSY 9; alexa Fluor 568/QSY 21; alexa Fluor 594/QSY 21; and Alexa Fluor 647/QSY 21. In some cases, the same quencher can be used for multiple dyes, e.g., a broad spectrum quencher, such as IowaQuenchers (Integrated DNAtechnologies, Coralville, IA) or Black Hole quenchers^TM(BHQ^TM；Sigma-Aldrich，St.Louis，MO)。

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

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

Examples of fluorescently labeled deoxyribonucleotides useful for preparing PCR probes for use in the methods described herein include Dinitrophenyl (DNP) -1' -dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, fluorescein-12-dUTP, Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, rhodamine Green-5-dUTP, Alexa Fluor532-5-dUTP, BODIPY TMR-14-dUTP, tetramethylrhodamine-6-dUTP, Alexa Fluor 546-14-dUTP, Alexa Fluor-5-dUTP, Tex Red-12-dUTP, Tex Red-5-dUTP, BODIPY TR-14-dUTP, Alexa Fluor-5-dFluP, BODIPY 568/630-dUTP, BODIPY 650/665-14-dUTP; AlexaFluor 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, dyes and other moieties, such as quenchers, are incorporated into polynucleotides, such as FRET probes, used in the methods described herein by 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 attached chemical moiety, such as a dye or quencher, and can be introduced into the polynucleotide, for example, by solid phase synthesis of the polynucleotide. In other embodiments, the modified nucleotide includes more than one reactive group that can be reacted with a dye or quencher before, during, or after introduction of the modified nucleotide into the nucleic acid. In particular embodiments, 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 moiety, such as uridine, adenosine, guanosine, and/or cytosine. In particular embodiments, the amine-modified nucleotide is selected from the group consisting of 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-bis- (ethylamine) ] -CTP; n6- (6-amino) hexyl-ATP; 8- [ (6-amino) hexyl ] -amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP. In some embodiments, nucleotides with different nucleobase moieties similar modifications, for example, 5- (3-amino allyl) -GTP instead of 5- (3-amino allyl) -UTP. Very polyamine modified nucleotides are available, for example, from Applied Biosystems, Sigma, Jena bioscience and TriLink.

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

In some embodiments, multiple target genes are detected in a single multiplex reaction. In some such embodiments, each probe targeting a unique amplicon can be spectrally differentiated when released from the probe, in which case each target gene is detected by a unique fluorescent signal. In some embodiments, the same fluorescent signal is used to detect more than two target genes, in which case the signal detection indicates the presence of one target gene or two.

One skilled in the art can select an appropriate detection method for the selected assay, for example, a real-time PCR assay. The detection method selected need not be the above method, and may be any method.

4.3 exemplary compositions and kits

In another aspect, a composition is provided. In some embodiments, compositions are 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 primers having: a contiguous nucleotide region having a sequence that is (i) at least 90%, at least 95%, or 100% identical to a region of the target gene, or (ii) at least 90%, at least 95%, or 100% complementary to a sequence of a region of contiguous 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 primer pairs suitable for amplifying a defined target gene region. A target gene-specific primer pair typically comprises a first primer comprising a sequence at least 90%, at least 95%, or 100% identical to a sequence of a region of a target gene and a second primer comprising a sequence at least 90%, at least 95%, or 100% complementary to a region of a target gene. Primer pairs are generally suitable for amplifying a region of a target gene that is 50 to 1500 nucleotides in length, 50 to 1000 nucleotides in length, 50 to 750 nucleotides in length, 50 to 500 nucleotides in length, 50 to 400 nucleotides in length, 50 to 300 nucleotides in length, 50 to 200 nucleotides in length, 50 to 150 nucleotides in length, 100 to 300 nucleotides in length, 100 to 200 nucleotides in length, or 100 to 150 nucleotides in length. Non-limiting exemplary primers, and primer pairs are shown in table 1.

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 probes having: a contiguous nucleotide region having a sequence that is (i) at least 90%, at least 95%, or 100% identical to a region of the target gene, or (ii) at least 90%, at least 95%, or 100% complementary to a sequence of a contiguous nucleotide region found in the target gene. Non-limiting exemplary target-specific probes are shown in table 1.

