Assay for detecting chlamydia trachomatis, neisseria gonorrhoeae, trichomonas vaginalis and mycoplasma genitalium

Abstract

The present invention relates to assays for the detection of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium. The present invention relates to methods, kits and compositions for amplifying and detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) in a sample comprising various combinations of forward oligonucleotide primers, reverse oligonucleotide primers and oligonucleotide probes.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a divisional application of the invention patent application entitled "Assay for Detecting Chlamydia Trachomatis, Neisseria Gonorrhoeae, Trichomonas Vaginalis and Mycoplasma Genitalum" with international application number 201880090983.9, which entered China with international application date of December 31, 2018. This application claims the benefit of U.S. Provisional Patent Application No. 62 / 615,057 filed on January 9, 2018, which is incorporated herein by reference.

[0002] Incorporation by reference of electronically submitted material Incorporated herein by reference in its entirety is the computer-readable nucleotide / amino acid sequence listing filed concurrently and identified as follows: a 3,590-byte ASCII (text) file created on December 31, 2018 and named "36075WO1ORD_ST25".

[0003] Technical Field and Background Art Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium are the most common non-viral sexually transmitted infections (STIs). Individuals infected by any of these pathogens are often asymptomatic or present similar symptoms. However, treatment regimens are different for each pathogen. Therefore, an accurate method for diagnosing each pathogen is needed to ensure that an appropriate treatment regimen is selected. Currently, clinicians who suspect STIs usually run an assay each time to detect each possible STI pathogen. In this regard, CT and NG are usually tested first, and only when those results are negative or the patient does not respond to antibiotic treatment, tests on TV or MG are performed. For asymptomatic cases, CT and NG are generally only evaluated as part of a routine annual screening program.

[0004] Nucleic acid tests (NAT) have been developed to detect each of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium, or a combination of CT and NG (see, e.g., Schachter J., West. J. Med., 153(5):523-34 (1990); Centers for Disease Control and Prevention, Sexually Transmitted Diseases Treatment Guidelines, MMWR Recomm Rep., 64(3) (2015); and Abbott REALTIME TM CT / NG Assay (Abbott Molecular, Des Plaines, IL). Nucleic acid testing is typically performed using PCR reagents provided in liquid form, which require frozen storage and batch testing, and for some tests, the turnaround time for sample preparation and real-time PCR can exceed several hours. NAT is also prone to handling errors such as contamination, and nucleic acid levels can drop below the limit of detection when the initial infection peak subsides, especially when pooled samples are tested.

[0005] Therefore, there remains a need for more sensitive detection methods and systems for simultaneously detecting multiple STI pathogens in a single assay, provided in a form that eliminates or reduces storage requirements and PCR reagent waste. The present disclosure provides such methods and systems. Summary of the invention The present disclosure provides a set of oligonucleotide sequences for amplifying and detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) nucleic acid sequences in a sample. The set comprises (a) a primer and probe set for amplifying and detecting at least a portion of Chlamydia trachomatis 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a first probe oligonucleotide sequence comprising SEQ ID NO: 3; (b) a primer and probe set for amplifying and detecting at least a portion of Neisseria gonorrhoeae opa gene, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 4, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 5, and a second probe oligonucleotide sequence comprising SEQ ID NO: 6; (c) a primer and probe set for amplifying and detecting at least a portion of Trichomonas vaginalis 18S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 7, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 8, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; and (d) a primer and probe set for amplifying and detecting at least a portion of Mycoplasma genitalium 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9. The invention also provides a method for detecting CT, NG, TV and MG in a sample using the oligonucleotide set described above.

[0007] The present disclosure also provides kits for detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) in a sample. The kit comprises (a) a primer and probe set for amplifying and detecting at least a portion of Chlamydia trachomatis 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a first probe oligonucleotide sequence comprising SEQ ID NO: 3; (b) a primer and probe set for amplifying and detecting at least a portion of the opa gene of Neisseria gonorrhoeae, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 4, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 5, and a second probe oligonucleotide sequence comprising SEQ ID NO: 6; (c) a primer and probe set for amplifying and detecting at least a portion of Trichomonas vaginalis 18S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 7, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 8, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; (d) a primer and probe set for amplifying and detecting at least a portion of the 23S rRNA of Mycoplasma genitalium, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; NO: 10, a forward primer oligonucleotide sequence comprising SEQ ID NO: 10, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 11, and a fourth probe oligonucleotide sequence comprising SEQ ID NO: 12; (e) reagents for amplifying and detecting nucleic acid sequences; and (f) instructions for use, wherein the probe oligonucleotide sequences each comprise a detectable label.

