1. FIELD OF THE INVENTION
[001] Compositions and methods for detecting Trichomonas vaginalis are
provided.
2. BACKGROUND
[002] The protozoan Trichomonas vaginalis is responsible for trichomoniasis,
which is a common sexually transmitted infection that can infect both men and women.
There are 7.4 million cases of trichomoniasis annually in the United States. Trichomoniasis
infections can be symptomatic or asymptomatic. See, e.g., Ginocchio et al, J. Clin.
Microbiol. 2012, 50: 2601-2608. In women, trichomoniasis is one of a range of conditions
that comprise vaginal discharge. See, e.g., Centers for Disease Control and Prevention
(CDC). CDC fact sheet: trichomoniasis. 2010. www.cdc.gov/std/trichomonas/STDFact-
Trichomoniasis.htm. Symptoms in females can include itching, burning, redness, or
soreness of the genitals, unusual odor, discomfort with urination, or a thin clear, white,
yellow, or green discharge. See id. In men, trichomoniasis may cause non-gonococcal
urethritis (NGU). Symptoms in males can include itching or burning inside the penis,
burning after ejaculation or urination, or penile discharge. See, e.g, Workowski et al.,
Centers for Disease Control and Prevention. Sexually transmitted disease treatment
guidelines, 2010. MMWR 2010;59 (RR-12):1-1 10; Centers for Disease Control and
Prevention. Biosafety in Microbiological and Biomedical laboratories.
www.cdc.gov/biosafety/publications/.
[003] Improved methods for detection of Trichomonas vaginalis (TV) are needed.
In particular, a highly specific, accurate, and sensitive urine- or swab-based diagnostic test
is needed.
3. SUMMARY
[004] In some embodiments, methods of detecting the presence or absence of
Trichomonas vaginalis (TV) in a sample from a subject are provided. In some
embodiments, methods of determining whether a subject has a Trichomonas vaginalis (TV)
infection are provided. In some embodiments, the methods comprise detecting the presence
or absence of the TV 40S ribosomal protein (Tv40Srp) gene or RNA in a sample from the
subject.
[005] In some embodiments, the subject has not previously been treated for TV
infection. In some embodiments, the subject has previously been treated for TV infection.
In some embodiments, the previous treatment comprised one or more doses of
metronidazole or tinidazole. In some embodiments, the subject does not have any
symptoms of TV infection. In some embodiments, the subject has one or more symptoms 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 itching, burning, redness, and/or soreness of the genitals;
unusual odor of the genitals; discomfort with urination; and a thin clear, white, yellow, or
green discharge. In some embodiments, the subject is pregnant. In some embodiments, the
subject is male and has one or more symptoms selected from itching and/or burning inside
the penis; burning after ejaculation and/or urination; and penile discharge.
[006] In some embodiments, the method comprises detecting an endogenous
control. In some embodiments, the endogenous control is a sample adequacy control. In
some embodiments, the endogenous control is a single-copy human gene. In some
embodiments, the endogenous control is selected from HMBS, GAPDH, beta actin, and beta
globin.
[007] 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.
[008] 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 an initial denaturation step
through a final extension step.
[009] In some embodiments, the the TV 40S ribosomal protein (Tv40Srp) gene
comprises the sequence of SEQ ID NO: 4. In some embodiments, the method comprises
contacting nucleic acids from the sample with a first primer pair for detecting the TV 40S
ribosomal protein (Tv40Srp) gene or RNA. In some embodiments, the method comprises
contacting 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.
[0010] In some embodiments, the first primer pair comprises a first primer and a
second primer, wherein the first primer comprises a sequence that is at least 90%, at least
95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of
SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at
least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 4. In some embodiments, the first primer and the second primer
each independently comprises 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its
complement. In some embodiments, the first primer pair comprises a first primer consisting
of 15 to 30 nucleotides and a second primer consisting of 15 to 30 nucleotides. In some
embodiments, the first primer pair comprises a first primer of SEQ ID NO: 1 and a second
primer of SEQ ID NO: 2. In some embodiments, the first primer pair produces an amplicon
that is 50 to 500 nucleotides long, 50 to 400 nucleotides long, 50 to 300 nucleotides long, 50
to 200 nucleotides long, 50 to 150 nucleotides long, 100 to 300 nucleotides long, 100 to 200
nucleotides long, or 100 to 150 nucleotides long.
[001 1] In some embodiments, the method comprises forming the Tv40Srp amplicon.
In some embodiments, the method comprises contacting the Tv40Srp amplicon with a first
probe capable of selectively hybridizing with the Tv40Srp amplicon. In some
embodiments, the first probe comprises a detectable label. In some embodiments, the first
probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the first
probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 4 or SEQ ID NO: 5. In some embodiments, the first probe comprises 0, 1, or 2
mismatches compared to SEQ ID NO: 4 or its complement or compared to SEQ ID NO: 5
or its complement. In some embodiments, the first probe consists of 15 to 30 nucleotides.
In some embodiments, the first probe has the sequence of SEQ ID NO: 3.
[0012] 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 with the engoenous control amplicon and/or contacting the
exogenous control amplicon with a third probe capable of selectively hybridizing with the
exoenous 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 labels of the second and third probes are detectably
different from the detectable label of the first probe. In some embodiments, the method
comprises detecting the Tv40Srp gene or RNA, an endogenous control, and an exogenous
control in a single multiplex reaction.
[0013] In some embodiments, the sample is selected from a urine sample, an
endocervical swab sample, a vaginal swab sample, and a urethral swab sample.
[0014] In some embodiments, compositions comprising a first primer pair for
detecting a Trichomonas vaginalis 40S ribosomal protein (Tv40Srp) gene or RNA are
provided. In some embodiments, the composition comprises a second primer pair for
detecting an endogenous control. In some embodiments, the endogenous control is a sample
adequacy control. In some embodiments, the endogenous control is selected from HMBS,
GAPDH, 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.
[0015] In some embodiments, the first primer pair comprises a first primer and a
second primer, wherein the first primer comprises a sequence that is at least 90%, at least
95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of
SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at
least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 4. In some embodiments, the first primer and the second primer
each independently comprises 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its
complement. In some embodiments, the first primer pair comprises a first primer consisting
of 15 to 30 nucleotides and a second primer consisting of 15 to 30 nucleotides. In some
embodiments, the first primer pair comprises a first primer of SEQ ID NO: 1 and a second
primer of SEQ ID NO: 2.
[0016] In some embodiments, the composition comprises a first probe capable of
selectively hybridizing to a Tv40Srp amplicon produced by the first primer pair. In some
embodiments, the first probe comprises a detectable label. In some embodiments, the first
probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the first
probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 4 or SEQ ID NO: 5. In some embodiments, the first probe comprises 0, 1, or 2
mismatches compared to SEQ ID NO: 4 or its complement or compared to SEQ ID NO: 5
or its complement. In some embodiments, the first probe consists of 15 to 30 nucleotides.
In some embodiments, the first probe has the sequence of SEQ ID NO: 3. In some
embodiments, the Tv40Srp amplicon has the sequence of SEQ ID NO: 5.
[0017] In some embodiments, the composition comprises a second probe capable of
selectively hybridizing to an endogenous control amplicon produced by the second primer
pair. In some embodiments, the endogenous control is a sample adequacy control. In some
embodiments, the endogenous control is selected from HMBS, GAPDH, beta actin, and beta
globin. In some embodiments, the composition comprises a third probe capable of
selectively hybridizing to an exogenous control amplicon produced by 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 a bacterial DNA.
[0018] In some embodiments, the composition is a lyophilized composition. In
some embodiments, the composition is in solution. In some embodiments, the composition
comprises nucleic acids from a sample from a subject being tested for the presence of
absence of Trichomonas vaginalis.
[0019] In some embodiments, kits are 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 compositions in the kit. In some embodiments, the endogenous control is a sample
adequacy control. In some embodiments, the endogenous control is selected from HMBS,
GAPDH, 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 from 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.
[0020] In some embodiments, the first primer pair comprises a first primer and a
second primer, wherein the first primer comprises a sequence that is at least 90%, at least
95%, or 100% identical to at least 15, at least 16, at least 17, at least 18, at least 19, at least
20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of
SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at least 90%, at
least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least 18, at least
19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25 contiguous
nucleotides of SEQ ID NO: 4. In some embodiments, the first primer and the second primer
each independently comprises 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its
complement. In some embodiments, the first primer pair comprises a first primer consisting
of 15 to 30 nucleotides and a second primer consisting of 15 to 30 nucleotides. In some
embodiments, the first primer pair comprises a first primer of SEQ ID NO: 1 and a second
primer of SEQ ID NO: 2.
[0021] In some embodiments, the kit comprises a first probe capable of selectively
hybridizing to a Tv40Srp amplicon produced by the first primer pair, wherein the first probe
is in the same or different composition from one or more of the primer pairs. In some
embodiments, the first probe comprises a detectable label. In some embodiments, the first
probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the first
probe comprises a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, at least 23, at least 24, or at least 25 contiguous nucleotides of SEQ ID
NO: 4 or SEQ ID NO: 5. In some embodiments, the first probe comprises 0, 1, or 2
mismatches compared to SEQ ID NO: 4 or its complement or compared to SEQ ID NO: 5
or its complement. In some embodiments, the first probe consists of 15 to 30 nucleotides.
In some embodiments, the first probe has the sequence of SEQ ID NO: 3. In some
embodiments, the Tv40Srp amplicon has the sequence of SEQ ID NO: 5.
[0022] In some embodiments, the kit comprises a second probe capable of
selectively hybridizing to an endogenous control amplicon produced by the second primer
pair, wherein the second probe is in the same or different composition from one or more of
the primer pairs. In some embodiments, the kit comprises a third probe capable of
selectively hybridizing to an exogenous control amplicon produced by the third primer pair,
wherein the third probe is in the same or different composition from one or more of the
primer pairs.
[0023] In some embodiments, the kit comprises dNTPs and/or a thermostable
polymerase. In some embodiments, the kit comprises one or more lyophilized
compositions.
[0024] In some embodiments, a primer is provided, wherein the primer consists of
the sequence of SEQ ID NO: 1, wherein the primer comprises at least one modified
nucleotide. In some embodiments, a primer is provided, wherein the primer consists of the
sequence of SEQ ID NO: 2, wherein the primer comprises at least one modified nucleotide.
In some embodiments, a probe is provided, wherein the probe consists of the sequence of
SEQ ID NO: 3, wherein the probe comprises at least one modified nucleotide and/or a
detectable label. In some embodiments, the probe comprises a fluorescent dye and a
quencher molecule. In some embodiments, the probe is a fluorescence resonance energy
transfer (FRET) probe. In some embodiments, the probe comprises at least one modified
nucleotide.
[0025] In some embodiments, a composition is provided, wherein the composition
comprises a first primer consisting of the sequence of SEQ ID NO: 2 and a second primer
consisting of the sequence of SEQ ID NO: 3, wherein the first primer and the second primer
each comprises at least one modified nucleotide. In some embodiments, the composition
comprises a probe consisting of the sequence of SEQ ID NO: 3, wherein the probe
comprises at least one modified nucleotide and/or a detectable label. In some embodiments,
the probe comprises a fluorescent dye and a quencher molecule. In some embodiments, the
probe is a fluorescence resonance energy transfer (FRET) probe. In some embodiments, the
probe comprises at least one modified nucleotide. In some embodiments, the composition is
a lyophilized composition. In some embodiments, the composition is in solution. In some
embodiments, the composition comprises nucleic acids of a sample from a subject.
[0026] Further embodiments and details of the inventions are described below.
4. DETAILED DESCRIPTION
4.1. Definitions
[0027] To facilitate an understanding of the present invention, a number of terms
and phrases are defined below:
[0028] As used herein, the terms "detect", "detecting" or "detection" may describe
either the general act of discovering or discerning or the specific observation of a detectably
labeled composition.
[0029] As used herein, the term "detectably different" refers to a set of labels (such
as dyes) that can be detected and distinguished simultaneously.
[0030] As used herein, the terms "patient" and "subject" are used interchangeably to
refer to a human. In some embodiments, the methods described herein may be used on
samples from non-human animals.
[0031] "Trichomonas vaginalis" refers to the protozoan responsible for
trichomoniasis, a common sexually transmitted infection that can infect both men and
women. Trichomoniasis may be symptomatic or asymptomatic. Symptoms of
trichomoniasis include, but are not limited to, vaginitis, urethritis, and cervicitis. Symptoms
in females include, but are not limited to, itching, burning, redness, or soreness of the
genitals, unusual odor, discomfort with urination, or a thin clear, white, yellow, or green
discharge. Symptoms in males include, but are not limited to, itching or burning inside the
penis, burning after ejaculation or urination, or penile discharge.
[0032] 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 that include any of the known base
analogs of DNA and RNA including, but not limited to, 4-acetylcytosine, 8-hydroxy-N6-
methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,
5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil,
dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-
methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,
7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-
2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and
2,6-diaminopurine.
[0033] As used herein, the term "oligonucleotide," refers to a single-stranded
polynucleotide having fewer than 500 nucleotides. In some embodiments, an
oligonucleotide is 8 to 200, 8 to 100, 12 to 200, 12 to 100, 12 to 75, or 12 to 50 nucleotides
long. Oligonucleotides may be referred to by their length, for example, a 24 residue
oligonucleotide may be referred to as a "24-mer."