In some embodiments, the compositions (including compositions described above that include more than one target gene-specific primer) comprise more than one probe for detecting a target gene. In some embodiments, the composition comprises a probe for detecting an endogenous control (such as SAC) and/or a probe for detecting an exogenous control (such as SPC).

In some embodiments, the composition is an aqueous composition. In some embodiments, the aqueous composition comprises buffering ingredients, such as phosphates, tris, HEPES, etc., and/or other ingredients, as discussed below. In some embodiments, the composition is dried, e.g., lyophilized, and suitable for reconstitution by the addition of a fluid. The dry composition may include more than one buffering ingredient and/or other ingredients.

In some embodiments, the composition further comprises one or more additional ingredients. Other ingredients include, but are not limited to, salts, such as NaCl, KCl, and MgCl₂polymerase enzymes including thermostable polymerases such as Taq, dNTPs, Bovine Serum Albumin (BSA), etc., reducing agents such as β -mercaptoethanol, EDTA, etc. those skilled in the art can select suitable composition components depending on the intended use of the composition.

In some embodiments, compositions comprising at least one polynucleotide for detecting at least one target gene are 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 isA probe, a molecular beacon, or a Scorpion probe.

In some embodiments, the compositions comprise 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 TV40S ribosomal protein (TV40Srp) gene. In some embodiments, the FRET probe is labeled with a donor/acceptor pair such that when the probe is digested during the PCR reaction, it produces a unique fluorescent emission associated with a specific target gene. In some embodiments, when the composition comprises a plurality of FRET probes, each probe is labeled with a different donor/acceptor pair, such that when the probes are digested during the PCR reaction, each generates a unique fluorescent emission associated with a specific probe sequence and/or target gene. In some embodiments, the sequence of the FRET probe is complementary to a target region of a target gene. In other embodiments, the FRET probe has a sequence comprising more than one base mismatch when compared to the sequence of the target gene that best matches the target region.

In some embodiments, a 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, or at least 90%, at least 95%, or 100% complementary to a region of the TV40S ribosomal protein (TV40Srp) gene. In some embodiments, at least 8, at least 9, at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 nucleotides of the FRET probe are present in, or complementary to, a region of the TV40S ribosomal protein (TV40Srp) gene. In some embodiments, the FRET probe comprises a sequence with one, two, or three mismatches when compared to the sequence or complement of the TV40S ribosomal protein (TV40Srp) gene.

In some embodiments, the kit comprises a polynucleotide as discussed above. In some embodiments, the kit comprises at least one primer and/or probe as discussed above. In some embodiments, the kit comprises at least one polymerase, such as a thermostable polymerase. In some embodiments, the kit comprises dntps. In some embodiments, the kits for the real-time PCR methods described herein comprise more than one target gene-specific FRET probe and/or more than one primer for amplifying a target gene.

In some embodiments, more than one primer and/or probe is "linear". By "linear" primer is meant a polynucleotide that is a single-stranded molecule and typically does not contain a short region of, for example, at least 3, 4 or5 contiguous nucleotides, which is complementary to another region within the same polynucleotide, such that the primer forms an inner duplex. In some embodiments, the primer for reverse transcription comprises a region of at least 4, such as at least 5, such as at least 6, such as at least 7 or more contiguous nucleotides at the 3 '-terminus 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 contiguous nucleotides at the 5' -terminus of the target gene.

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

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

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 the nucleotide analogs of other stable duplexes described above. In some embodiments, the probes and/or primers used in the compositions described herein comprise all nucleotide analogs. In some embodiments, the probe and/or primer comprises more than one nucleotide analogue of a stable duplex, such as an LNA analogue, in the region of complementarity.

In some embodiments, the kits for the real-time PCR methods described herein further comprise reagents for reverse transcription and amplification reactions. In some embodiments, the kit comprises an enzyme such as a thermostable DNA polymerase, such as Taq polymerase. In some embodiments, the kit further comprises deoxyribonucleotide triphosphates (dntps) for amplification. In other embodiments, the kit comprises a buffer optimized for hybridization specific for the probe and primer.