[0008] The present disclosure also provides compositions for detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV), and Mycoplasma genitalium (MG) in a sample. The composition comprises (a) a primer and probe set for amplifying and detecting at least a portion of Chlamydia trachomatis 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a first probe oligonucleotide sequence comprising SEQ ID NO: 3; (b) a primer and probe set for amplifying and detecting at least a portion of the opa gene of Neisseria gonorrhoeae, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 4, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 5, and a second probe oligonucleotide sequence comprising SEQ ID NO: 6; (c) a primer and probe set for amplifying and detecting at least a portion of Trichomonas vaginalis 18S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 7, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 8, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; and (d) a primer and probe set for amplifying and detecting at least a portion of the 23S rRNA of Mycoplasma genitalium, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9. NO: 10, a forward primer oligonucleotide sequence comprising SEQ ID NO: 10, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 11, and a fourth probe oligonucleotide sequence comprising SEQ ID NO: 12; wherein each of the probe oligonucleotide sequences comprises a detectable label. DETAILED DESCRIPTION

[0009] The present disclosure provides a group of oligonucleotides, which are used to amplify and detect four non-viral sexually transmitted pathogens in samples: Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium. As used herein, the term "oligonucleotide" refers to a short nucleic acid sequence comprising about 2 to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100 nucleotides, or a range defined by any of the aforementioned values). As used herein, the terms "nucleic acid" and "polynucleotide" refer to a polymeric form of nucleotides of any length, ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of a molecule, and therefore include double-stranded and single-stranded DNA and double-stranded and single-stranded RNA. As an equivalent, the term includes analogs of RNA or DNA prepared by nucleotide analogs and modified polynucleotides (e.g., methylated and / or capped polynucleotides). Nucleic acids are typically linked via phosphate bonds to form nucleic acid sequences or polynucleotides, although many other linkages are known in the art (eg, phosphorothioate, boranophosphate, etc.).

[0010] Oligonucleotide can be single-stranded or double-stranded, or can contain the part of double-stranded and single-stranded sequence.Oligonucleotide can be DNA, genome and complementary DNA (cDNA), RNA or hybrid, wherein said nucleic acid can contain the combination of deoxyribonucleotide and ribonucleotide, and base comprises the combination of uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine and isoguanine.Oligonucleotide can be obtained by chemical synthesis method or recombinant method.Specific oligonucleotide sequence can contain variant (for example, codon replaces), allele, straight homologue, single nucleotide polymorphism (SNP) and complementary sequence and the sequence clearly pointed out of its conservative modification.

[0011] Chlamydia trachomatis Chlamydia is a non-motile, Gram-negative, obligate intracellular parasite of eukaryotic cells that forms inclusion bodies in the cytoplasm of host cells. Chlamydia trachomatis (CT) is the pathogen of sexually transmitted disease (STD) Chlamydia. Chlamydia infection of the urogenital tract is associated with salpingitis, ectopic pregnancy and tubal factor infertility in women, and non-gonococcal urethritis and epididymitis in men (see, e.g., Schachter, J., supra, Cates et al., Am. J. Obstet. Gynecol., (6 Pt 2): 1771-1781 (1991); and Berger et al., J. Urol., 121 (6): 750-754 (1979)). The female genital site most commonly affected by Chlamydia in women is the cervix, but infection may be asymptomatic and, if untreated, may ascend to the uterus, fallopian tubes, and ovaries, causing pelvic inflammatory disease (PID) (Brunham et al., N. Eng. J. Med., 311(1):1-6 (1984)). Newborns born to infected mothers may develop inclusion conjunctivitis, nasopharyngeal infections, and pneumonia due to CT (Alexander, ER and HR Harrison, Rev. Infect. Dis., 5(4):713-719 (1983)). Patients infected with CT may be co-infected with Neisseria gonorrhoeae (NG), the causative agent of gonorrhea. In addition, patients with treatment indications for gonorrhea rather than chlamydia often carry CT (Lyss et al., Ann. Intern. Med., 139(3):178-185 (2003)). Chlamydia infection may not adequately respond to recommended regimens for treating NG. Therefore, dual therapy for gonococcal and chlamydial infections is generally recommended in patients treated for gonorrhea unless chlamydial infection has been excluded (MMWR Sexually Transmitted Disease Treatment Guidelines, Morb. Mortal Wkly. Rep. [Series Online]; 51(RR-06) (2002)).