[0034] As used herein, the term "complementary" to a target gene (or target region
thereof), and the percentage of "complementarity" of the probe sequence to the target gene
sequence is the percentage "identity" to the sequence of target gene or to the reverse
complement of the sequence of the target gene. In determining the degree of
"complementarity" between probes used in the compositions described herein (or regions
thereof) and a target gene, such as those disclosed herein, the degree of "complementarity"
is expressed as the percentage identity between the sequence of the probe (or region thereof)
and sequence of the target gene or the reverse complement of the sequence of the target
gene that best aligns therewith. The percentage is calculated by counting the number of
aligned bases that are identical as between the 2 sequences, dividing by the total number of
contiguous 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 indicated otherwise. 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.
[0035] A "primer" or "probe" as used herein, refers to an oligonucleotide that
comprises a region that is 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 an mRNA (or a DNA
reverse-transcribed from an 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 a primer or
probe comprises a region that is "complementary to at least x contiguous nucleotides of a
target molecule," the primer or probe is at least 95% complementary to at least x contiguous
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.
[0036] The term "nucleic acid amplification," encompasses any means by which
at least a part of at least one target nucleic acid is reproduced, typically in a templatedependent
manner, including without limitation, a broad range of techniques for amplifying
nucleic acid sequences, either linearly or exponentially. Exemplary means for performing
an amplifying step include polymerase chain reaction (PCR), ligase chain reaction (LCR),
ligase detection reaction (LDR), multiplex ligation-dependent probe amplification (MLPA),
ligation followed by Q-replicase amplification, primer extension, strand displacement
amplification (SDA), hyperbranched strand displacement amplification, multiple
displacement amplification (MDA), nucleic acid strand-based amplification (NASBA), twostep
multiplexed amplifications, rolling circle amplification (RCA), and the like, including
multiplex versions and combinations thereof, for example but not limited to, OLA/PCR,
PCR/OLA, LDR/PCR, PCR/PCR/LDR, PCR/LDR, LCR/PCR, PCR/LCR (also known as
combined chain reaction—CCR), digital amplification, and the like. Descriptions of such
techniques can be found in, among other sources, Ausbel et al; PCR Primer: A Laboratory
Manual, Diffenbach, Ed., Cold Spring Harbor Press (1995); The Electronic Protocol Book,
Chang Bioscience (2002); Msuih et al, J . Clin. Micro. 34:501-07 (1996); The Nucleic Acid
Protocols Handbook, R. Rapley, ed., Humana Press, Totowa, N.J. (2002); Abramson et al.,
Curr Opin Biotechnol. 1993 Feb.;4(l):41-7, U.S. Pat. No. 6,027,998; U.S. Pat. No.
6,605,451, Barany et al, PCT Publication No. WO 97/31256; Wenz et al, PCT Publication
No. WO 01/92579; Day et al, Genomics, 29(1): 152-162 (1995), Ehrlich et al, Science
252:1643-50 (1991); Innis et al, PCR Protocols: A Guide to Methods and Applications,
Academic Press (1990); Favis et al, Nature Biotechnology 18:561-64 (2000); and Rabenau
et al., Infection 28:97-102 (2000); Belgrader, Barany, and Lubin, Development of a
Multiplex Ligation Detection Reaction DNA Typing Assay, Sixth International Symposium
on Human Identification, 1995 (available on the world wide web at:
promega.com/geneticidproc/ussymp6proc/blegrad.html); LCR Kit Instruction Manual, Cat.
#200520, Rev. #050002, Stratagene, 2002; Barany, Proc. Natl. Acad. Sci. USA 88:188-93
(1991); Bi and Sambrook, Nucl. Acids Res. 25:2924-2951 (1997); Zirvi et al, Nucl. Acid
Res. 27:e40i-viii (1999); Dean et al, Proc Natl Acad Sci USA 99:5261-66 (2002); Barany
and Gelfand, Gene 109:1-11 (1991); Walker et al, Nucl. Acid Res. 20:1691-96 (1992);
Polstra et al, BMC Inf. Dis. 2:18- (2002); Lage et al, Genome Res. 2003 Feb.;13(2):294-
307, and Landegren et al, Science 241:1077-80 (1988), Demidov, V., Expert Rev Mol
Diagn. 2002 Nov.;2(6):542-8., Cook et al, J Microbiol Methods. 2003 May;53(2): 165-74,
Schweitzer et al, Curr Opin Biotechnol. 2001 Feb.;12(l):21-7, U.S. Pat. No. 5,830,71 1,
U.S. Pat. No. 6,027,889, U.S. Pat. No. 5,686,243, PCT Publication No. WO0056927A3, and
PCT Publication No. WO9803673A1.
[0037] In some embodiments, amplification comprises at least one cycle of the
sequential procedures of: annealing at least one primer with complementary or substantially
complementary sequences in at least one target nucleic acid; synthesizing at least one strand
of nucleotides in a template-dependent manner using a polymerase; and denaturing the
newly-formed nucleic acid duplex to separate the strands. The cycle may or may not be
repeated. Amplification can comprise thermocycling or can be performed isothermally.
[0038] Unless otherwise indicated, the term "hybridize" is used herein refer to
"specific hybridization" which is the binding, duplexing, or hybridizing of a nucleic acid
molecule preferentially to a particular nucleotide sequence, in some embodiments, under
stringent conditions. The term "stringent conditions" refers to conditions under which a
probe will hybridize preferentially to its target sequence, and to a lesser extent to, or not at
all to, other sequences. A "stringent hybridization" and "stringent hybridization wash
conditions" in the context of nucleic acid hybridization (e.g., as in array, Southern, or
Northern hybridization) are sequence-dependent and are different under different
environmental parameters. An extensive guide to the hybridization of nucleic acids is found
in, e.g., Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology-
Hybridization with Nucleic Acid Probes part I, Ch. 2, "Overview of principles of
hybridization and the strategy of nucleic acid probe assays," Elsevier, NY ("Tijssen").
Generally, highly stringent hybridization and wash conditions for filter hybridizations are
selected to be about 5° C. lower than the thermal melting point (Tm) for the specific
sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic
strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched
probe. Very stringent conditions are selected to be equal to the Tm for a particular probe.
Dependency of hybridization stringency on buffer composition, temperature, and probe
length are well known to those of skill 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).
[0039] A "sample," as used herein, includes urine samples (including samples
derived from urine samples), endocervical swabs, and patient-collected vaginal swabs, and
other types of human samples. In some embodiments, a urine sample is a "first catch" urine
sample, which is a sample taken as a subject first begins to urinate. As used herein, urine
samples include, but are not limited to, whole urine, a sample comprising cells from a urine
sample, a sample comprising the 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, a urine sample comprises a buffer, such as a preservative. In some
embodiments, a sample is a human sample other than a urine sample, such as an
endocervical swab or a vaginal swab, including a patient-collected vaginal swab, and a
urethral swab. In some embodiments, a swab sample comprises a buffer, such as a
preservative.
[0040] An "endogenous control," as used herein refers to a moiety that is naturally
present in the sample to be used for detection. In some embodiments, an endogenous
control is a "sample adequacy control" (SAC), which may be used to determine whether
there was sufficient sample used in the assay, or whether the sample comprised sufficient
biological material, such as cells. In some embodiments, an SAC is a single copy human
gene. In some embodiments, an endogenous control, such as an SAC, is selected that can be
detected in the same manner as the target gene is detected and, in some embodiments,
simultaneously with the target gene.
[0041] An "exogenous control," as used herein, refers to a moiety that is added to a
sample or to an assay, such as a "sample processing control" (SPC). In some embodiments,
an exogenous control is included with the assay reagents. An exogenous control is typically
selected that is not expected to be present in the sample to be used for detection, or is
present at very low levels in the sample such that the amount of the moiety naturally present
in the sample is either undetectable or is detectable at a much lower level than the amount
added to the sample as an exogenous control. In some embodiments, an exogenous control
comprises a nucleotide sequence that is not expected to be present in the sample type used
for detection of the target gene. In some embodiments, an exogenous control comprises a
nucleotide sequence that is not known to be present in the species from whom the sample is
taken. In some embodiments, an exogenous control comprises a nucleotide sequence from a
different species than the subject from whom the sample was taken. In some embodiments,
an exogenous control comprises a nucleotide sequence that is not known to be present in
any species. In some embodiments, an exogenous control is selected that can be detected in
the same manner as the target gene is detected and, in some embodiments, simultaneously
with the target gene. In some embodiments, an exogenous control is a bacterial DNA. In
some embodiments, the bacterium is a species not expected to be found in the sample type
being tested.
[0042] In the sequences herein, "U" and "T" are used interchangeably, such that
both letters indicate a uracil or thymine at that position. One skilled in the art will
understand from the context and/or intended use whether a uracil or thymine is intended
and/or should be used at that position in the sequence. For example, one skilled in the art
would understand that native RNA molecules typically include uracil, while native DNA
molecules typically include thymine. Thus, where an RNA sequence includes "T", one
skilled in the art would understand that that position in the native RNA is likely a uracil.
[0043] In the present disclosure, "a sequence selected from" encompasses both "one
sequence selected from" and "one or more sequences 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 chosen.
4.2. Detecting Trichomonas vaginalis
[0044] The present inventors have developed an assay for detecting Trichomonas
vaginalis (TV). In some embodiments, the assay comprises detecting the TV 40S ribosomal
protein (Tv40Srp) gene. In some embodiments, the assay comprises detecting RNA
transcribed from the TV 40S ribosomal protein (Tv40Srp) gene. The present assay relies on
the polymerase chain reaction (PCR), and can be carried out in a substantially automated
manner using a commercially available nucleic acid amplification system. Exemplary
nonlimiting nucleic acid amplification systems that can be used to carry out the methods of
the invention include the GeneXpert® system, a GeneXpert® Infinity system, and a
Smartcycler System (Cepheid, Sunnyvale, CA). The present assay can be completed in
under 3 hours, and in some embodiments, under 2 hours, using an automated system, for
example, the GeneXpert® system.
4.2.1. General methods
[0045] Compositions and methods for detecting Trichomonas vaginalis (TV) are
provided. In some embodiments, the method comprises detecting the TV 40S ribosomal
protein (Tv40Srp) gene.
[0046] In some embodiments, a method of detecting Trichomonas vaginalis (TV) in
a subject comprises detecting the presence of the TV 40S ribosomal protein (Tv40Srp) gene
in a sample from the subject. In some embodiments, the sample is selected from a urine
sample, an endocervical swab, and a vaginal swab. In some embodiments, the urine sample
is a first catch urine sample.
[0047] In some embodiments, a method of detecting TV further comprises detecting
at least one endogenous control, such as a sample adequacy control (SAC). In some
embodiments, a method of detecting TV further comprises detecting at least one exogenous
control, such as a sample processing control (SPC). In some embodiments, a method of
detecting TV further comprises detecting at least one endogenous control and at least one
exogenous control.
[0048] In some embodiments, a method of detecting TV comprises detecting the TV
40S ribosomal protein (Tv40Srp) gene in a sample. In some embodiments, a method of
detecting TV further comprises detecting a sample adequacy control (SAC), such as a single
copy human gene. In some embodiments, a method of detecting TV further comprises
detecting a sample processing control (SPC), such as an exogenously added bacterial DNA.
In some embodiments, a method of detecting TV further comprises detecting an SAC and an
SPC.
[0049] In the present disclosure, the term "target gene" is used for convenience to
refer to the TV 40S ribosomal protein (Tv40Srp) gene, and also to exogenous and/or
endogenous controls. Thus, it is to be understood that when a discussion is presented in
terms of a target gene, that discussion is specifically intended to encompass the TV 40S
ribosomal protein (Tv40Srp) gene, the endogenous control(s) (e.g., SAC), and the
exogenous control(s) (e.g., SPC).
[0050] In some embodiments, the presence of the TV 40S ribosomal protein
(Tv40Srp) gene is detected in a urine sample. In some embodiments, the target gene is
detected in a urine sample to which a buffer (such as a preservative) has been added. In
some embodiments, the buffer is added to a 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 buffenurine. In some embodiments, the presence of the TV 40S
ribosomal protein (Tv40Srp) gene is detected in an endocervical swab sample or a vaginal
swab sample. In some embodiments, the vaginal swab is a patient-collected vaginal swab.
In some embodiments, the target gene is detected in an endocervical swab sample or a
vaginal swab sample that has been placed in a buffer (such as a preservative). In some
embodiments, a swab is placed in 1 mL, 2 mL, 2.5 mL of buffer.
[0051] In some embodiments, detection of the TV 40S ribosomal protein (Tv40Srp)
gene in a sample from a subject indicates the presence of Trichomonas vaginalis in the
subject. In some embodiments, the detecting is done quantitatively. In other embodiments,
the detecting is done qualitatively. In some embodiments, detecting a target gene comprises
forming a complex comprising a polynucleotide and a nucleic acid selected from a target
gene, a DNA amplicon of a target gene, and a complement of a target gene. In some
embodiments, detecting a target gene comprises PCR. In some embodiments, detecting a
target gene comprises quantitative PCR or real-time PCR. In some embodiments, a sample
adequacy control (SAC) and/or a sample processing control (SPC) is detected in the same
assay as the target gene. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp)
gene is detected, TV is considered to be detected even if the SPC and/or SAC are not
detected in the assay. In some embodiments, if the TV 40S ribosomal protein (Tv40Srp)
gene is not detected, TV is considered to be not detected only if the SPC and SAC are also
detected in the assay.