Kits generally comprise a package having one or more containers holding reagents, either as one or more separate compositions or, optionally, as a mixture in which the reagents are allowed to be compatible. The kit may also include other material or materials that may be desirable from a user's perspective, such as one or more buffers, one or more diluents, one or more standards, and/or any other material for sample processing, washing, or any other step of performing an assay.

The kit preferably includes instructions for performing one or more of the methods described herein. Instructions included in the kit may be attached to packaging materials or may be included as a pharmaceutical instruction. The instructions are typically handwritten or printed material, but they are not limited thereto. The present invention contemplates any medium that can store the instructions and communicate them to the end user. Such media include, but are not limited to, electronic storage media (e.g., diskette, magnetic tape, magnetic cartridge, magnetic chip), optical media (e.g., CD ROM), and the like. As used herein, the term "instructions for use" may include the address of a website that provides the instructions for use.

In some embodiments, the kit may comprise more than oneThe above reagents provided in the sample cartridge. These cartridges allow extraction, amplification and detection to be performed in the self-contained "lab-in-a-cartridge" (see, e.g., U.S. Pat. Nos. 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 reagents for measuring genomic 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 embodiments, any of the kits described herein can include a urine sample and/or a swab container for collecting a urethral swab sample, a vaginal swab sample, or an endocervical swab sample.

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

5. Examples of the embodiments

5.1. Example 1: detection of Trichomonas vaginalis

The assay was designed to detect the gene for Trichomonas Vaginalis (TV)40S ribosomal protein (TV40Srp) by PCR using the primers and probes shown in table 1. In addition to TV-specific primers and probes, primers and probes are included that detect a single copy of the human gene that serves as a Sample Adequacy Control (SAC) target. Also included are primers and probes that detect bacterial genes that are included in the multiplex reaction as Sample Processing Control (SPC) targets.

Table 1: primer and probe sequences

The final primer and probe compositions of the multiplex assay are shown in table 2.

Table 2: concentration of primers and probes

F1 and F2 are detectably different dyes that can be detected and distinguished simultaneously in multiple reactions. Each probe also comprises a quencher (e.g., Q1, supra).

Each reaction contained 42-58mM KCl, 3.5-5.0mM MgCl₂250-350. mu.M dNTPs, 50mM Tris, pH8.6, and 0.01% sodium azide. AptaTaq (0.27-0.37 units/. mu.l; Roche) was used for the amplification.

For each sample to be tested, approximately 7mL of first, freshly voided (voided) urine is added to 1mL of buffer (preferably within 2 hours of sample collection). Either a physician-collected endocervical swab or a self-collected (in a clinical setting) vaginal swab was immediately placed in 2.5mL of buffer.

500 μ L of buffered urine or swab sample is loaded intoCartridge for analysis. The sample is mixed with a lysis reagent to release the nucleic acids.After lysis, nucleic acids released from the sample are captured on the DNA-binding substrate. The nucleic acids are eluted from the substrate and used to recover the reagents for real-time PCR (described above). The reaction cycles used were: in thatUse in a systemCylinder, 95 ℃ for 1 minute, followed by 40 cycles of 92.5 ℃ for 5 seconds, at 68 ℃ for 20 seconds.

The results of the measurements are as 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 assay results and interpretation

5.2 example 2: clinical Properties

The performance characteristics of the Xpert TV assay were evaluated in 13 institutions in the united states. Due to the low prevalence of Trichomonas vaginalis and the difficulty of obtaining fresh Trichomonas vaginalis-positive samples from male subjects, the sample populations used in this study were provided using artificial male urine samples.

Subjects include asymptomatic and symptomatic, sexually active males and females with informed consent, who observe: OB/GYN, Sexually Transmitted Diseases (STD), adolescents, public health facilities, and family planning clinics.

Study samples consisted of male urine, female urine, cervical swabs and patient-collected vaginal swabs (collected in a clinical setting) which were expected to be collected. An artificial male urine sample is included to supplement the male sample size.

Comparing Xpert TV assay performance with the use of transcription-mediated assays (Trichomonas vaginalis assay, GenProbe Hologic, San Diego, USA) for detection of Trichomonas vaginalis ribosomal RNA by FDA-specific in vitro quantitative nucleic acid amplification comparator assay. Samples with differential results between the Xpert TV assay and the comparator assay were analyzed using two-way sequencing of separate repeated genomic DNA sequences. See Bandea, et al, journal of clinical microbiology.2013, 51 (4): 1298-1300.