[0012] Neisseria gonorrhoeae Neisseria gonorrhoeae (NG) is a non-flagellated, Gram-negative, oxidase-positive diplococcus and is the causative agent of gonorrhea, one of the most common STDs in the United States, with an estimated 700,000 new NG infections occurring each year (Weinstock et al., Perspect Sex Reprod Health, 36(1):6-10 (2004)). In men, gonococcal infection usually causes acute anterior urethritis with purulent exudate (Hook EW and Hansfield HH., Gonococcal infection in the adult, In: Holmes KK, Mardh PA, Sparling PF, Lemon SM, Stamm WE, Piot P, Wasserheit J (eds.), Sexually Transmitted Diseases, 3rd ed. New York, NY: McGraw-Hill Book Co., pp. 451-66 (1999); and Sparling PF, Handsfield HH, Neisseria gonorrhoeae. In: Mandell GL, Bennett JE, Dolin R (eds.), Mandell, Douglas, and Bennet's Principles and Practice of Infectious Diseases. Diseases, 5th ed. Philadelphia, PA: Churchill Livingstone, Inc., pp. 2242-58 (2000). In women, infection is most often found in the cervix, but the vagina and uterus may also be infected. The infection is usually asymptomatic, especially in women. If left untreated, local complications of gonococcal infection may occur, including PID or acute salpingitis in women and epididymitis in men (Hook and Hansfield, supra; and Sparling et al., supra). Rarely, disseminated gonococcal infection (DGI) may occur in untreated patients (Eisenstein, BI and ATMasi, Semin. Arthritis Rheum., 10(3):155-172 (1981)).

[0013] Trichomonas vaginalis Trichomonas vaginalis (TV) is an anaerobic protozoan parasite and the causative agent of trichomoniasis. The US Centers for Disease Control and Prevention (CDC) estimates that 3.7 million people are infected with TV, making it the most common curable sexually transmitted infection in the United States (Centers for Disease Control and Prevention. Sexually Transmitted Diseases Treatment Guidelines, 2015. MMWR Recomm Rep., 64 (3) (2015)). In women, infection with TV can cause vaginitis, urethritis and cervicitis, and is associated with PID, tubal infertility, premature birth, low birth weight and premature rupture of membranes (Centers for Disease Control and Prevention. Sexually Transmitted Diseases Treatment Guidelines, MMWR Recomm Rep., 64 (3) (2015); Kissinger, P., BMC Infect. Dis., 15: 307 (2015)). Women with TV infection are more susceptible to HIV infection and are at higher risk of transmitting HIV to sexual partners (Van Der Pol et al., J. Infect. Dis., 197:548-554 (2008); and Kissinger, P. and A. Adamski, Sex Transm. Infect., 89:426-433 (2013)). In men, TV infection can cause non-gonococcal urethritis (NGU), epididymitis, or prostatitis (Van Der Pol et al., supra, and Kissinger and Adamski, supra). 70-85% of patients infected with TV are asymptomatic. Due to adverse events associated with infection, screening of asymptomatic patients at high risk of infection, including patients with multiple sexual partners, illicit drug use, or a history of STD infection, may be considered. Microscopic evaluation of wet mounts and TV cultures are frequently used to diagnose TV infection; however, nucleic acid amplification testing has become the preferred method for TV detection due to its superior sensitivity (Centers for Disease Control and Prevention. Sexually Transmitted Diseases Treatment Guidelines, MMWR Recomm Rep., 64(3) (2015)).

[0014] Mycoplasma genitalium Mycoplasma genitalium (MG) is a small sexually transmitted bacterium that colonizes the urogenital tract of both men and women. It is recognized as the cause of nongonococcal urethritis (NGU) in men, accounting for 15-20% of NGU and 20-25% of non-chlamydial NGU (Anagrius et al., Sex Transm. Infect., 81(6):458-62 (2005)). In women, MG infection is detected in 10-30% of cervicitis cases, with infection being more common in women with cervicitis than in women without cervicitis (Centers for Disease Control and Prevention. Sexually Transmitted Diseases Treatment Guidelines, MMWR Recomm Rep., 64(3) (2015), Anagrius et al., supra, and Lusk et al., Sex Transm. Infect., 87:107-109 (2011)). Recent evidence also indicates an association between MG infection and PID, premature birth, and infertility (Lis et al., Clin. Infect. Disease, 61(3):418-426 (2015)). Identifying MG infection is often a challenge because most cases are asymptomatic or cause symptoms similar to other STIs. Treatment regimens that are effective for other STIs generally show lower efficacy for MG infection (Centers for Disease Control and Prevention. Sexually Transmitted Diseases Treatment Guidelines, MMWR Recomm Rep., 64(3) (2015)).