[0052] In some embodiments, the presence of the TV 40S ribosomal protein
(Tv40Srp) gene can be measured in samples collected at one or more times from a subject to
monitor treatment for TV infection in the subject. Treatments include, but are not limited
to, a single dose or multiple doses of metronidazole or tinidazole. In some embodiments, a
subject with a history of TV infection is monitored for recurrence of TV by detecting the
presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene at regular or semiregular
intervals. In some such embodiments, the patient is monitored by detecting the
presence or absence of the TV 40S ribosomal protein (Tv40Srp) gene at least once per
month, at least once every two months, at least once every three months, at least once every
four months, at least once every five months, at least once every six months, at least once
every nine months, at least once per year, or at least once every two years.
[0053] In some embodiments, the present assay may be used as part of routine
and/or preventative healthcare for a subject. That is, in some embodiments, the present
assay may be used to test an individual for TV infection whether or not the individual has
exhibited symptoms of TV infection or has a history of TV infection. In some
embodiments, the present assay is used to detect TV infection in subjects who are pregnant
and/or who are attempting to become pregnant. In some instances, pregnant women with
TV are more likely to experience pre-term delivery and/or have babies with low birth
weight (less than 5.5 pounds).
[0054] In some embodiments, a sample to be tested is a urine sample (such as a first
catch urine sample), or is derived from a urine sample. In some embodiments, a buffer
(such as 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 sample collection.
[0055] In some embodiments, a 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 the 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 sample collection.
[0056] In some embodiments, less than 5 ml, less than 4 ml, less than 3 ml, less than
2 ml, less than 1 ml, or less than 0.75 ml of urine are used in the present methods. In some
embodiments, 0.1 ml to 1ml of urine is used in the present methods.
[0057] In some embodiments, the sample to be tested is another bodily fluid, such
as blood, sputum, mucus, saliva, vaginal or penile discharge, semen, etc.
[0058] The clinical sample to be tested is, in some embodiments, fresh (i.e., never
frozen). In other embodiments, the sample is a frozen specimen. In some embodiments, the
sample is a tissue sample, such as a formalin-fixed paraffin embedded sample. In some
embodiments, the sample is a liquid cytology sample.
[0059] In some embodiments, the sample to be tested is obtained from an individual
who has one or more symptoms of TV infection. Nonlimiting exemplary symptoms of TV
infection include vaginitis, urethritis, and cervicitis; in females: itching, burning, redness, or
soreness of the genitals, unusual odor, discomfort with urination, and a thin clear, white,
yellow, or green discharge; and in males: itching or burning inside the penis, burning after
ejaculation or urination, and penile discharge. In some embodiments, the sample to be
tested is obtained from an individual who has previously been diagnosed with TV infection.
In some such embodiments, the individual is monitored for recurrence of TV infection.
[0060] In some embodiments, methods described herein can be used for routine
screening of healthy individuals with no risk factors. In some embodiments, methods
described herein are used to screen asymptomatic individuals, for example, during routine
or preventative care. In some embodiments, methods described herein are used to screen
women who are pregnant or who are attempting to become pregnant.
[0061] In some embodiments, the methods described herein can be used to assess
the effectiveness of a treatment for TV infection in a patient.
[0062] In some embodiments, use of the the TV 40S ribosomal protein (Tv40Srp)
gene for detecting TV infection is provided. In some embodiments, use of the the TV 40S
ribosomal protein (Tv40Srp) gene for monitoring recurrence of TV infection is provided.
[0063] In any of the embodiments described herein, the TV 40S ribosomal protein
(Tv40Srp) gene may be detected in the same assay reaction as a sample processing control
(SPC) and/or sample adequacy control (SAC).
[0064] In some embodiments, a method of facilitating detection of TV infection in a
subject is provided. Such methods comprise detecting the presence or absence of the TV
40S ribosomal protein (Tv40Srp) gene in a sample from the subject. In some embodiments,
information concerning the presence or absence of the TV 40S ribosomal protein (Tv40Srp)
gene in the sample from the subject is communicated to a medical practitioner. A "medical
practitioner," as used herein, refers to an individual or entity that diagnoses and/or treats
patients, such as a hospital, a clinic, a physician's office, a physician, a nurse, or an agent of
any of the aforementioned entities and individuals. In some embodiments, detecting the
presence or absence of TV 40S ribosomal protein (Tv40Srp) gene is carried out at a
laboratory that has received the subject's sample from the medical practitioner or agent of
the medical practitioner. The laboratory carries out the detection by any method, including
those described herein, and then communicates the results to the medical practitioner. A
result is "communicated," as used herein, when it is provided by any means to the medical
practitioner. In some embodiments, such communication may be oral or written, may be by
telephone, in person, by e-mail, by mail or other courier, or may be made by directly
depositing the information into, e.g., a database accessible by the medical practitioner,
including databases not controlled by the medical practitioner. In some embodiments, the
information is maintained in electronic form. In some embodiments, the information can be
stored in a memory or other computer readable medium, such as RAM, ROM, EEPROM,
flash memory, computer chips, digital video discs (DVD), compact discs (CDs), hard disk
drives (HDD), magnetic tape, etc.
[0065] 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 TV 40S ribosomal protein (Tv40Srp) gene in the sample. In
some embodiments, the method further comprises receiving a communication from the
laboratory that indicates the presence or absence of the TV 40S ribosomal protein (Tv40Srp)
gene in the sample. A "laboratory," as used herein, is any facility that detects the target
gene in a sample by any method, including the methods described herein, and communicates
the result to a medical practitioner. In some embodiments, a laboratory is under the control
of a medical practitioner. In some embodiments, a laboratory is not under the control of the
medical practitioner.
[0066] When a laboratory communicates the result of detecting the presence or
absence of the TV 40S ribosomal protein (Tv40Srp) gene to a medical practitioner, in some
embodiments, the laboratory indicates whether or not the TV 40S 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," and the like.
[0067] As used herein, when a method relates to detecting TV, determining the
presence of TV, monitoring for TV, and/or diagnosing TV infection, the method includes
activities in which the steps of the method are carried out, but the result is negative for the
presence of TV. That is, detecting, determining, monitoring, and diagnosing TV or TV
infection include instances of carrying out the methods that result in either positive or
negative results.
[0068] In some embodiments, at least one endogenous control (e.g., an SAC) and/or
at least one exogenous control (e.g., an SPC) are detected simultaneously with the TV 40S
ribosomal protein (Tv40Srp) gene in a single reaction.
4.2.2. Exemplary controls
[0069] In some embodiments, an assay described herein comprises detecting the TV
40S ribosomal protein (Tv40Srp) gene and at least one endogenous control. In some
embodiments, the endogenous control is a sample adequacy control (SAC). In some such
embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is not detected in a sample,
and the SAC is also not detected in the sample, the assay result is considered "invalid"
because the sample may have been insufficient. While not intending to be bound by any
particular theory, an insufficient sample may be too dilute, contain too little cellular
material, contain an assay inhibitor, etc. In some embodiments, the failure to detect an SAC
may indicate that the assay reaction failed. In some embodiments, an endogenous control
(such as an SAC) is a single-copy human gene. Nonlimiting exemplary SACs include
human hydroxymethyl-bilane synthase (HMBS), glyceraldehyde-3 -phosphate
dehydrogenase (GAPDH), beta actin, beta2-microglobin, cyclooxygenase 1, hypoxanthine
phosphoribosyl-transferase, porphobilinogen deaminase, and the transferrin receptor.
[0070] In some embodiments, an assay described herein comprises detecting the TV
40S ribosomal protein (Tv40Srp) gene and at least one exogenous control. In some
embodiments, the exogenous control is a sample processing control (SPC). In some such
embodiments, if the TV 40S ribosomal protein (Tv40Srp) gene is not detected in a sample,
and the SPC is also not detected in the sample, the assay result is considered "invalid"
because there may have been an error in sample processing, including but not limited to,
failure of the assay. Nonlimiting exemplary errors in sample processing include, inadequate
sample processing, the presence of an assay inhibitor, compromised reagents, etc. In some
embodiments, an exogenous control (such as an SPC) is added to a sample. In some
embodiments, an exogenous control (such as an SPC) is added during performance of an
assay, such as with one or more buffers or reagents. In some embodiments, when a
GeneXpert® system is to be used, the SPC is included in the GeneXpert® cartridge. In
some embodiments, an exogenous control (such as an SPC) is a DNA sequence that is not
expected to be present in the sample being assayed. Nonlimiting exemplary SPCs include
bacterial genes not expected to be present in the sample being assayed.
[0071] In some embodiments, an endogenous control and/or an exogenous control is
detected contemporaneously, such as in the same assay, as detection of the TV 40S
ribosomal protein (Tv40Srp) gene in a sample. In some embodiments, an assay comprises
reagents for detecting the TV 40S ribosomal protein (Tv40Srp) gene, an exogenous control,
and an endogenous control simultaneously in the same assay reaction. In some such
embodiments, for example, an assay reaction comprises a primer set for amplifying the TV
40S ribosomal protein (Tv40Srp) gene, a primer set for amplifying an endogenous control,
and a primer set for amplifying an exogenous control, and labeled probes for detecting the
amplification products (such as, for example, TaqMan® probes).
4.2.3. Exemplary sample preparation
4.2.3.1. Exemplary buffers
[0072] In some embodiments, a buffer is added to a urine sample. In some
embodiments, the buffer is added within one hour, two hours, three hours, or six hours of
the time the urine sample was collected (e.g., voided). In some embodiments, a 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.
[0073] In some embodiments, a swab sample is placed in a buffer. In some
embodiments, the swab sample is placed in the buffer within one hour, two hours, three
hours, or six hours of the time the swab sample was collected. In some embodiments, the
swab sample is placed in a buffer within one hour, two hours, three hours, or six hours
before the sample is analyzed by the methods described herein.
[0074] Non-limiting exemplary commercial buffers include PreservCyt
(Hologic, Bedford, MA), SurePath (BD, Franklin Lakes, NJ), and CyMol (Copan
Diagnostics, Murrietta, CA).
4.2.3.2. Exemplary DNA preparation
[0075] Sample DNA can be prepared by any appropriate method. In some
embodiments, target DNA is prepared by contacting a sample with a lysis buffer and
binding DNA to a DNA binding substrate, such as a glass or silica substrate. The binding
substrate may have any suitable form, such as a particulate, porous solid, or membrane
form. For example, the support may comprise hydroxycellulose, glass fiber, cellulose,
nitrocellulose, zirconium hydroxide, titanium (IV) oxide, silicon dioxide, zirconium silicate,
or silica particles (e.g., see U.S. Patent No. 5,234,809). Many such DNA binding substrates
are known in the art.
[0076] In some embodiments, DNA is detected in a lysate without first isolating
or separating the DNA. In some some embodiments, the sample is subject to a lysis step to
release the DNA. Non-limiting exemplary lysis methods include sonication (for example,
for 2-15 seconds, 8-18 mih at 36 kHz); chemical lysis, for example, using a detergent; and
various commercially available lysis reagents. In some embodiments, DNA is detected are
measured in a sample in which DNA has been isolated or separated from at least some other
cellular components.
[0077] When the methods discussed herein indicate that a target gene is
detected, such detection may be carried out on a complement of a target gene instead of, or
in addition to, the target gene sequence shown herein. In some embodiments, when the
complement of a target gene is detected, a polynucleotide for detection is used that is
complementary to the complement of the target gene. In some some embodiments, a
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
[0078] As described above, methods are presented for detecting Trichomonas
vaginalis. The methods comprise detecting the presence of the TV 40S ribosomal protein
(Tv40Srp) gene in a sample from a subject. In some embodiments, the method further
comprises detecting at least one endogenous control (such as an SAC) and/or at least one
exogenous control (such as an SPC). In some embodiments, detection of the TV 40S
ribosomal protein (Tv40Srp) gene indicates the presence of TV, even if the endogenous
control and/or exogenous control is not detected in the assay. In some embodiments, if the
TV 40S ribosomal protein (Tv40Srp) gene is not detected, the result is considered to be
negative for TV only if the controls are detected. In some embodiments, if the TV 40S
ribosomal protein (Tv40Srp) gene is not detected, the result is considered to be negative for
TV only if the endogenous control and exogenous control are detected.
[0079] Any analytical procedure capable of permitting specific detection of a target
gene may be used in the methods herein presented. Exemplary nonlimiting analytical
procedures include, but are not limited to, nucleic acid amplification methods, PCR
methods, isothermal amplification methods, and other analytical detection methods known
to those skilled in the art.