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

Results from the Xpert TV assay were compared to the comparator assay and differential bidirectional sequencing was performed. The sensitivity and specificity of gender, sample type and symptom status are provided in table 4.

5.3 example 3: detection limit

Analysis of sensitivity or detection Limit (LoD) of the Xpert TV assay assessed using two Trichomonas vaginalis strains, one susceptible to Metronidazole (Trichomonas vaginalis)30001^TM) And a metronidazole resistant (Trichomonas vaginalis)30238^TM). Both strains were tested in Trichomonas vaginalis-negative pooled Male Urine (MU) mixed with buffer and Trichomonas vaginalis-negative pooled Vaginal Swabs (VS) in buffer.

Detection limit (LoD) was assessed in 20 replicates over three days at a minimum of five concentrations for each strain and sample type. LoD was assessed by logistic regression. LoD is defined as the lowest concentration at which the lowest number of cells/mL that can be repeatedly distinguished from a negative sample with 95% confidence or 19 of 20 replicates are positive. The study was performed with 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 is 6 cells/mL. The proposed LoD was verified by analyzing at least 20 replicates diluted to the estimated LoD concentration.

Table 5: trichomonas vaginalis detection limit using Xpert TV

5.4 example 4: measurement of reproducibility

A group of eight samples with different concentrations of trichomonas vaginalis were tested on 12 different days by two different operators at each of three sites (8 samples x1 times/day x 12 days x2 operators x3 sites). Three batches of Xpert TV assays were used at each of the 3 test sites. The Xpert TV assay was performed according to the Xpert TV assay procedure. The results are summarized in table 6.

The reproducibility of the Xpert TV assay was also evaluated with respect to the fluorescence signal expressed as Ct values for each detected target. Mean, Standard Deviation (SD), and Coefficient of Variation (CV) between sites, batches, days, operators, and within-assay for each panel member are provided in table 7.

5.5. Example 5: analytical containment

Analytical inclusion of the Xpert TV assay was assessed by testing 17 trichomonas vaginalis strains in triplicate at a concentration no greater than the 3x analytical limit of detection (3x LoD). Each strain was tested in trichomonas vaginalis-negatively pooled Vaginal Swabs (VS) in buffer, and Male Urine (MU) mixed with buffer. See table 8. Under the study conditions, all strains reported the TV-detected ED results. The Xpert TV assay showed 100% inclusion in both sample types.

Table 8: analysis containment table

5.6. Example 6: analysis of specificity

A group of 47 organisms (including bacteria, fungi and viruses common in the urogenital tract, and other protozoa closely related to trichomonas) were tested using the Xpert TV assay. At 1x 10⁷Each bacterial or fungal strain is tested at cfu/mL or higher. Strains that did not produce countable clones were diluted to 0.5McFarland units, corresponding approximately to 1.5X108cfu/mL for E.coli. The virus strain was purchased as a heat-inactivated stock from ZeptoMetrix corp and was purchased at 1x 10⁶U/mL or 10⁶genome/mL test. In the growth cultureProtozoa were cultured in culture medium, visually counted by light microscope and counted at 1x 10⁶cell/mL assay. The tests were performed in triplicate. The results of the tested organism and Xpert TV assays are listed in table 9.

One organism, Trichomonas stomachica (Trichomonas tenax), reported 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: analysis specificity table

5.7. Example 7: interfering substances

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

The material was diluted in pooled negative vaginal swab matrix and pooled negative male urine matrix, respectively. The substances were tested in the same matrix loaded with Trichomonas vaginalis cells at no more than three times the limit of detection for each sample type. Eight replicates of each set of negative and positive samples were tested using the Xpert TV assay and compared to results obtained in controls of the same samples without the addition of potential interfering substances. The substances and test concentrations are listed in tables 10 and 11.

No invalid results were reported in the test with the substance diluted in the negative urine matrix under the study conditions; as expected, all test reports did not detect TV. Assay interference was observed in a test with 0.75% v/v blood diluted in a positive urine matrix and 1.8mg/mL azithromycin. No false negative results were reported for the test with 0.5% v/v blood and 1mg/mL azithromycin.