[0015] Primer and probe oligonucleotides Oligonucleotides are used in various applications in biotechnology, such as artificial gene synthesis, as polymerase chain reaction (PCR) primers, DNA sequencing, and as molecular probes. In one embodiment, oligonucleotides as described herein can be used as primers for nucleic acid amplification or probes for nucleic acid hybridization and detection. As used herein, the terms "primer", "primer sequence" and "primer oligonucleotide" refer to such oligonucleotides, when placed in the presence of nucleotides and reagents for nucleic acid polymerization (e.g., DNA dependency or RNA dependency polymerase), the oligonucleotides can serve as the synthesis starting point of the primer extension product as the complementary chain of nucleic acid (all types of DNA or RNA). Primers can be single-stranded or double-stranded. If double-stranded, then before being used to prepare extension products, primers can first be processed (e.g., denaturation) to allow the separation of their chains. Such denaturation steps are usually performed using heating, but alternatively alkali can be used to carry out, followed by neutralization. The primers of the present disclosure can have any suitable size, and desirably comprise, consist of or consist of about 15 to 50 nucleotides, about 20 to 40 nucleotides, or about 22 to 30 nucleotides. In addition to those described herein, the primers of the present disclosure can also contain other nucleotides. For example, depending on the type of amplification process adopted, the primer can include, for example, a restriction endonuclease recognition site (see, for example, U.S. Patents 5,270,184 and 5,455,166) of the target binding sequence 5', or an RNA polymerase promoter connected to the target binding sequence of the primer. A "forward primer" is a primer that hybridizes (or anneals) with a target nucleic acid sequence (for example, a template strand) for amplification. A "reverse primer" is a primer that hybridizes (or anneals) with the complementary strand of the target sequence during amplification. The forward primer hybridizes with the target sequence 5' relative to the reverse primer.

[0016] The terms "probe", "probe sequence" and "probe oligonucleotide" refer to oligonucleotides that can selectively hybridize to at least a portion of a target sequence (e.g., a portion of a target sequence that has been amplified) under appropriate amplification conditions. In general, probe sequences are identified as "complementary" (i.e., complementary to the coding strand or sense strand (+)) or "reverse complementary" (i.e., complementary to the antisense strand (-)). The probe can be single-stranded or double-stranded. If double-stranded, the probe oligonucleotide sequence can include a first nucleic acid sequence comprising a detectable label, and a second nucleic acid sequence comprising a quencher portion, as described in U.S. Patent No. 9,388,455. The probes of the present disclosure can have any suitable size, and desirably comprise, consist essentially of, or consist of about 10-50 nucleotides, about 12-35 nucleotides, or about 14-25 nucleotides.

[0017] As used herein, the terms "set", "primer set", "probe set" and "primer and probe set" refer to two or more oligonucleotide primers that together are capable of initiating amplification of a target sequence or target nucleic acid of interest (e.g., a target sequence within CT, NG, TV or MG); and / or at least one probe that can detect a target sequence or target nucleic acid. In certain embodiments, the term "primer set" refers to a pair of primers that includes a forward primer (or 5' (upstream) primer) that hybridizes to the 5' end of a target sequence or target nucleic acid to be amplified, and a reverse primer (or 3' (downstream) primer) that hybridizes to the complement of the target sequence or target nucleic acid to be amplified. Such primer sets or primer pairs are particularly useful in PCR amplification reactions.

[0018] The oligonucleotide sets described herein can be used to amplify and detect one or more target nucleic acid sequences from CT, NG, TV and MG in a sample. The terms "target sequence" and "target nucleic acid" are used interchangeably herein and refer to a specific nucleic acid sequence, the presence or absence of which is to be detected by the disclosed method. In the context of the present disclosure, the target sequence preferably includes a nucleic acid sequence to which one or more primers will hybridize and from which amplification will be initiated. The target sequence may also include a probe hybridization region with which the probe can form a stable hybrid under appropriate hybridization conditions. The target sequence may be single-stranded or double-stranded, and more than one target sequence may be amplified and detected. The primer and probe sequences described herein may target any suitable nucleic acid sequence or combination of sequences present in the genomes of CT, NG, TV and MG.