[0080] In some embodiments, the method of detecting a target gene, such as the TV
40S ribosomal protein (Tv40Srp) gene, comprises amplifying the gene and/or a complement
thereof. Such amplification can be accomplished by any method. Exemplary methods
include, but are not limited to, isothermal amplification, real time PCR, endpoint PCR, and
amplification using T7 polymerase from a T7 promoter annealed to a DNA, such as
provided by the SenseAmp Plus™ Kit available at Implen, Germany.
[0081] When a target gene is amplified, in some embodiments, an amplicon of the
target gene is formed. An amplicon may be single stranded or double-stranded. In some
embodiments, when an amplicon is single-stranded, the sequence of the amplicon is related
to the target gene in either the sense or antisense orientation. In some embodiments, an
amplicon of a target gene is detected rather than the target gene itself. Thus, when the
methods discussed herein indicate that a target gene is detected, such detection may be
carried out on an amplicon of the target gene instead of, or in addition to, the target gene
itself. In some embodiments, when the amplicon of the target gene is detected rather than
the target gene, a polynucleotide for detection is used that is complementary to the
complement of the target gene. In some embodiments, when the amplicon of the target gene
is detected rather than the target gene, a polynucleotide for detection is used that is
complementary to the target gene. Further, in some embodiments, multiple polynucleotides
for detection may be used, and some polynucleotides may be complementary to the target
gene and some polynucleotides may be complementary to the complement of the target
gene.
[0082] In some embodiments, the method of detecting the TV 40S ribosomal protein
(Tv40Srp) gene comprises PCR, as described below. In some embodiments, detecting one
or more target genes comprises real-time monitoring of a PCR reaction, which can be
accomplished by any method. Such methods include, but are not limited to, the use of
TaqMan®, molecular beacons, or Scorpion probes (i.e., energy transfer (ET) probes, such as
FRET probes) and the use of intercalating dyes, such as SYBR green, EvaGreen, thiazole
orange, YO-PRO, TO-PRO, etc.
[0083] Nonlimiting exemplary conditions for amplifying a target gene are as
follows. An exemplary cycle comprises an initial denaturation at 90°C to 100°C for 30
seconds to 5 minutes, followed by cycling that comprises denaturation at 90°C to 100°C for
1 to 10 seconds, followed by annealing and amplification at 60°C to 75°C for 10 to 30
seconds. A further exemplary cycle comprises 1 minute at 95°C, followed by up to 40
cycles of 5 seconds at 92.5°C, 20 seconds at 68°C. In some embodiments, for the first
cycle following the initial denaturation step, the cycle denaturation step is omitted. In some
embodiments, Taq polymerase is used for amplification. In some embodiments, the cycle is
carried out 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 is
used with a hot start function. In some embodiments, the amplification reaction occurs in a
GeneXpert® cartridge, and amplification of the TV 40S ribosomal protein (Tv40Srp) gene,
an endogenous control, and an exogenous control occurs in the same reaction. In some
embodiments, detection of the TV 40S ribosomal protein (Tv40Srp) gene occurs in less than
3 hours, less than 2.5 hours, less than 2 hours, less than 1 hour, or less than 30 minutes from
initial denaturation through the last extension.
[0084] In some embodiments, detection of a target gene comprises forming a
complex comprising a polynucleotide that is complementary to a target gene or to a
complement thereof, and a nucleic acid selected from the target gene, a DNA amplicon of
the target gene, and a complement of the target gene. Thus, in some embodiments, the
polynucleotide forms a complex with a target gene. In some embodiments, the
polynucleotide forms a complex with a complement of the target gene. In some
embodiments, the polynucleotide forms a complex with a DNA amplicon of the target gene.
When a double-stranded DNA amplicon is part of a complex, as used herein, the complex
may comprise one or both strands of the DNA amplicon. Thus, in some embodiments, a
complex comprises only one strand of the DNA amplicon. In some embodiments, a
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, complement of the target gene, or DNA amplicon of the
target gene. The polynucleotide, in some embodiments, is a primer or probe.
[0085] In some embodiments, a method comprises detecting the complex. In some
embodiments, the complex does not have to be associated at the time of detection. That is,
in some embodiments, a complex is formed, the complex is then dissociated or destroyed in
some manner, and components from the complex are detected. An example of such a
system is a TaqMan® assay. In some embodiments, when the polynucleotide is a primer,
detection of the complex may comprise amplification of the target gene, a complement of
the target gene, or a DNA amplicon of the target gene.
[0086] In some embodiments the analytical method used for detecting at least one
target gene in the methods set forth herein includes real-time quantitative PCR. In some
embodiments, the analytical method used for detecting at least one target gene includes the
use of a TaqMan probe. The assay uses energy transfer ("ET"), such as fluorescence
resonance energy transfer ("FRET"), to detect and quantitate the synthesized PCR product.
Typically, the TaqMan® probe comprises a fluorescent dye molecule coupled to the 5'-end
and a quencher molecule coupled to the 3'-end, such that the dye and the quencher are in
close proximity, allowing the quencher to suppress the fluorescence signal of the dye via
FRET. When the polymerase replicates the chimeric amplicon template to which the
TaqMan® probe is bound, the 5'-nuclease of the polymerase cleaves the probe, decoupling
the dye and the quencher so that the dye signal (such as fluorescence) is detected. Signal
(such as fluorescence) increases with each PCR cycle proportionally to the amount of probe
that is cleaved.
[0087] In some embodiments, a target gene is considered to be detected if any
signal is generated from the TaqMan probe during the PCR cycling. For example, in some
embodiments, if the PCR includes 40 cycles, if a signal is generated at any cycle during the
amplification, the target gene is considered to be present and detected. In some some
embodiments, if no signal is generated by the end of the PCR cycling, the target gene is
considered to be absent and not detected.
[0088] In some embodiments, quantitation of the results of real-time PCR assays is
done by constructing a standard curve from a nucleic acid of known concentration and then
extrapolating quantitative information for target genes of unknown concentration. In some
embodiments, the nucleic acid used for generating a standard curve is a DNA (for example,
an endogenous control, or an exogenous control). In some embodiments, the nucleic acid
used for generating a standard curve is a purified double-stranded plasmid DNA or a singlestranded
DNA generated in vitro.
[0089] In some embodiments, in order for an assay to indicate that TV is not present
in a sample, the Ct values for an endogenous control (such as an SAC) and/or an exogenous
control (such as an SPC) must be within a previously-determined valid range. That is, in
some embodiments, the absence of TV cannot be confirmed unless the controls are detected,
indicating that the assay was successful. Ct values are inversely proportional to the amount
of nucleic acid target in a sample.
[0090] In some embodiments, a threshold Ct (or a "cutoff Ct") value for a target
gene (including an endogenous control and/or exogenous control), below which the gene is
considered to be detected, has previously been determined. In some embodiments, a
threshold Ct is determined using substantially the same assay conditions and system (such
as a GeneXpert®) on which the samples will be tested.
[0091] In addition to the TaqMan assays, other real-time PCR chemistries useful
for detecting and quantitating PCR products in the methods presented herein include, but are
not limited to, Molecular Beacons, Scorpion probes and intercalating dyes, such as SYBR
Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc., which are discussed below.
[0092] In various embodiments, real-time PCR detection is utilized to detect, in a
single multiplex reaction, the TV 40S ribosomal protein (Tv40Srp) gene, an endogenous
control, and an exogenous control. In some multiplex embodiments, a plurality of probes,
such as TaqMan® probes, each specific for a different target, is used. In some embodiments,
each target gene-specific probe is spectrally distinguishable from the other probes used in
the same multiplex reaction.
[0093] Real-time PCR is performed using any PCR instrumentation available in the
art. Typically, instrumentation used in 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.
[0094] In some embodiments, detection and/or quantitation of real-time PCR
products is accomplished using a dye that binds to double-stranded DNA products, such as
SYBR Green, EvaGreen, thiazole orange, YO-PRO, TO-PRO, etc. In some embodiments,
the analytical method used in the methods described herein is a DASL® (DNA-mediated
Annealing, Selection, Extension, and Ligation) Assay. In some embodiments, the analytical
method used for detecting and quantifying the target genes in the methods described herein
is a bead-based flow cytometric assay. See Lu J . et al. (2005) Nature 435:834-838, which is
incorporated herein by reference in its entirety. An example of a bead-based flow
cytometric assay is the xMAP® technology of Luminex, Inc. See www.luminexcorp.com/
technology/index.html. In some embodiments, the analytical method used for detecting and
quantifying the levels of the at least one target gene in the methods described herein is by
gel electrophoresis and detection with labeled probes (e.g., probes labeled with a radioactive
or chemiluminescent label), such as by northern blotting. In some embodiments, exemplary
probes contain one or more affinity-enhancing nucleotide analogs as discussed below, such
as locked nucleic acid ("LNA") analogs, which contain a bicyclic sugar moiety instead of
deoxyribose or ribose sugars. See, e.g., Varallyay, E. et al. (2008) Nature Protocols
3(2): 190-196, which is incorporated herein by reference in its entirety. In some
embodiments, detection and quantification of one or more target genes is accomplished
using microfluidic devices and single-molecule detection.
[0095] Optionally, the sample DNA is modified before hybridization. The target
DNA/probe duplex is then passed through channels in a microfluidic device and that
comprise detectors that record the unique signal of the 3 labels. In this way, individual
molecules are detected by their unique signal and counted. 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 Systems
[0096] In some embodiments, gene expression is detected using an automated
sample handling and/or analysis platform. In some embodiments, commercially available
automated analysis platforms are utilized. For example, in some embodiments, the
GeneXpert® system (Cepheid, Sunnyvale, CA) is utilized.
[0097] The present invention is illustrated for use with 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. One of
skill in the art recognizes that any number of platforms and methods may be utilized.
[0098] The GeneXpert® utilizes a self-contained, single use cartridge. Sample
extraction, amplification, and detection may all carried out within this self-contained
"laboratory in a cartridge." (See e.g., US Patents 5,958,349, 6,403,037, 6,440,725,
6,783,736, 6,818,185; each of which is herein incorporated by reference in its entirety.)
[0099] Components of the cartridge include, but are not limited to, processing
chambers containing reagents, filters, and capture technologies useful to extract, purify, and
amplify target nucleic acids. A valve enables fluid transfer from chamber to chamber and
contain nucleic acids lysis and filtration components. An optical window enables real-time
optical detection. A reaction tube enables very rapid thermal cycling.
[00100] In some embodiments, the GenXpert® system includes a plurality of
modules for scalability. Each module includes a plurality of cartridges, along with sample
handling and analysis components.
[00101] After the sample is added to the cartridge, the sample is contacted with
lysis buffer and released DNA is bound to a DNA-binding substrate such as a silica or glass
substrate. The sample supernatant is then removed and the DNA eluted in an elution buffer
such as a Tris/EDTA buffer. The eluate may then be processed in the cartridge to detect
target genes 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.
[00102] In some embodiments, PCR is used to amplify and analyze the
presence of the target genes. In some embodiments, the PCR uses Taq polymerase with hot
start function, such as AptaTaq (Roche). In some embodiments, the initial denaturation is at
90°C to 100°C for 30 seconds to 5 minutes; the cycling denaturation temperature is 90°C to
100°C for 1 to 10 seconds; the cycling anneal and amplification temperature is 60°C to
75°C for 10 to 30 seconds; and up to 50 cycles are performed.
[00103] In some embodiments, a double-denature method is used to amplify low
copy number targets. A double-denature method comprises, in some embodiments, a first
denaturation step followed by addition of primers and/or probes for detecting target genes.
All or a substantial portion of the DNA-containing sample (such as a DNA eluate) is then
denatured a second time before, in some instances, a portion of the sample is aliquotted for
cycling and detection of the target genes. While not intending to be bound by any particular
theory, the double-denature protocol may increase the chances that a low copy number
target gene (or its complement) will be present in the aliquot selected for cycling and
detection because the second denaturation effectively doubles the number of targets (i.e., it
separates the target and its complement into two separate templates) before an aliquot is
selected for cycling. In some embodiments, the first denaturation step comprises heating to
a temperature of 90°C to 100°C for a total time of 30 seconds to 5 minutes. In some
embodiments, the second denaturation step comprises heating to a temperature of 90°C to
100°C for a total time of 5 seconds to 3 minutes. In some embodiments, the first
denaturation step and/or the second denaturation step is carried out by heating aliquots of
the sample separately. In some embodiments, each aliquot may be heated for the times
listed above. As a non-limiting example, a first denaturation step for a DNA-containing
sample (such as a DNA eluate) may comprise heating at least one, at least two, at least
three, or at least four aliquots of the sample separately (either sequentially or
simultaneously) to a temperature of 90°C to 100°C for 60 seconds each. As a non-limiting
example, a second denaturation step for a DNA-containing sample (such as a DNA eluate)
containing enzyme, primers, and probes may comprise heating at least one, at least two, at
least three, or at least four aliquots of the eluate separately (either sequentially or
simultaneously) to a temperature of 90°C to 100°C for 5 seconds each. In some
embodiments, an aliquot is the entire DNA-containing sample (such as a DNA eluate). In
some embodiments, an aliquot is less than the entire DNA-containing sample (such as a
DNA eluate).