Under the conditions studied, no invalid results were reported in the test with the negative swab matrix diluted in the pool; as expected, all test reports did not detect TV.

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

Table 10: possible interfering substances in urine samples

Table 11: possible interfering substances in swab samples

5.8. Example 8: pollution caused by carrying

Study was carried out in bufferHigh (10) in the sample⁶Individual cells/mL) composition of repeated tests of TV-negative vaginal swab pools in buffer samples treated in the same GeneXpert module immediately after TV-positive vaginal swab pool. Study of TV-negative vaginal swab pool in buffer sample treated in the same GeneXpert Module followed by high in buffer sample only (10)⁶Individual cells/mL) composition of TV positive vaginal swab pool. The test protocol was repeated 20 more times on two GeneXpert modules for a total of 82 runs, resulting in 40 positive and 42 negative samples. All 40 positive samples were correctly reported as TV detected and all 42 negative samples were correctly reported as no TV detected.

5.9. Example 9: surrogate primers and probes tested for detection of TV

To develop the TV assay described herein, four different forward primers, two different reverse primers, and two different probes for detecting the TV40S ribosomal protein (TV40Srp) gene were tested for sensitivity and specificity in the assay (e.g., cross-reactivity with other species). Table 12 shows the primers and probes tested.

Table 12: alternative primer and probe sequences

The TV positive ALT1 was found to cross-react with human five trichomonas (Pth), another closely related trichomonas found in the human gut. When the TV forward ALT1 was used for assays with 1000 copies of TV and assays with 500,000 copies of Pth, a Ct of 30.7 for TV and a Ct of 26.3 for Pth were detected. The TV forward ALT2 was less sensitive than the final design, detecting TV at a higher Ct value of 31.5. Similarly, the TV forward ALT3 was less sensitive than the final design, and TV was also detected at higher Ct values. The TV reverse ALT1 also resulted in a less sensitive assay, with TV detected at higher Ct values. Finally, the TV probe ALT1 was not as sensitive and consistent as the final design.

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

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

Tables of certain sequences

Sequence listing

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Claims

1. A method of detecting the presence or absence of Trichomonas Vaginalis (TV) in a sample from a subject, the method comprising detecting the presence or absence of the TV40S ribosomal protein (TV40Srp) gene or RNA in the sample.

2. A method of determining whether a subject has a Trichomonas Vaginalis (TV) infection, the method comprising detecting the presence or absence of a TV40S ribosomal protein (TV40Srp) gene or RNA in a sample from the subject.

3. The method of claim 1 or claim 2, wherein the subject has not previously been treated for TV infection.

4. The method of claim 1 or claim 2, wherein the subject was previously treated for TV infection.

5. The method of claim 4, wherein the prior treatment comprises one or more doses of metronidazole or tinidazole.

6. The method of any one of the preceding claims, wherein the subject does not have any symptoms of TV infection.

7. The method of any one of claims 1 to 5, wherein the subject has more than one symptom of TV infection.

8. The method of claim 7, wherein the subject has one or more symptoms selected from the group consisting of vaginitis, urethritis, and cervicitis.

9. The method of claim 7 or claim 8, wherein the subject is female and has one or more symptoms selected from: itching, burning, redness, and/or pain of the genitals; genital odor; discomfort with urination; and thin clear, white, yellow or green excreta.

10. The method of any one of the preceding claims, wherein the subject is pregnant.

11. The method of claim 7 or claim 8, wherein the subject is male and has one or more symptoms selected from: itching and/or burning within the penis; cauterizing after ejaculation and/or urination; and penile waste.

12. The method of any one of the preceding claims, wherein the method comprises detecting an endogenous control.

13. The method of claim 12, wherein the endogenous control is a sample sufficiency control.

14. The method of claim 12 or claim 13, wherein the endogenous control is a single copy of a human gene.

15. The method of claim 14, wherein the endogenous control is selected from the group consisting of HMBS, GAPDH, beta actin, and beta globin.

16. The method of any one of the preceding claims, wherein the method comprises detecting an exogenous control.

17. The method of claim 16, wherein the exogenous control is a sample processing control.

18. The method of claim 16 or claim 17, wherein the exogenous control comprises a DNA sequence that is not expected to be present in the sample.