[0019] The genome of Chlamydia trachomatis comprises a 1.0 Mb chromosome and a 7.5 kb plasmid, which has been found to be highly conserved between strains, with few insertions / deletions, and no variably present genomic islands have been identified to date (Stephens et al., Science, 282:754-759 (1998); Carlson et al., Infect Immun., 73:6407-6418 (2005); Thomson et al., Genome Res., 18:161-171 (2008); Seth-Smith et al., BMC Immunol., 23 ... Genomics, 10:239 (2009); Jeffrey et al., Infect. Immun., 78:2544-2553 (2010); Unemo et al., Microbiology, 156:1394-1404 (2010); Somboonna et al., MBio, 2:e00045-11 (2011); Harris et al., Nat Genet., 44:364-366 (2012); and Seth-Smith et al., Genome Res.; 23(5):855-866 (2013)). Chlamydia trachomatis strains have traditionally been classified into serovars based on the major outer membrane protein (MOMP), which represents the major surface antigen (Stephens et al., J. Immunol., 128: 1083-1089 (1982); and Wang et al., J. Infect. Dis., 152: 791-800 (1985)). Currently, genotyping of the ompA gene encoding MOMP is more commonly performed (Pedersen et al., FEMS Immunol. Med. Microbiol., 55: 120-130 (2009)). OmpA genotype AC has been associated with trachoma, the leading cause of infectious blindness worldwide, genotype DK has been associated with urogenital tract infections, and genotypes L1-L3 have been associated with the disease lymphogranuloma venereum (LGV) (Seth-Smith et al., Genome Res.; 23(5):855-866 (2013)).

[0020] The oligonucleotide sets described herein may comprise, consist essentially of, or consist of any number of primer and probe oligonucleotides to amplify and detect any suitable number of CT nucleic acid sequences. In one embodiment, the oligonucleotide sets described herein comprise, consist essentially of, or consist of two or more primers that amplify at least a portion of the 23S rRNA of the CT genome to produce a single CT amplicon; and at least one probe that hybridizes to the single CT amplicon. This oligonucleotide set is different from other CT assays (e.g., REALTIMETM The CT / NG assay (Abbott Molecular, Des Plaines, IL) targets CT cryptic plasmid DNA. Targeting ribosomal RNA increases CT analytical sensitivity because ribosomal RNA copy numbers far exceed the number of cryptic plasmid copies per organism. In this regard, the CT primer and probe sets were designed to target sequences of 23S ribosomal RNA that are highly conserved among all CT serovars but do not cross-react with RNAs derived from commensal and closely related bacterial species.

[0021] A "portion" of a nucleic acid sequence comprises at least ten nucleotides (e.g., about 10 to about 5000 nucleotides). Preferably, a "portion" of a nucleic acid sequence comprises 10 or more (e.g., 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, or 100 or more) nucleotides, but less than 5,000 (e.g., 4900 or less, 4000 or less, 3000 or less, 2000 or less, 1000 or less, 800 or less, 500 or less, 300 or less, or 100 or less) nucleotides. As used herein, the term "amplicon" refers to the product of a natural or artificial amplification reaction.

[0022] Neisseria gonorrhoeae contains a circular genome of approximately 2.2 Mb that encodes approximately 2,000 genes (Marri et al., PLoS ONE, 5(7):el 1835 (2010)). The core Neisseria genome - a set of genes present in all Neisseria species - consists of 896 genes, most of which display housekeeping functions. The protein encoded by the opacity (Opa) gene is responsible for the opaque colony phenotype caused by tight junctions between neighboring Neisseria species and is also responsible for tight adhesion to host cells. The oligonucleotide sets described herein may comprise, consist essentially of, or consist of any number of primers and probe oligonucleotides to amplify and detect any suitable number of NG nucleic acid sequences. In one embodiment, the oligonucleotide sets described herein comprise, consist essentially of, or consist of two or more primers that amplify at least a portion of the NG opa gene to produce a single NG amplicon; and at least one probe that hybridizes to the single NG amplicon.

[0023] The genome of Trichomonas vaginalis is about 160Mb, two-thirds of which is composed of repeats and transposable elements (Carlton et al., Science, 315(5809):207-212(2007)). The TV genome contains about 60,000 protein-coding genes, which is one of the highest coding capacities in eukaryotes (Carlton et al., supra). Introns have been identified in 65 genes. Transfer RNAs (tRNAs) for all 20 amino acids have been identified in the TV genome, and about 250 ribosomal DNA (rDNA) units have been located in one of the six Trichomonas vaginalis chromosomes. The oligonucleotide set described herein may comprise, consist essentially of, or consist of any number of primers and probe oligonucleotides to amplify and detect any suitable number of TV nucleic acid sequences. In one embodiment, the oligonucleotide set described herein comprises, consists essentially of, or consists of: two or more primers that amplify at least a portion of the 18S rRNA of the TV genome to produce a single TV amplicon; and at least one probe that hybridizes to the single TV amplicon.