[00104] In some embodiments, target genes in a DNA-containing sample, such as
a DNA eluate, are detected using the following protocol: One or more aliquots of the DNAcontaining
sample are heated separately to 95°C for 60 seconds each. The enzyme and
primers and probes are added to the DNA-containing sample and one or more aliquots are
heated separately to 95°C for 5 seconds each. At least one aliquot of the DNA-containing
sample containing enzyme, primers, and probes is then heated to 94°C for 60 seconds. The
aliquot is then cycled 45 times with the following 2-step cycle: (1) 94°C for 5 seconds, (2)
66°C for 30 seconds.
[00105] The present invention is not limited to particular primer and/or probe
sequences. Exemplary amplification primers and detection probes are described in the
Examples.
[00106] In some embodiments, an off-line centrifugation is used, for example,
with samples with low cellular content. The sample, with or without a buffer added, is
centrifuged and the supernatant removed. The pellet is then resuspended in a smaller
volume of either supernatant or the buffer. The resuspended pellet is then analyzed as
described herein.
4.2.6. Exemplary Data Analysis
[00107] In some embodiments, the presence of TV is detected if the Ct value
for the TV 40S ribosomal protein (Tv40Srp) gene is below a certain threshold. In some
embodiments the valid range of Ct values is 9 to 39.9 Ct. In some such embodiments, if no
amplification above background is observed from the TV-specific primers after 40 cycles,
the sample is considered to be negative for TV.
[00108] In some embodiments, a computer-based analysis program is used to
translate the raw data generated by the detection assay into data of predictive value for a
clinician. The clinician can access the predictive data using any suitable means. Thus, in
some embodiments, the present invention provides the further benefit that the clinician, who
is not likely to be trained in genetics or molecular biology, need not understand the raw
data. The data is presented directly to the clinician in its most useful form. The clinician is
then able to immediately utilize the information in order to optimize the care of the subject.
[00109] The present invention contemplates any method capable of receiving,
processing, and transmitting the information to and from laboratories conducting the assays,
information provides, medical personal, and subjects. For example, in some embodiments
of the present invention, a sample (e.g., a biopsy or a serum or urine sample) is obtained
from a subject and submitted to a profiling service (e.g., clinical lab at a medical facility,
genomic profiling business, etc.), located in any part of the world (e.g., in a country
different than the country where the subject resides or where the information is ultimately
used) to generate 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 the profiling
center, or subjects may collect the sample themselves (e.g., a urine sample) and directly
send it to a profiling center. Where the sample comprises previously determined biological
information, the information may be directly sent to the profiling service by the subject
(e.g., an information card containing the information may be scanned by a computer and the
data transmitted to a computer of the profiling center using an electronic communication
systems). Once received by the profiling service, the sample is processed and a profile is
produced (i.e., expression data), specific for the diagnostic or prognostic information desired
for the subject.
[001 10] The profile data is then prepared in a format suitable for interpretation
by a 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,
with or without recommendations for particular treatment options. The data may be
displayed to the clinician by any suitable method. For example, in some embodiments, the
profiling service generates a report that can be printed for the clinician (e.g. , at the point of
care) or displayed to the clinician on a computer monitor.
[001 11] In some embodiments, the information is first analyzed at the point of
care or at a regional facility. The raw data is then sent to a central processing facility for
further analysis and/or to convert the raw data to information useful for a clinician or
patient. The central processing facility provides the advantage of privacy (all data is stored
in a central facility with uniform security protocols), speed, and uniformity of data analysis.
The central processing facility can then control the fate of the data following treatment of
the subject. For example, using an electronic communication system, the central facility can
provide data to the clinician, the subject, or researchers.
[001 12] In some embodiments, the subject is able to directly access the data
using the electronic communication system. The subject may chose further intervention or
counseling based on the results. In some embodiments, the data is used for research use.
For example, the data may be used to further optimize the inclusion or elimination of
markers as useful indicators of a particular condition or stage of disease or as a companion
diagnostic to determine a treatment course of action.
4.2.7. Exemplary polynucleotides
[00113] In some embodiments, polynucleotides are provided. In some
embodiments, synthetic polynucleotides are provided. Synthetic polynucleotides, as used
herein, refer to polynucleotides that have been synthesized in vitro either chemically or
enzymatically. Chemical synthesis of polynucleotides includes, but is not limited to,
synthesis using polynucleotide synthesizers, such as OligoPilot (GE Healthcare), ABI 3900
DNA Synthesizer (Applied Biosystems), and the like. Enzymatic synthesis includes, but is
not limited, to producing polynucleotides by enzymatic amplification, e.g., PCR. A
polynucleotide may comprise one or more nucleotide analogs (i.e., modified nucleotides)
discussed herein.
[00114] In some embodiments, a polynucleotide is provided that comprises a
region that is at least 90%, at least 95%, or 100% identical to, or at least 90%, at least 95%,
or 100% complementary to, at least 8, at least 9, at least 10, at least 11, at least 12, at least
13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at elast
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 the TV 40S ribosomal protein (Tv40Srp) gene.
In some embodiments, a polynucleotide is provided that comprises a region that is at least
90%, at least 95%, or 100% identical to, or complementary to, a span of 6 to 100, 8 to 100,
8 to 75, 8 to 50, 8 to 40, or 8 to 30 contiguous nucleotides of the TV 40S ribosomal protein
(Tv40Srp) gene. Nonlimiting exemplary polynucleotides are shown in Table 1.
[00115] In various embodiments, a polynucleotide comprises fewer than 500,
fewer than 300, fewer than 200, fewer than 150, fewer than 100, fewer than 75, fewer than
50, fewer than 40, or fewer than 30 nucleotides. In various embodiments, a polynucleotide
is between 6 and 200, between 8 and 200, between 8 and 150, between 8 and 100, between
8 and 75, between 8 and 50, between 8 and 40, between 8 and 30, between 15 and 100,
between 15 and 75, between 15 and 50, between 15 and 40, or between 15 and 30
nucleotides long.
[00116] In some embodiments, the polynucleotide is a primer. In some
embodiments, the primer is labeled with a detectable moiety. In some embodiments, a
primer is not labeled. A primer, as used herein, is a polynucleotide that is capable of
selectively hybridizing to a target gene or to an amplicon that has been amplified from a
target gene (collectively referred to as "template"), and, in the presence of the template, a
polymerase and suitable buffers and reagents, can be extended to form a primer extension
product.
[00117] 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 directly detectable moieties, such as fluorescent dyes, and indirectly
detectable moieties, such as members of binding pairs. When the detectable moiety is a
member of a binding pair, in some embodiments, the probe can be detectable by incubating
the probe with a detectable label bound to the second member of the binding pair. In some
embodiments, a probe is not labeled, such as when a probe is a capture probe, e.g., on a
microarray or bead. In some embodiments, a probe is not extendable, e.g., by a polymerase.
In other embodiments, a probe is extendable.
[00118] In some embodiments, the polynucleotide is a FRET probe that in
some embodiments is labeled at the 5'-end with a fluorescent dye (donor) and at the 3'-end
with a quencher (acceptor), a chemical group that absorbs (i.e., suppresses) fluorescence
emission from the dye when the groups are in close proximity (i.e., attached to the same
probe). Thus, in some embodiments, the emission spectrum of the dye should overlap
considerably with the absorption spectrum of the quencher. In other embodiments, the dye
and quencher are not at the ends of the FRET probe.
4.2.7.1. Exemplary polynucleotide modifications
[00119] In some embodiments, the methods of detecting at least one target
gene described herein employ one or more polynucleotides that have been modified, such as
polynucleotides comprising one or more affinity-enhancing nucleotide analogs. Modified
polynucleotides useful in the methods described herein include primers for reverse
transcription, PCR amplification primers, and probes. In some embodiments, the
incorporation of affinity-enhancing nucleotides increases the binding affinity and specificity
of a polynucleotide for its target nucleic acid as compared to polynucleotides that contain
only deoxyribonucleotides, and allows for the use of shorter polynucleotides or for shorter
regions of complementarity between the polynucleotide and the target nucleic acid.
[00120] In some embodiments, affinity-enhancing nucleotide analogs include
nucleotides comprising one or more base modifications, sugar modifications and/or
backbone modifications.
[00121] In some embodiments, modified bases for use in affinity-enhancing
nucleotide analogs include 5-methylcytosine, isocytosine, pseudoisocytosine, 5-
bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, 2-
chloro-6-aminopurine, xanthine and hypoxanthine.
[00122] In some embodiments, affinity-enhancing nucleotide analogs include
nucleotides having modified sugars such as 2'-substituted sugars, such as 2'-0-alkyl-ribose
sugars, 2'-amino-deoxyribose sugars, 2'-fluoro- deoxyribose sugars, 2'-fluoro-arabinose
sugars, and 2'-0-methoxyethyl-ribose (2'MOE) sugars. In some embodiments, modified
sugars are arabinose sugars, or d-arabino-hexitol sugars.
[00123] In some embodiments, affinity-enhancing nucleotide analogs include
backbone modifications such as the use of peptide nucleic acids (PNA; e.g., an oligomer
including nucleobases linked together by an amino acid backbone). Other backbone
modifications include phosphorothioate linkages, phosphodiester modified nucleic acids,
combinations of phosphodiester and phosphorothioate nucleic acid, methylphosphonate,
alkylphosphonates, phosphate esters, alkylphosphonothioates, phosphoramidates,
carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters,
methylphosphorothioate, phosphorodithioate, p-ethoxy, and combinations thereof.
[00124] In some embodiments, a polynucleotide includes at least one affinityenhancing
nucleotide analog that has a modified base, at least nucleotide (which may be the
same nucleotide) that has a modified sugar, and/or at least one intemucleotide linkage that is
non-naturally occurring.
[00125] In some embodiments, an affinity-enhancing nucleotide analog
contains a locked nucleic acid ("LNA") sugar, which is a bicyclic sugar. In some
embodiments, a polynucleotide for use in the methods described herein comprises one or
more nucleotides having an LNA sugar. In some embodiments, a polynucleotide contains
one or more regions consisting of nucleotides with LNA sugars. In other embodiments, a
polynucleotide contains nucleotides with LNA sugars interspersed with
deoxyribonucleotides. See, e.g., Frieden, M. et al. (2008) Curr. Pharm. Des. 14(1 1):1 138-
1142.
4.2.7.2. Exemplary primers
[00126] In some embodiments, a primer is provided. In some embodiments, a
primer is at least 90%, at least 95%, or 100% identical to, or at least 90%, at least 95%, or
100% complementary to, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13,
at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at elast 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 the TV 40S ribosomal protein (Tv40Srp) gene. In
some embodiments, a primer is provided that comprises a region that is at least 90%, at least
95%, or 100% identical to, or complementary to, a span of 6 to 100, 8 to 100, 8 to 75, 8 to
50, 8 to 40, or 8 to 30 contiguous nucleotides of the TV 40S ribosomal protein (Tv40Srp)
gene. Nonlimiting exemplary primers are shown in Table 1. In some embodiments, a
primer may also comprise portions or regions that are not identical or complementary to the
target gene. In some embodiments, a region of a primer that is at least 90%, at least 95%, or
100% identical or complementary to a target gene is contiguous, such that any region of a
primer that is not identical or complementary to the target gene does not disrupt the identical
or complementary region.
[00127] In some embodiments, a primer comprises a portion that is at least
90%, at least 95%, or 100% identical to a region of a target gene. In some such
embodiments, a primer that comprises a region that is at least 90%, at least 95%, or 100%
identical to a region of the target gene is capable of selectively hybridizing to an amplicon
that has been 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 conditions of the particular assay being used.
[00128] 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 it will hybridize to another nucleic acid present in the same
sample that has a different nucleotide sequence in the hybridizing region. Exemplary
hybridization conditions are discussed herein, for example, in the context of a reverse
transcription reaction or a PCR amplification reaction. In some embodiments, a
polynucleotide will hybridize to a particular nucleic acid in a sample with at least 10-fold
greater affinity than it will hybridize to another nucleic acid present in the same sample that
has a different nucleotide sequence in the hybridizing region.
[00129] In some embodiments, a primer comprises a detectable moiety.
[00130] In some embodiments, primer pairs are provided. Such primer pairs
are designed to amplify a portion of a target gene, such as the TV 40S ribosomal protein
(Tv40Srp) gene, or an endogenous control such as a sample adequacy control (SAC), or an
exogenous control such as a sample processing control (SPC). In some embodiments, a
primer pair is designed to produce an amplicon that is 50 to 1500 nucleotides long, 50 to
1000 nucleotides long, 50 to 750 nucleotides long, 50 to 500 nucleotides long, 50 to 400
nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotides long, 50 to 150
nucleotides long, 100 to 300 nucleotides long, 100 to 200 nucleotides long, or 100 to 150
nucleotides long. Nonlimiting exemplary primer pairs are shown in Table 1.
4.2.7.3. Exemplary probes
[0013] ] In various embodiments, methods of detecting the presence of
Trichomonas vaginalis comprise hybridizing nucleic acids of a sample with a probe. In
some embodiments, the probe comprises a portion that is complementary to a target gene,
such as the TV 40S ribosomal protein (Tv40Srp) gene, or an endogenous control such as a
sample adequacy control (SAC), or an exogenous control such as a sample processing
control (SPC). In some embodiments, the probe comprises a portion that is at least 90%, at
least 95%, or 100% identical to a region of the target gene. In some such embodiments, a
probe that is at least 90%, at least 95%, or 100% complementary to a target gene is
complementary to a sufficient portion of the target gene such that it selectively hybridizes to
the target gene under the conditions of the particular assay being used. In some
embodiments, a probe that is complementary to a 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 contiguous nucleotides of the target gene.