19. The method of any one of claims 16 to 18, wherein the exogenous control is a bacterial gene.

20. The method of any one of the preceding claims, wherein the method comprises PCR.

21. The method of claim 20, wherein the method comprises quantitative PCR.

22. The method of claim 20 or claim 21, wherein the PCR reaction takes less than 2 hours from the initial denaturation step through the final extension step.

23. The method of any one of the preceding claims, wherein the TV40S ribosomal protein (TV40Srp) gene comprises the amino acid sequence of SEQ ID NO: 4.

24. The method of any one of the preceding claims, wherein the method comprises contacting nucleic acids from the sample with a first primer pair for detecting the TV40S ribosomal protein (TV40Srp) gene or RNA.

25. The method of claim 24, wherein the method comprises contacting nucleic acids from the sample with a second primer pair for detecting an endogenous control.

26. The method of claim 24 or claim 25, wherein the method comprises contacting nucleic acids from the sample with a third primer pair for detecting an exogenous control.

27. The method of any one of claims 24 to 26, wherein the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence identical to SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100%.

28. The method of claim 27, wherein the amino acid sequence of SEQ ID NO: 4 or its complement, each of the first primer and the second primer independently comprises 0, 1, or 2 mismatches.

29. The method of any one of claims 24 to 28, wherein the first primer pair comprises a first primer consisting of 15 to 30 nucleotides and a second primer consisting of 15 to 30 nucleotides.

30. The method of any one of claims 24 to 29, wherein the first primer pair comprises SEQ ID NO: 1 and SEQ ID NO: 2.

31. The method of any one of claims 24 to 30, wherein the first primer pair produces an amplicon that is 50 to 500 nucleotides in length, 50 to 400 nucleotides in length, 50 to 300 nucleotides in length, 50 to 200 nucleotides in length, 50 to 150 nucleotides in length, 100 to 300 nucleotides in length, 100 to 200 nucleotides in length, or 100 to 150 nucleotides in length.

32. The method of claim 31, wherein the method comprises forming a Tv40Srp amplicon.

33. The method of claim 32, wherein the method contacts the Tv40Srp amplicon with a first probe capable of selectively hybridizing to the Tv40Srp amplicon.

34. The method of claim 33, wherein the first probe comprises a detectable label.

35. The method of claim 34, wherein the first probe comprises a fluorescent dye and a quencher molecule.

36. The method of any one of claims 33 to 35, wherein the first probe comprises SEQ ID NO: 4 or SEQ ID NO: 5, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100% identical or complementary sequence.

37. The method of claim 36, wherein the amino acid sequence of SEQ ID NO: 4 or the complement thereof, or a sequence that is identical to SEQ ID NO: 5 or its complement, the first probe comprises 0, 1, or 2 mismatches.

38. The method of any one of claims 33 to 37, wherein the first probe consists of 15 to 30 nucleotides.

39. The method of any one of claims 33 to 38, wherein the first probe has the sequence of SEQ ID NO: 3.

40. The method of any one of claims 32 to 39, wherein the method comprises forming an endogenous control amplicon and/or an exogenous control amplicon.

41. The method of claim 40, wherein the method comprises contacting the endogenous control amplicon with a second probe capable of selectively hybridizing to the endogenous control amplicon and/or contacting the exogenous control amplicon with a third probe capable of selectively hybridizing to the exogenous control amplicon.

42. The method of claim 41, wherein the second and third probes each comprise a detectable label, wherein the detectable labels may be the same or different.

43. The method of claim 42, wherein the detectable labels of the second and third probes are detectable differently from the detectable label of the first probe.

44. The method of any one of the preceding claims, wherein the method comprises detecting the Tv40Srp gene or RNA, an endogenous control, and an exogenous control in a single multiplex reaction.

45. The method of any one of the preceding claims, wherein the sample is selected from the group consisting of a urine sample, an endocervical swab sample, a vaginal swab sample, and a urethral swab sample.

46. A composition comprising a first primer pair for detecting a trichomonas vaginalis 40S ribosomal protein (Tv40Srp) gene or RNA.