[0024] The genome of M. genitalium consists of 525 genes (New Scientist, 215(2875):19(2012)) in a circular DNA of 580,070 base pairs (Fraser et al., Science, 270(5235):397-403(1995)), making it one of the smallest genomes of a self-replicating organism. As such, the MG genome is thought to be very close to the minimal set of genes required to sustain bacterial life. Approximately 382 of the 482 M. genitalium protein-coding genes have been identified as essential, while genes encoding proteins of unknown function make up 28% of the essential protein-coding gene set (Glass et al., Proc. Natl. Acad. Sci. USA, 103(2):425-430(2006)). The oligonucleotide sets described herein may comprise, consist essentially of, or consist of any number of primer and probe oligonucleotides to amplify and detect any suitable number of MG nucleic acid sequences. In one embodiment, the oligonucleotide sets described herein comprise, consist essentially of, or consist of: two or more primers that amplify at least a portion of the 23S rRNA of the MG genome to produce a single MG amplicon; and at least one probe that hybridizes to the single MG amplicon.

[0025] In one embodiment, the oligonucleotide sets described herein comprise, consist essentially of, or consist of: (a) a primer and probe set that amplifies and detects at least a portion of Chlamydia trachomatis 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a first probe oligonucleotide sequence comprising SEQ ID NO: 3; (b) a primer and probe set that amplifies and detects at least a portion of Neisseria gonorrhoeae opa gene, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 4, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 5, and a second probe oligonucleotide sequence comprising SEQ ID NO: 6; (c) a primer and probe set that amplifies and detects at least a portion of Trichomonas vaginalis 18S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 7, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 8, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; and (d) a primer and probe set that amplifies and detects Mycoplasma genitalium 23S A primer and probe set for detecting at least a portion of rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 10, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 11, and a fourth probe oligonucleotide sequence comprising SEQ ID NO: 12, wherein each of the probe oligonucleotide sequences comprises a detectable label. The aforementioned oligonucleotide set is also referred to as ALINITY TM mSTI.

[0026] In contrast to other commercially available STI nucleic acid tests that only detect and quantify one or two STI pathogens at a time, the oligonucleotide panels described herein allow for simultaneous detection of the four most common non-viral STI pathogens in a single test on a single sample. When combined with internal control and cell control primer and probe sets, as described herein, the oligonucleotide panels allow for more accurate detection of CT, NG, TV, and MG in a single assay. In addition, the oligonucleotide panels and detection methods described herein allow for sample-to-result analysis in less time than other STI detection systems known in the art.

[0027] Any one or combination of oligonucleotides described herein may be modified in any suitable manner to stabilize or enhance the binding affinity (also referred to as the "melting temperature" or "T") of the primer or probe oligonucleotide for its target. m”). In this regard, the oligonucleotide sequence as described herein may comprise one or more modified oligonucleotide bases. For example, the oligonucleotide sequence may comprise one or more propyne-modified bases, wherein the oligonucleotide comprises an alkyne of the chemical formula CH3C≡CH. The one or more propyne-modified bases may include, for example, 5-(1-propynyl)-2'-deoxyuridine (pdU) and / or 5-(1-propynyl)-2'-deoxycytidine (pdC).

[0028] Any oligonucleotide sequence described herein can comprise, consist essentially of, or consist of the complement of any sequence disclosed herein. As used herein, the term "complement" or "complementary sequence" refers to a nucleic acid sequence that forms a stable duplex with an oligonucleotide as described herein via Watson-Crick base pairing rules, and typically shares about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% about 95%, about 96%, about 97%, about 98% or about 99% greater identity with the oligonucleotide of the present invention. Nucleic acid sequence identity can be determined using any suitable mathematical algorithm or computer software known in the art, for example, CLUSTAL-W, T-Coffee and ALIGN (for alignment of nucleic acid and amino acid sequences), BLAST programs (e.g., BLAST 2.1, BL2SEQ and later versions) and FASTA programs (e.g., FASTA3x, FASTM and SSEARCH) (for sequence alignment and sequence similarity searches). Sequence alignment algorithms are also disclosed in, for example, Altschul et al., J. Molecular Biol., 215(3):403-410 (1990); Beigert et al., Proc. Natl. Acad. Sci. USA, 106(10):3770-3775 (2009), Durbin et al., eds., Biological Sequence Analysis: Probalistic Models of Proteins and Nucleic Acids, Cambridge University Press, Cambridge, UK (2009); Soding, Bioinformatics, 21(7):951-960 (2005); Altschul et al., Nucleic Acids Res., 25(17):3389-3402 (1997); and Gusfield, Algorithms on Strings, Trees and Sequences, Cambridge University Press, Cambridge UK (1997)).