Nonlimiting exemplary probes are shown in Table 1. A probe that is at least 90%, at least
95%, or 100% complementary to a target gene may also comprise portions or regions that
are not complementary to the target gene. In some embodiments, a region of a probe that is
at least 90%, at least 95%, or 100% complementary to a target gene is contiguous, such that
any region of a probe that is not complementary to the target gene does not disrupt the
complementary region.
[00132] 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, such as the TV 40S
ribosomal protein (Tv40Srp) gene, or an endogenous control such as a sample adequacy
control (SAC), or an exogenous control such as a sample processing control (SPC). In some
such embodiments, a probe that comprises a region that is at least 90%, at least 95%, or
100% identical to a region of the target gene is capable of selectively hybridizing to an
amplicon that has been 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 conditions of the
particular assay being used. In some embodiments, a probe that is complementary to a
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 contiguous
nucleotides of the amplicon. A probe that is at least 90%, at least 95%, or 100%
complementary to an amplicon may also comprise portions or regions that are not
complementary to the amplicon. In some embodiments, a region of a probe that is at least
90%, at least 95%, or 100% complementary to an amplicon is contiguous, such that any
region of a probe that is not complementary to the amplicon does not disrupt the
complementary region.
[00133] In some embodiments, the method of detecting one or more target
genes comprises: (a) amplifying a region of the target gene; and (b) detecting the amplified
region using real time PCR and a detection probe (which may be simultaneous with the
amplification step (a)).
[00134] As described above, in some embodiments, real time PCR detection
may be performed using a FRET probe, which includes, but is not limited to, a TaqMan®
probe, a Molecular beacon probe and a Scorpion probe. In some embodiments, the real time
PCR detection is performed with a TaqMan® probe, i.e., a linear probe that typically has a
fluorescent dye covalently bound at one end of the DNA and a quencher molecule
covalently bound elsewhere, such as at the other end of, the DNA. The FRET probe
comprises a sequence that is 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 produces a fluorescence signal. In some embodiments, the amount of target gene in the
sample is proportional to the amount of fluorescence measured during amplification.
[00135] The TaqMan® probe typically comprises a region of contiguous
nucleotides having a sequence that is at least 90%, at least 95%, or 100% identical or
complementary to a region of a target gene such that the probe is selectively hybridizable to
a PCR amplicon of a region of the target gene. In some embodiments, the probe comprises
a region of at least 6 contiguous nucleotides having a sequence that is fully complementary
to or identically present in a region of a 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 a
target gene to be detected.
[00136] In some embodiments, the region of the amplicon that has a sequence
that is at least 90%, at least 95%, or 100% complementary to the TaqMan® probe sequence
is at or near the center of the amplicon molecule. In some embodiments, there are
independently at least 2 nucleotides, such as at least 3 nucleotides, such as at least 4
nucleotides, such as at least 5 nucleotides of the amplicon at the 5'-end and at the 3'-end of
the region of complementarity.
[00137] In some embodiments, Molecular Beacons can be used to detect PCR
products. Like TaqMan® probes, Molecular Beacons use FRET to detect a PCR product via
a probe having a fluorescent dye and a quencher attached at the ends of the probe. Unlike
TaqMan® probes, Molecular Beacons remain intact during the PCR cycles. Molecular
Beacon probes form a stem-loop structure when free in solution, thereby allowing the dye
and quencher to be in close enough proximity to cause fluorescence quenching. When the
Molecular Beacon hybridizes to a target, the stem-loop structure is abolished so that the dye
and the quencher become separated in space and the dye fluoresces. Molecular Beacons are
available, e.g., from Gene Link™ (see www.genelink.com/newsite/products/mbintro.asp).
[00138] In some embodiments, Scorpion probes can be used as both
sequence-specific primers and for PCR product detection. Like Molecular Beacons,
Scorpion probes form a stem-loop structure when not hybridized to a target nucleic acid.
However, unlike Molecular Beacons, a Scorpion probe achieves both sequence-specific
priming and PCR product detection. A fluorescent dye molecule is attached to the 5'-end of
the Scorpion probe, and a quencher is attached elsewhere, such as to the 3'-end. The 3'
portion of the probe is complementary to the extension product of the PCR primer, and this
complementary portion is linked to the 5'-end of the probe by a non-amplifiable moiety.
After the Scorpion primer is extended, the target-specific sequence of the probe binds to its
complement within the extended amplicon, thus opening up the stem-loop structure and
allowing the dye on the 5'-end to fluoresce and generate a signal. Scorpion probes are
available from, e.g, Premier Biosoft International (see
www.premierbiosoft.com/tech_notes/Scorpion.html).
[00139] In some embodiments, labels that can be used on the FRET probes
include colorimetric and fluorescent dyes such as Alexa Fluor dyes, BODIPY dyes, such as
BODIPY FL; Cascade Blue; Cascade Yellow; coumarin and its derivatives, such as 7-
amino-4-methylcoumarin, aminocoumarin and hydroxycoumarin; cyanine dyes, such as
Cy3 and Cy5; eosins and erythrosins; fluorescein and its derivatives, such as fluorescein
isothiocyanate; macrocyclic chelates of lanthanide ions, such as Quantum Dye™; Marina
Blue; Oregon Green; rhodamine dyes, such as rhodamine red, tetramethylrhodamine and
rhodamine 6G; Texas Red; fluorescent energy transfer dyes, such as thiazole orangeethidium
heterodimer; and, TOTAB.
[00140] Specific examples of dyes include, but are not limited to, those
identified above and the following: Alexa Fluor 350, Alexa Fluor 405, Alexa Fluor 430,
Alexa Fluor 488, Alexa Fluor 500. Alexa Fluor 514, Alexa Fluor 532, Alexa Fluor 546,
Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 610, Alexa Fluor 633,
Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and, Alexa Fluor 750;
amine-reactive BODIPY dyes, such as BODIPY 493/503, BODIPY 530/550, BODIPY
558/568, BODIPY 564/570, 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'-Tetrabromosulfonefluorescein, and
TET.
[00141] Examples of dye/quencher pairs (i.e., donor/acceptor pairs) include,
but are not limited to, fluorescein/tetramethylrhodamine; IAEDANS/fluorescein;
EDANS/dabcyl; fluorescein/fluorescein; BODIPY FL/BODIPY FL; fluorescein/QSY 7 or
QSY 9 dyes. When the donor and acceptor are the same, FRET may be detected, in some
embodiments, by fluorescence depolarization. Certain specific examples of dye/quencher
pairs (i.e., donor/acceptor pairs) include, but are not limited to, Alexa Fluor 350/Alexa
Fluor488; Alexa Fluor 488/Alexa Fluor 546; Alexa Fluor 488/Alexa Fluor 555; Alexa Fluor
488/Alexa Fluor 568; Alexa Fluor 488/Alexa Fluor 594; Alexa Fluor 488/Alexa Fluor 647;
Alexa Fluor 546/Alexa Fluor 568; Alexa Fluor 546/Alexa Fluor 594; Alexa Fluor 546/Alexa
Fluor 647; Alexa Fluor 555/Alexa Fluor 594; Alexa Fluor 555/Alexa Fluor 647; Alexa Fluor
568/Alexa Fluor 647; Alexa Fluor 594/Alexa Fluor 647; Alexa Fluor 350/QSY35; Alexa
Fluor 350/dabcyl; Alexa Fluor 488/QSY 35; Alexa Fluor 488/dabcyl; Alexa Fluor 488/QSY
7 or QSY 9; Alexa Fluor 555/QSY 7 or QSY9; 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
instances, the same quencher may be used for multiple dyes, for example, a broad spectrum
quencher, such as an Iowa Black® quencher (Integrated DNA Technologies, Coralville, IA)
or a Black Hole Quencher™ (BHQ™; Sigma-Aldrich, St. Louis, MO).
[00142] In some embodiments, for example, in a multiplex reaction in which
two or more moieties (such as amplicons) are detected simultaneously, each probe
comprises a detectably different dye such that the dyes may 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.
[00143] Specific examples of fluorescently labeled ribonucleotides useful in
the preparation of PCR probes for use in some embodiments of the methods described
herein are available from Molecular Probes (Invitrogen), and these include, Alexa Fluor
488-5-UTP, Fluorescein- 12-UTP, BODIPY FL-14-UTP, BODIPY TMR-14-UTP,
Tetramethylrhodamine-6-UTP, Alexa Fluor 546-14-UTP, Texas Red-5-UTP, and BODIPY
TR-14-UTP. Other fluorescent ribonucleotides are available from Amersham Biosciences
(GE Healthcare), such as Cy3-UTP and Cy5-UTP.
[00144] Examples of fluorescently labeled deoxyribonucleotides useful in the
preparation of PCR probes for use in the methods described herein include Dinitrophenyl
(DNP)-l'-dUTP, Cascade Blue-7-dUTP, Alexa Fluor 488-5-dUTP, Fluorescein- 12-dUTP,
Oregon Green 488-5-dUTP, BODIPY FL-14-dUTP, Rhodamine Green-5-dUTP, Alexa
Fluor 532-5-dUTP, BODIPY TMR-14-dUTP, Tetramethylrhodamine-6-dUTP, Alexa Fluor
546-14-dUTP, Alexa Fluor 568-5-dUTP, Texas Red-12-dUTP, Texas Red-5-dUTP,
BODIPY TR-14-dUTP, Alexa Fluor 594-5-dUTP, BODIPY 630/650- 14-dUTP, BODIPY
650/665- 14-dUTP; Alexa Fluor 488-7-OBEA-dCTP, Alexa Fluor 546-16-OBEA-dCTP,
Alexa Fluor 594-7-OBEA-dCTP, Alexa Fluor 647-12-OBEA-dCTP. Fluorescently labeled
nucleotides are commercially available and can be purchased from, e.g., Invitrogen.
[00145] In some embodiments, dyes and other moieties, such as quenchers,
are introduced into polynucleotide used in the methods described herein, such as FRET
probes, via modified nucleotides. A "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 chemical moiety, such as a dye or quencher, covalently attached,
and can be introduced into a polynucleotide, for example, by way of solid phase synthesis of
the polynucleotide. In other embodiments, the modified nucleotide includes one or more
reactive groups that can react with a dye or quencher before, during, or after incorporation
of the modified nucleotide into the nucleic acid. In specific embodiments, the modified
nucleotide is an amine-modified nucleotide, i.e., a nucleotide that has been 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 specific
embodiments, the amine-modified nucleotide is selected from 5-(3-aminoallyl)-UTP; 8-[(4-
amino)butyl]-amino-ATP and 8-[(6-amino)butyl]-amino-ATP; N6-(4-amino)butyl-ATP,
N6-(6-amino)butyl-ATP, N4-[2,2-oxy-bis-(ethylamine)]-CTP; N6-(6-Amino)hexyl-ATP; 8-
[(6-Amino)hexyl]-amino-ATP; 5-propargylamino-CTP, 5-propargylamino-UTP. In some
embodiments, nucleotides with different nucleobase moieties are similarly modified, for
example, 5-(3-aminoallyl)-GTP instead of 5-(3-aminoallyl)-UTP. Many amine modified
nucleotides are commercially available from, e.g., Applied Biosystems, Sigma, Jena
Bioscience and TriLink.
[00146] Exemplary detectable moieties also include, but are not limited to,
members of binding pairs. In some such embodiments, a first member of a binding pair is
linked to a polynucleotide. The second member of the binding pair is linked to a detectable
label, such as a fluorescent label. When the polynucleotide linked to the first member of the
binding pair is incubated with the second member of the binding pair linked to the
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, etc.
[00147] In some embodiments, multiple target genes are detected in a single
multiplex reaction. In some such embodiments, each probe that is targeted to a unique
amplicon is spectrally distinguishable when released from the probe, in which case each
target gene is detected by a unique fluorescence signal. In some embodiments, two or more
target genes are detected using the same fluorescent signal, in which case detection of that
signal indicates the presence of either of the target genes or both.
[00148] One skilled in the art can select a suitable detection method for a
selected assay, e.g., a real-time PCR assay. The selected detection method need not be a
method described above, and may be any method.
4.3. Exemplary compositions and kits
[00149] In another aspect, compositions are provided. In some embodiments,
compositions are provided for use in the methods described herein.
[00150] In some embodiments, compositions are provided that comprise at
least one target gene-specific primer. The term "target gene-specific primer" encompasses
primers that have a region of contiguous nucleotides having a sequence that is (i) at least
90%, at least 95%, or 100% identical to a region of a target gene, or (ii) at least 90%, at least
95%, or 100% complementary to the sequence of a region of contiguous nucleotides found
in a target gene. In some embodiments, a composition is provided that comprises at least
one pair of target gene-specific primers. The term "pair of target gene-specific primers"
encompasses pairs of primers that are suitable for amplifying a defined region of a target
gene. A pair of target gene-specific primers typically comprises a first primer that
comprises a sequence that is at least 90%, at least 95%, or 100% identical to the sequence of
a region of a target gene and a second primer that comprises a sequence that is at least 90%,
at least 95%, or 100% complementary to a region of a target gene. A pair of primers is
typically suitable for amplifying a region of a target gene that is 50 to 1500 nucleotides
long, 50 to 1000 nucleotides long, 50 to 750 nucleotides long, 50 to 500 nucleotides long,
50 to 400 nucleotides long, 50 to 300 nucleotides long, 50 to 200 nucleotides long, 50 tO
150 nucleotides long, 100 to 300 nucleotides long, 100 to 200 nucleotides long, or 100 to
150 nucleotides long. Nonlimiting exemplary primers, and pairs of primers, are shown in
Table 1.