47. The composition of claim 39, wherein the composition comprises a second primer pair for detecting an endogenous control.

48. The composition of claim 40, wherein the endogenous control is a sample sufficiency control.

49. The composition of claim 40, wherein the endogenous control is selected from HMBS, GAPDH, beta actin, and beta globin.

50. The composition of any one of claims 39 to 42, comprising a third primer pair for detecting an exogenous control.

51. The composition of claim 43, wherein the exogenous control is a sample processing control.

52. The composition of claim 43, wherein the exogenous control is a bacterial gene.

53. The composition of any one of claims 46 to 52, wherein the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence identical to SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100%.

54. The composition of claim 53, wherein the amino acid sequence of SEQ ID NO: 4 or its complement, each of the first and second primers independently comprises 0, 1, or 2 mismatches.

55. The composition of any one of claims 46 to 54, wherein the first primer pair comprises a first primer consisting of 15 to 30 nucleotides and a second primer consisting of 15 to 30 nucleotides.

56. The composition of any one of claims 46 to 55, wherein the first primer pair comprises SEQ ID NO: 1 and SEQ ID NO: 2.

57. The composition of any one of claims 46 to 56, wherein the composition comprises a first probe capable of selectively hybridizing to the Tv40Srp amplicon generated by the first primer pair.

58. The composition of claim 57, wherein the first probe comprises a detectable label.

59. The composition of claim 58, wherein the first probe comprises a fluorescent dye and a quencher molecule.

60. The composition of any one of claims 57-59, wherein the first probe comprises a sequence identical to SEQ ID NO: 4 or SEQ ID NO: 5, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100% identical or complementary sequence.

61. The composition of claim 60, wherein the amino acid sequence of SEQ ID NO: 4 or the complement thereof or to SEQ ID NO: 5 or its complement, the first probe comprises 0, 1, or 2 mismatches.

62. The composition of any one of claims 57-61, wherein the first probe consists of 15 to 30 nucleotides.

63. The composition of any one of claims 57-62, wherein the first probe has the sequence of SEQ ID NO: 3.

64. The composition of any one of claims 57-63, wherein the Tv40Srp amplicon has the amino acid sequence of SEQ ID NO: 5.

65. The composition of any one of claims 46 to 64, wherein the composition comprises a second probe capable of selectively hybridizing to an endogenous control amplicon produced by a second primer pair.

66. The composition of claim 65, wherein said endogenous control is a sample sufficiency control.

67. The composition of claim 65, wherein the endogenous control is selected from the group consisting of HMBS, GAPDH, beta actin, and beta globin.

68. The composition of any one of claims 46 to 67, wherein the composition comprises a third probe capable of selectively hybridizing to an exogenous control amplicon generated by the third primer pair.

69. The composition of claim 68, wherein the exogenous control is a sample processing control.

70. The composition of claim 68, wherein the exogenous control comprises a DNA sequence that is not expected to be present in the sample.

71. The composition of any one of claims 68-70, wherein the exogenous control is bacterial DNA.

72. The composition of any one of claims 46 to 71, wherein the composition is a lyophilized composition.

73. The composition of any one of claims 46 to 71, wherein the composition is in solution.

74. The composition of claim 73, wherein the composition comprises nucleic acids from a sample from a subject being tested for the presence or absence of Trichomonas vaginalis.

75. A kit comprising a first primer pair for detecting a trichomonas vaginalis 40S ribosomal protein (Tv40Srp) gene or RNA.

76. The kit of claim 75, wherein 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 composition in the kit.

77. The kit of claim 76, wherein the endogenous control is a sample sufficiency control.

78. The kit of claim 76, wherein the endogenous control is selected from the group consisting of HMBS, GAPDH, beta actin, and beta globin.

79. The kit of any one of claims 75 to 78, comprising a third primer pair for detecting an exogenous control, wherein the third primer pair is in the same or different composition as the primer pair for detecting Tv40Srp and the second primer pair.

80. The kit of claim 79, wherein the exogenous control is a sample processing control.

81. The kit of claim 79 or claim 80, wherein the exogenous control comprises a DNA sequence that is not expected to be present in the sample.