[0029] The oligonucleotides described herein can be prepared using any suitable method, various of which are known in the art (see, e.g., Sambrook et al., Molecular Cloning. A Laboratory Manual, 1989, 2. Supp. Ed., Cold Spring Harbour Laboratory Press: New York, NY; MA Innis (ed.), PCR Protocols. A Guide to Methods and Applications, Academic Press: New York, NY (1990); P. Tijssen, Hybridization with Nucleic Acid Probes-Laboratory Techniques in Biochemistry and Molecular Biology (Parts I and II), Elsevier Science (1993); MA Innis (ed.), PCR Strategies, Academic Press: New York, NY (1995); FM Ausubel (ed.), Short Protocols in Molecular Biology, John Wiley and Sons, 1996). Wiley & Sons: Secaucus, NJ (2002); Narang et al., Meth. Enzymol., 68:90-98 (1979); Brown et al., Meth. Enzymol., 68:109-151 (1979); and Belousov et al., Nucleic Acids Res., 25:3440-3444 (1997)). Primer pairs can also be designed using various tools, such as the Primer-BLAST tool provided by the National Center of Biotechnology Information (NCBI). Oligonucleotide synthesis can be performed on an oligonucleotide synthesizer, such as those commercially available from PerkinElmer / Applied Biosystems, Inc. (Foster City, CA), DuPont (Wilmington, DE), or Milligen (Bedford, MA).Alternatively, oligonucleotides can be custom made and obtained from various commercial sources well known in the art, including, for example, Midland Certified Reagent Company (Midland, TX), Eurofins Scientific (Louisville, KY), BioSearch Technologies, Inc. (Novato, CA), etc. Oligonucleotides can be purified using any suitable method known in the art, such as native acrylamide gel electrophoresis, anion exchange HPLC (see, e.g., Pearson et al., J. Chrom., 255: 137-149 (1983)), and reverse phase HPLC (see, e.g., McFarland et al., Nucleic Acids Res., 7: 1067-1080 (1979)).

[0030] The sequences of primers and probes can be verified using any suitable sequencing method known in the art, including but not limited to chemical degradation (see, e.g., Maxam et al., Methods of Enzymology, 65:499-560 (1980)), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (see, e.g., Pieles et al., Nucleic Acids Res., 21:3191-3196 (1993)), mass spectrometry after a combination of alkaline phosphatase and exonuclease digestion (Wu et al. Anal. Biochem., 290:347-352 (2001)), and the like.

[0031] The primers and probe oligonucleotides described herein desirably comprise a melting temperature (T) in the range of 45°C to 80°C. M ). According to the present disclosure, the oligonucleotides specifically hybridize to the target nucleic acid sequence without showing significant hybridization to non-target (i.e., non-CT, non-NG, non-TV or non-MG nucleic acids). In addition, the oligonucleotides are selected so that they hybridize to conserved regions in the CT, NG, TV and MG genomes, thereby minimizing mismatches with the target sequence. Furthermore, the oligonucleotides are selected so that they show a minimal likelihood of dimer formation and contain minimal sequence repeats. Such properties can be determined by methods known in the art, for example, using computer modeling programs PrimerAnalysis Software (published by National Biosciences, Inc., Plymouth, MN).

[0032] Detectable Label Any one or more of the primers and probe oligonucleotide sequences described herein may include a detectable label so that after being combined with another entity (e.g., an amplified product or an amplicon), a primer and / or a probe may be visualized. As used herein, the term "detectable label" refers to a portion or compound that generates a signal, and the signal is measurable, and its intensity is related to the amount of the entity combined therewith (e.g., proportional). Any suitable detectable label may be used, which may be conjugated or connected to an oligonucleotide to detect the combination of an oligonucleotide with a target sequence, many of which are known in the art. In one embodiment, a detectable label may be detected indirectly. An indirect detectable label is typically a specific binding member used in combination with a "conjugate", which is attached or coupled to a direct detectable label. The coupling chemistry for synthesizing such a conjugate is well known in the art, and is designed so that the specific binding properties of the specific binding members and the detectable properties of the label remain intact. As used herein, "specific binding members" and "conjugates" refer to two members of a pair, i.e., two different molecules, wherein a specific binding member specifically binds to a polynucleotide of the present invention, and a "conjugate" specifically binds to a specific binding member. The binding between the two members of the pair is generally chemical or physical in nature. Examples of such binding pairs include, but are not limited to, antigen and antibody, avidin / streptavidin and biotin, hapten and antibody specific for the hapten, complementary nucleotide sequences, enzyme cofactor / substrate and enzyme, and the like.