[00151] In some embodiments, a composition comprises at least one pair of
target gene-specific primers. In some embodiments, a composition additionally comprises a
pair of target gene-specific primers for amplifying an endogenous control (such as an SAC)
and/or one pair of target gene-specific primers for amplifying an exogenous control (such as
an SPC).
[00152] In some embodiments, a composition comprises at least one target
gene-specific probe. The term "target gene-specific probe" encompasses probes that have a
region of contiguous nucleotides having a sequence that is (i) at least 90%, at least 95%, or
100% identical to a region of a target gene, or (ii) at least 90%, at least 95%, or 100%
complementary to the sequence of a region of contiguous nucleotides found in a target gene.
Nonlimiting exemplary target-specific probes are shown in Table 1.
[00153] In some embodiments, a composition (including a composition
described above that comprises one or more pairs of target gene-specific primers) comprises
one or more probes for detecting the target genes. In some embodiments, a composition
comprises a probe for detecting an endogenous control (such as an SAC) and/or a probe for
detecting an exogenous control (such as an SPC).
[00154] In some embodiments, a composition is an aqueous composition. In
some embodiments, the aqueous composition comprises a buffering component, such as
phosphate, tris, HEPES, etc., and/or additional components, as discussed below. In some
embodiments, a composition is dry, for example, lyophilized, and suitable for reconstitution
by addition of fluid. A dry composition may include one or more buffering components
and/or additional components.
[00155] In some embodiments, a composition further comprises one or more
additional components. Additional components include, but are not limited to, salts, such as
NaCl , KCl,and MgC¾; polymerases, including thermostable polymerases such as Taq;
dNTPs; bovine serum albumin (BSA) and the like; reducing agents, such as b-
mercaptoethanol; EDTA and the like; etc. One skilled in the art can select suitable
composition components depending on the intended use of the composition.
[00156] In some embodiments, compositions are provided that comprise at
least one polynucleotide for detecting at least one target gene. In some embodiments, the
polynucleotide is used as a primer for a reverse transcriptase reaction. In some
embodiments, the polynucleotide is used as a primer for amplification. In some
embodiments, the polynucleotide is used as a primer for PCR. In some embodiments, the
polynucleotide is used as a probe for detecting at least one target gene. In some
embodiments, the polynucleotide is detectably labeled. In some embodiments, the
polynucleotide is a FRET probe. In some embodiments, the polynucleotide is a TaqMan®
probe, a Molecular Beacon, or a Scorpion probe.
[00157] In some embodiments, a composition comprises at least one FRET
probe having a sequence that is at least 90%, at least 95%, or 100% identical, or at least
90%, at least 95%, or 100% complementary, to a region of, the TV 40S ribosomal protein
(Tv40Srp) gene. In some embodiments, a FRET probe is labeled with a donor/acceptor pair
such that when the probe is digested during the PCR reaction, it produces a unique
fluorescence emission that is associated with a specific target gene. In some embodiments,
when a composition comprises multiple FRET probes, each probe is labeled with a different
donor/acceptor pair such that when the probe is digested during the PCR reaction, each one
produces a unique fluorescence emission that is 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 that comprises one or more base mismatches when compared to the
sequence of the best-aligned target region of a target gene.
[00158] 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 TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, at least 8,
at least 9, at least 10, at least 11, at least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
nucleotides of the FRET probe are identically present in, or complementary to a region of,
the TV 40S ribosomal protein (Tv40Srp) gene. In some embodiments, the FRET probe has
a sequence with one, two or three base mismatches when compared to the sequence or
complement of the TV 40S ribosomal protein (Tv40Srp) gene.
[00159] In some embodiments, a kit comprises a polynucleotide discussed
above. In some embodiments, a kit comprises at least one primer and/or probe discussed
above. In some embodiments, a kit comprises at least one polymerase, such as a
thermostable polymerase. In some embodiments, a kit comprises dNTPs. In some
embodiments, kits for use in the real time PCR methods described herein comprise one or
more target gene-specific FRET probes and/or one or more primers for amplification of
target genes.
[00160] In some embodiments, one or more of the primers and/or probes is
"linear". A "linear" primer refers to a polynucleotide that is a single stranded molecule, and
typically does not comprise a short region of, for example, at least 3, 4 or 5 contiguous
nucleotides, which are complementary to another region within the same polynucleotide
such that the primer forms an internal duplex. In some embodiments, the primers for use in
reverse transcription comprise 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'-end that has a sequence that is
complementary to 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'-end of a target gene.
[00161] In some embodiments, a kit comprises one or more pairs of linear
primers (a "forward primer" and a "reverse primer") for amplification of a target gene.
Accordingly, in some embodiments, a 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 location in the target gene. Furthermore, in some embodiments, a second primer
comprises a region of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at
least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at
least 22, at least 23, at least 24, or at least 25 contiguous nucleotides having a sequence that
is at least 90%, at least 95%, or 100% complementary to the sequence of a region of at least
8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16,
at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24,
or at least 25 contiguous nucleotides at a second location in the target gene, such that a PCR
reaction using the two primers results in an amplicon extending from the first location of the
target gene to the second location of the target gene.
[00162] In some embodiments, the kit comprises at least two, at least three, or
at least four sets of primers, each of which is for amplification of a different target gene,
such as an endogenous control and/or an exogenous control.
[00163] In some embodiments, probes and/or primers for use in the
compositions described herein comprise deoxyribonucleotides. In some embodiments,
probes and/or primers for use in the compositions described herein comprise
deoxyribonucleotides and one or more nucleotide analogs, such as LNA analogs or other
duplex-stabilizing nucleotide analogs described above. In some embodiments, probes
and/or primers for use in the compositions described herein comprise all nucleotide analogs.
In some embodiments, the probes and/or primers comprise one or more duplex-stabilizing
nucleotide analogs, such as LNA analogs, in the region of complementarity.
[00164] In some embodiments, the kits for use in real time PCR methods
described herein further comprise reagents for use in the reverse transcription and
amplification reactions. In some embodiments, the kits comprise enzymes such as heat
stable DNA polymerases, such as Taq polymerase. In some embodiments, the kits further
comprise deoxyribonucleotide triphosphates (dNTP) for use in amplification. In further
embodiments, the kits comprise buffers optimized for specific hybridization of the probes
and primers.
[00165] A kit generally includes a package with one or more containers holding
the reagents, as one or more separate compositions or, optionally, as an admixture where the
compatibility of the reagents will allow. The kit can also include other material(s) that may
be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or
any other material useful in sample processing, washing, or conducting any other step of the
assay.
[00166] Kits preferably include instructions for carrying out one or more of the
methods described herein. Instructions included in kits can be affixed to packaging material
or can be included as a package insert. While the instructions are typically written or
printed materials they are not limited to such. Any medium capable of storing such
instructions and communicating them to an end user is contemplated by this invention.
Such media include, but are not limited to, electronic storage media (e.g., magnetic discs,
tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the
term "instructions" can include the address of an internet site that provides the instructions.
[00167] In some embodiments, the kit can comprise the reagents described above
provided in one or more GeneXpert®Sample cartridge(s). These cartridges permit
extraction, amplification, and detection to be be carried out within this self-contained
"laboratory in a cartridge." (See e.g., US Patents 5,958,349, 6,403,037, 6,440,725,
6,783,736, 6,818,185; each of which is herein incorporated by reference in its entirety.)
Reagents for measuring genomic copy number level and detecting a pathogen could be
provided in separate cartridges within a kit or these reagents (adapted for multiplex
detection) could be provide in a single cartridge.
[00168] Any of the kits described here can include, in some embodiments, a
receptacle for a urine sample and/or a swab for collecting a urethral swab sample, a vaginal
swab sample, or an endocervical swab sample.
[00169] The following examples are for illustration purposes only, and are not
meant to be limiting in any way.
5. EXAMPLES
5.1. Example 1: Detection of Trichomonas vaginalis
[00170] An assay was designed to detect the gene for the 40S ribosomal
protein (Tv40Srp) of Trichomonas vaginalis (TV) by PCR, using the primers and probe
shown in Table 1. In addition to the TV-specific primers and probe, primers and probe were
included to detect a single-copy human gene used as a sample adequacy control (SAC)
target. Primers and probe were also included to detect a bacterial gene, which was included
in the multiplex reaction as a sample processing control (SPC) target.
Table 1: Primer and probe sequences
[00171] The final primer and probe compositions of the multiplex assay are
shown in Table 2.
Table 2: Primer and probe concentrations
Fl and F2 are detectably different dyes that can be detected and distinguished
simultaneously in a multiplex reaction. Each probe also comprises a quencher (e.g., Ql,
above).
[00172] Each reaction contained 42-58 mM KC1, 3.5-5.0 mM MgCl2, 250-350
mM dNTPs, 50 mM Tris, pH 8.6, and 0.01% sodium azide. AptaTaq (0.27-0.37 units/ mΐ ;
Roche) was used for amplification.
[00173] For each sample to be tested, approximately 7 mL of first catch,
voided urine was added to 1mL of buffer, preferably within 2 hours of sample collection.
Physician-collected endocervical swabs or self-collected (in a clinical setting) vaginal swabs
were immediately placed into 2.5mL of buffer.
[00174] 500 mL of buffered urine or swab sample was loaded into a
GeneXpert® cartridge for analysis. The sample was mixed with a lysis reagent to release
nucleic acids. After lysis, the released nucleic acid from the sample was captured on a
DNA-binding substrate. The nucleic acid was eluted from the substrate and used to
reconstitute the reagents used for real-time PCR (described above). The reaction cycle used
was: 1 minute at 95°C, followed by up to 40 cycles of 5 seconds at 92.5°C, 20 seconds at
68°C using a GeneXpert® cartridge in a GeneXpert® system.
[00175] The results of the assay were interpreted as shown in Table 3. The
valid range of Ct values for the TV, SAC, and SPC targets were 9-39.9 Ct.
Table 3 : Xpert TV assay results and interpretation
5.2. Example 2: Clinical performance
[00176] Performance characteristics of the Xpert TV Assay were evaluated at
13 institutions in the U.S. Due to the low prevalence of Trichomonas vaginalis and the
difficulty in obtaining fresh Trichomonas vaginalis-positive specimens from male subjects,
the specimen population for this study was supplemented with contrived male urine
specimens.
[00177] Subjects included consenting asymptomatic and symptomatic,
sexually active males and females seen in locations including, but not limited to: OB/GYN,
sexually transmitted disease (STD), teen, public health, and family planning clinics.
[00178] The study specimens consisted of prospectively collected male urine,
female urine, endocervical swabs, and patient-collected vaginal swabs (collected in a
clinical setting). Contrived male urine specimens were included to supplement the male
sample size.
[00179] The Xpert TV Assay performance was compared to an FDA-cleared
in vitro qualitative nucleic acid amplification comparator assay that detects the ribosomal
RNA of Trichomonas vaginalis using a transcription-mediated assay (APTIMA®
Trichomonas vaginalis assay, GenProbe Hologic, San Diego, USA). Samples with
discrepant results between the Xpert TV Assay and the comparator assay were analysed
with bi-directional sequencing of a separate repetitive genomic DNA sequence. See
Bandea, et al., Jornal of Clinical Microbiology. 2013, 51(4): 1298-1300.
[00180] Of the Xpert TV Assays runs performed with eligible specimens,
97.3% (5327/5474) of these specimens were successful on the first attempt. The remaining
147 gave indeterminate results on the first attempt (91 ERROR, 44 INVALID and 12 NO
RESULT). One hundred nineteen of the 147 specimens yielded valid results after a single
retest; 17 of the specimens were indeterminate on the second attempt and 11 specimens
were not retested. The overall assay success rate was 99.5% (5446/5474).
[00181] Results from the Xpert TV Assay were compared to the comparator
assay, with bi-directional sequencing of discrepants. Sensitivity and specificity by gender,
specimen type and symptom status are presented in Table 4.