82. The kit of any one of claims 79 to 81, wherein the exogenous control is a bacterial gene.

83. The kit of any one of claims 75 to 82, wherein the first primer pair comprises a first primer and a second primer, wherein the first primer comprises a sequence identical to SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at least 95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 consecutive nucleotides of SEQ ID NO: 4, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100%.

84. The kit of claim 83, wherein the variant of SEQ ID NO: 4 or its complement, each of the first and second primers independently comprises 0, 1, or 2 mismatches.

85. The kit of any one of claims 75 to 84, wherein the first primer pair comprises a first primer consisting of 15 to 30 nucleotides and a second primer consisting of 15 to 30 nucleotides.

86. The kit of any one of claims 75 to 85, wherein the primer pair for detecting the Tv40Srp gene or RNA comprises the nucleotide sequence of SEQ ID NO: 1 and SEQ ID NO: 2.

87. The kit of any one of claims 75 to 86, wherein said kit comprises a first probe capable of selectively hybridizing to a Tv40Srp amplicon generated by a first primer pair, wherein said first probe is in the same or different composition as one or more of said primer pairs.

88. The kit of claim 87, wherein the first probe comprises a detectable label.

89. The kit of claim 88, wherein the first probe comprises a fluorescent dye and a quencher molecule.

90. The kit of any one of claims 87-89, wherein the first probe comprises a sequence identical to SEQ ID NO: 4 or SEQ ID NO: 5, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of at least 90%, at least 95%, or 100% identical or complementary sequence.

91. The kit of claim 90, wherein the nucleic acid sequence identical to SEQ ID NO: 4 or the complement thereof or to SEQ ID NO: 5 or its complement, the first probe comprises 0, 1, or 2 mismatches.

92. The kit of any one of claims 87 to 91, wherein the first probe consists of 15 to 30 nucleotides.

93. The kit of any one of claims 87-92, wherein the first probe has the sequence of SEQ ID NO: 3.

94. The kit of any one of claim 87 or claim 93, wherein the Tv40Srb amplicon has the amino acid sequence of SEQ ID NO: 5.

95. The kit of any one of claims 75 to 94, wherein the kit comprises a second probe capable of selectively hybridizing to an endogenous control amplicon generated by a second primer pair, wherein the second probe is in the same or different composition as one or more of the primer pairs.

96. The kit of any one of claims 75 to 95, wherein the kit comprises a third probe capable of selectively hybridizing to an exogenous control amplicon generated by a third primer pair, wherein the third probe is in the same or different composition as one or more of the primer pairs.

97. The kit of any one of claims 75 to 96, wherein the kit comprises dNTPs and/or a thermostable polymerase.

98. The kit of any one of claims 75 to 97, wherein the kit comprises more than one lyophilized composition.

99. Consisting of SEQ ID NO: 1, wherein the primer comprises at least one modified nucleotide.

100. Consisting of SEQ ID NO: 2, wherein the primer comprises at least one modified nucleotide.

101, SEQ ID NO: 3, wherein the probe comprises at least one modified nucleotide and/or a detectable label.

102. The probe of claim 101, wherein the probe comprises a fluorescent dye and a quencher molecule.

103. The probe of claim 102, which is a Fluorescence Resonance Energy Transfer (FRET) probe.

104. The probe of any one of claims 101 to 103, wherein the probe comprises at least one modified nucleotide.

105. A composition comprising a polypeptide consisting of SEQ ID NO: 2 and a first primer consisting of the sequence of SEQ ID NO: 3, wherein the first primer and the second primer each comprise at least one modified nucleotide.

106. The composition of claim 105, wherein the composition comprises a polypeptide consisting of SEQ ID NO: 3, wherein the probe comprises at least one modified nucleotide and/or a detectable label.

107. The composition of claim 106, wherein the probe comprises a fluorescent dye and a quencher molecule.

108. The composition of claim 106, wherein the probe is a Fluorescence Resonance Energy Transfer (FRET) probe.

109. The composition of any one of claims 106 to 108, wherein the probe comprises at least one modified nucleotide.

110. The composition of any one of claims 105-109, wherein the composition is a lyophilized composition.

111. The composition of any one of claims 105 to 109, wherein the composition is in solution.

112. The composition of claim 111, wherein the composition comprises nucleic acids from a sample from a subject.