Atty Docket No. CEPHD-33923/WO-l/PRI
Table 4 : Xpert TV assay vs. reference NAAT test plus sequencing
TP = true positive, FP = false positive, TN = true negative, FN. = false negative, PC-VS = patient-collected vaginal swab,
ES = endocervical swab, CS = contrived specimens, UR = urine
5.3. Example 3: Limit of detection
[00182] The analytical sensitivity or limit of detection (LoD) of the Xpert TV
Assay was assessed using two Trichomonas vaginalis strains, one metronidazole susceptible
(Tvaginalis ATCC® 30001™), and one metronidazole resistant (Tvaginalis ATCC®
30238™). Both strains were tested in T. vaginalis-negative pooled male urine (MU) mixed
with buffer and T. vaginalis-negative pooled vaginal swab (VS) in buffer
[00183] The limit of detection (LoD) was estimated by testing replicates of 20
at a minimum of five concentrations for each strain and sample type over three days. LoDs
were estimated by logistic regression. The LoD is defined as the lowest number of cells/mL
that can be reproducibly distinguished from negative samples with 95% confidence or the
lowest concentration at which 19 of 20 replicates were positive. The study was performed
with two different lots of Xpert TV reagents and the claimed LoD for each strain is the
higher of the two determinations (Table 5). For swab samples in buffer, the limit of
detection is 5 cells/mL. For urine samples in buffer, the limit of detection is 6 cells/mL. The
claimed LoDs were verified by analyzing at least 20 replicates diluted to the estimated LoD
concentrations.
Table 5: Limit of detection of Trichomonas vaginalis using Xpert TV
5.4. Example 4: Assay reproducibility
[00184] A panel of eight specimens with varying concentrations of
Trichomonas vaginalis was tested on 12 different days by two different operators, at each of
three sites (8 specimens x 1 times/day x 12 days x 2 operators x 3 sites). Three lots of Xpert
TV Assay were used at each of the 3 testing sites. Xpert TV Assays were performed
according to the Xpert TV Assay procedure. Results are summarized in Table 6.
[00185] The reproducibility of the Xpert TV Assay was also evaluated in
terms of the fluorescence signal expressed in Ct values for each target detected. The mean,
standard deviation (SD), and coefficient of variation (CV) between-sites, between-lots,
between-days, between-operator, and within-assay for each panel member are presented in
Table 7.
Atty Docket No. CEPHD-33923/WO-l/PRI
Summary of reproducibility results
a . FS = female swab matrix; UR = male urine matrix
Atty Docket No. CEPHD-33923/WO-l/PRI
Summary of reproducibility data
a. Results with non-zero Ct values out of 144
5.5. Example 5: Analytical inclusivity
[00186] The analytical inclusivity of the Xpert TV Assay was evaluated by
testing 17 T. vaginalis strains in triplicate at a concentration no greater than 3x analytical
limit of detection (3x LoD). Strains were tested in T. vaginalis -negative pooled vaginal
swab (VS) in buffer, and male urine (MU) mixed with buffer. See Table 8. Under the
conditions of this study, all strains reported TV DETECTED results. The Xpert TV assay
demonstrated 100% inclusivity in both sample types.
Table 8: Analytical inclusivity panel
5.6. Example 6: Analytical specificity
[00187] A panel of 47 organisms, including bacteria, fungi, and viruses
commonly found in the urogenital tract, as well as other closely related protozoans to
Trichomonas were tested with the Xpert TV Assay. Each bacterial or fungal strain was
tested at 1 x 107 cfu/mL or greater. Strains which did not produce countable colonies were
diluted to 0.5 McFarland units, approximately equivalent to1.5 x 108 cfu per mL for E. coli.
Viral strains were purchased as heat inactivated stocks from ZeptoMetrix Corp. and tested at
1 x 106 U/mL or 106 genomes/mL. Protozoans were cultured in growth media, visually
enumerated by light microscopy and tested at 1 x 106 cells/mL. Tests were performed in
triplicate. The organisms tested and the Xpert TV assay results are listed in Table 9.
[00188] One organism, Trichomonas tenax, reported a TV DETECTED result
with the Xpert TV assay. Under the conditions of this study, the analytical specificity of the
Xpert TV Assay was 98%.
Table 9 : Analytical specificity panel
5.7. Example 7: Interfering substances
[00189] In a non-clinical study, potentially interfering endogenous and
exogenous substances that may be within the urogenital tract and present in endocervical
and vaginal swab or first catch urine samples were evaluated with the Xpert TV Assay.
[00190] Substances were individually diluted into a pooled negative vaginal
swab matrix and a pooled negative male urine matrix. The substances were also tested in the
same matrices spiked with T. vaginalis cells at no greater than three times the limit of
detection for the respective sample type. Eight replicates of each set of negative and positive
samples were tested with the Xpert TV assay and compared to the results obtained in a
control of the same sample without the potential interfering substance added. The
substances and test concentrations are listed in Table 10 and Table 11.
[00191] Under the conditions of the study, in tests with the substances diluted
into negative urine matrix no invalid results were reported; all tests reported TV NOT
DETECTED as expected. Assay interference was observed in tests with blood at 0.75% v/v
and azithromycin at 1.8 mg/mL diluted into positive urine matrix. False negative results
were not reported for tests with blood at 0.5% v/v and azithromycin at 1 mg/mL.
[00192] Under the conditions of the study, in tests with the substances diluted
into pooled negative swab matrix no invalid results were reported; all tests reported TV
NOT DETECTED as expected.
[00193] In testing of substances diluted into pooled positive swab matrix, no
false negative TV results were reported. Testing with all the substances reported TV
DETECTED results as expected.
Table 10: Potentially interfering substances in urine samples
Table 11: Potentially interfering substances in swab samples
5.8. Example 8: Carry-over contamination
[00194] The study consisted of repeated tests of a TV-negative vaginal swab
pool in buffer sample processed within the same GeneXpert module immediately following
a high (106 cells/mL) TV positive vaginal swab pool in buffer sample. The study consisted
of a TV-negative vaginal swab pool in buffer sample processed within the same GeneXpert
module immediately followed by a high (106 cells/mL) TV positive vaginal swab pool in
buffer sample. This testing scheme was repeated a further 20 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 TV NOT DETECTED.
5.9. Example 9: Alternate primers and probes tested to detect TV
[00195] To develop the TV assay described herein, four different forward
primer, two different reverse primers, and two different probes for detecting the TV 40S
ribosomal protein (Tv40Srp) gene were tested for sensitivity and specificity (e.g., crossreactivity
with other species) in the assay. Table 12 shows the tested primers and probes.
Table 12: Alternate primer and probe sequences
[00196] It was found that TV forward ALTl cross-reacted with Pentatric
homonas hominis (Pth), another closely related trichomonad found in human gut. When TV
forward ALTl was used in an assay with 1000 copies of TV and an assay with 500,000
copies of Pth, TV was detected with a Ct of 30.7 and Pth was detected with a Ct of 26.3.
TV forward ALT2 was less sensitive than the final design, detecting TV with a higher Ct
value of 31.5. Similarly, TV forward ALT3 was less sensitive than the final design, also
detecting TV with a higher Ct value. TV reverse ALTl also resulted in a less sensitive
assay, detecting TV with a higher Ct value. Finally, TV probe ALTl was less sensitive and
less consistent than the final design.
[00197] All publications, patents, patent applications and other documents
cited in this application are hereby incorporated by reference in their entireties for all
purposes to the same extent as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by reference for all purposes.
[00198] While various specific embodiments have been illustrated and
described, it will be appreciated that changes can be made without departing from the spirit
and scope of the invention(s).

WHAT IS CLAIMED IS:
1. A method of detecting the presence or absence of Trichomonas vaginalis (TV) in a
sample from a subject comprising detecting the presence or absence of the TV 40S ribosomal
protein (Tv40Srp) gene or RNA in the sample.
2. A method of determining whether a subject has a Trichomonas vaginalis (TV)
infection comprising detecting the presence or absence of the TV 40S 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 has previously been treated for
TV infection.
5. The method of claim 4, wherein the previous treatment comprised 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 one or more
symptoms of TV infection.
8. The method of claim 7, wherein the subject has one or more symptoms selected from
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 soreness of the genitals; unusual
odor of the genitals; discomfort with urination; and a thin clear, white, yellow, or green
discharge.
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 inside the penis; burning after ejaculation
and/or urination; and penile discharge.
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 adequacy
control.
14. The method of claim 12 or claim 13, wherein the endogenous control is a single-copy
human gene.
15. The method of claim 14, wherein the endogenous control is selected from 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 an initial denaturation step through a final extension step.
23. The method of any one of the preceding claims, wherein the the TV 40S ribosomal
protein (Tv40Srp) gene comprises the 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 TV 40S
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 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 contiguous
nucleotides of SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at
least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 4.
28. The method of claim 27, wherein the first primer and the second primer each
independently comprises 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its
complement.
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 a
first primer of SEQ ID NO: 1 and a second primer of 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 long, 50 to 400 nucleotides long, 50 to 300 nucleotides
long, 50 to 200 nucleotides long, 50 to 150 nucleotides long, 100 to 300 nucleotides long, 100
to 200 nucleotides long, or 100 to 150 nucleotides long.
32. The method of claim 31, wherein the method comprises forming the Tv40Srp
amplicon.
33. The method of claim 32, wherein the method comprises contacting the Tv40Srp
amplicon with a first probe capable of selectively hybridizing with 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 a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 4 or SEQ ID NO: 5.
37. The method of claim 36, wherein the first probe comprises 0, 1, or 2 mismatches
compared to SEQ ID NO: 4 or its complement or compared to SEQ ID NO: 5 or its
complement.
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 with the engoenous
control amplicon and/or contacting the exogenous control amplicon with a third probe
capable of selectively hybridizing with the exoenous control amplicon.
42. The method of claim 41, wherein the second probe and the third probe 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 detectably different 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 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 adequacy
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
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
contiguous nucleotides of SEQ ID NO: 4, and wherein the second primer comprises a
sequence that is at least 90%, at least 95%, or 100% complementary to at least 15, at least 16,
at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or
at least 25 contiguous nucleotides of SEQ ID NO: 4.
54. The composition of claim 53, wherein the first primer and the second primer each
independently comprises 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its
complement.
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 a first primer of SEQ ID NO: 1 and a second primer of 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 a Tv40Srp amplicon produced 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 to 59, wherein the first probe comprises a
sequence that is at least 90%, at least 95%, or 100% identical or complementary to at least 15,
at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at
least 24, or at least 25 contiguous nucleotides of SEQ ID NO: 4 or SEQ ID NO: 5.
61. The composition of claim 60, wherein the first probe comprises 0, 1, or 2 mismatches
compared to SEQ ID NO: 4 or its complement or compared to SEQ ID NO: 5 or its
complement.
62. The composition of any one of claims 57 to 61, wherein the first probe consists of 15
to 30 nucleotides.
63. The composition of any one of claims 57 to 62, wherein the first probe has the
sequence of SEQ ID NO: 3.
64. The composition of any one of claims 57 to 63, wherein the Tv40Srp amplicon has
the 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 the second primer pair.
66. The composition of claim 65, wherein the endogenous control is a sample adequacy
control.
67. The composition of claim 65, wherein the endogenous control is selected from
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 produced 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 to 70, wherein the exogenous control is a
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 of 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 compositions in the kit.
77. The kit of claim 76, wherein the endogenous control is a sample adequacy control.
78. The kit of claim 76, wherein the endogenous control is selected from 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 from
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 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 contiguous
nucleotides of SEQ ID NO: 4, and wherein the second primer comprises a sequence that is at
least 90%, at least 95%, or 100% complementary to at least 15, at least 16, at least 17, at least
18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, or at least 25
contiguous nucleotides of SEQ ID NO: 4.
84. The kit of claim 83, wherein the first primer and the second primer each
independently comprises 0, 1, or 2 mismatches compared to SEQ ID NO: 4 or its
complement.
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 a first primer of SEQ ID NO: 1 and a second primer of
SEQ ID NO: 2.
87. The kit of any one of claims 75 to 86, wherein the kit comprises a first probe capable
of selectively hybridizing to a Tv40Srp amplicon produced by the first primer pair, wherein
the first probe is in the same or different composition from one or more of the 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 to 89, wherein the first probe comprises a sequence
that is at least 90%, at least 95%, or 100% identical or complementary to at least 15, at least
16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least
24, or at least 25 contiguous nucleotides of SEQ ID NO: 4 or SEQ ID NO: 5.
91. The kit of claim 90, wherein the first probe comprises 0, 1, or 2 mismatches compared
to SEQ ID NO: 4 or its complement or compared to SEQ ID NO: 5 or its complement.
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 to 92, wherein the first probe has the sequence of SEQ
ID NO: 3.
94. The kit of any one of claims 87 or claim 93, wherein the Tv40Srb amplicon has the
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 produced by the second
primer pair, wherein the second probe is in the same or different composition from 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 produced by the third primer
pair, wherein the third probe is in the same or different composition from 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 one or more
lyophilized compositions.
99. A primer consisting of the sequence of SEQ ID NO: 1, wherein the primer comprises
at least one modified nucleotide.
100. A primer consisting of the sequence of SEQ ID NO: 2, wherein the primer
comprises at least one modified nucleotide.
101. A probe consisting of the sequence of SEQ ID NO: 3, wherein the probe
comprises at least one modified nucleotide and/or a detectable label.
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 first primer consisting of the sequence of SEQ ID
NO: 2 and a second primer consisting of the sequence of SEQ ID NO: 3, wherein the first
primer and the second primer each comprises at least one modified nucleotide.
106. The composition of claim 105, wherein the composition comprises a probe
consisting of the sequence of SEQ ID NO: 3, wherein the probe comprises at least one
modified nucleotide and/or a detectable label.
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 to 109, wherein the composition is
a lyophilized composition.