[0033] In another embodiment, the detectable label can be directly detected. Such direct detectable labels include, for example, radioisotopes, fluorophores, chemiluminescent bodies, enzymes, colloidal particles, fluorescent microparticles, intercalating dyes (e.g., SYBRGreen or ethidium bromide), etc. In one embodiment, the detectable label can be a fluorophore, such as a fluorescein family dye, a polyhalogenated fluorescein family dye, a hexachlorofluorescein family dye, a coumarin family dye, a rhodamine family dye, a cyanine family dye, an oxazine family dye, a thiazine family dye, a square acid cyanine family dye, a chelated lanthanide family dye, an azo family dye, a triphenylmethane family dye, or Family dyes. Examples of fluorophores include, but are not limited to, FAM TM 、HEX TM , JOE TM 、NED TM , ROX TM 、TAMRA TM 、TET TM 、TEXAS and Those skilled in the art will appreciate that directly detectable labels may require additional components, such as substrates, triggering agents, light, etc., to enable detection of the label. Methods for labeling oligonucleotides such as probes are well known in the art and are described, for example, in: LJKricka, Ann.Clin.Biochem., 39:114-129 (2002); van Gijlswijk et al., Expert Rev. Mol.Diagn., 1:81-91 (2001); Joos et al., J.Biotechnol., 35:135-153 (1994); Smith et al., Nucl.Acids Res., 13:2399-2412 (1985); Connoly et al., Nucl.Acids Res., 13:4485-4502 (1985); Broker et al., Nucl.Acids Res., 5:363-384 (1978); Bayer et al., Methods of Biochem. Analysis, 26:1-45 (1980); Langer et al., Proc. Natl. Acad. Sci. USA, 78:6633-6637 (1981); Richardson et al., Nucl. Acids Res., 11:6167-6184 (1983); Brigati et al., Virol., 126:32-50 (1983); Tchen et al., Proc. Natl. Acad. Sci. USA, 81:3466-3470 (1984); Landegent et al., Exp. Cell Res., 15:61-72 (1984); AH Hopman et al., Exp. Cell Res., 169:357-368 (1987); and Temsamani et al., Mol. Biotechnol., 5:223-232 (1996).

[0034] In another embodiment, any one or more of the primers and probe oligonucleotide sequences described herein may also include a quencher portion. When the detectable label (e.g., fluorophore) and the quencher portion are kept in close proximity, for example, at the end of the probe, the quencher portion prevents detection of the signal (e.g., fluorescence) from the detectable label. When the two portions are physically separated, for example, after being cut by a DNA polymerase, the signal becomes detectable. The quencher may be selected from any suitable quencher known in the art, such as a BLACK HOLE 1 BLACK HOLE 2 IOWA FQ and IOWA RQ. For example, the oligonucleotide probe can comprise a FAM fluorophore and a BHQ-1 quencher.

[0035] The probe oligonucleotide sequence in the oligonucleotide sequence group described herein each desirably comprises a detectable label. Each probe can be labeled with the same detectable label or different detectable labels. When the probe comprises the same detectable label (e.g., FAM), in the real-time PCR process, the amplified portion of CT 23S rRNA, NG opa gene, TV 18S rRNA, and MG 23S rRNA is detected as a single signal. When each probe comprises different detectable labels, the amplified portion of CT 23S rRNA, NG opa gene, TV 18S rRNA, and MG 23S rRNA is detected as four separate signals.

[0036] The choice of a particular labeling technique will depend on several factors, such as the ease and cost of the labeling method, the spectral spacing between the different detectable labels used, the desired quality of sample labeling, the effect of the detectable moiety on the hybridization reaction (e.g., on the rate and / or efficiency of the hybridization process), the nature of the amplification method used, the nature of the detection system, the nature and intensity of the signal generated by the detectable label, and the like.

[0037] Internal Control The oligonucleotide sets described herein for detecting CT, NG, TV and MG may further comprise primers and probe oligonucleotide sequences for amplifying and detecting internal control (IC) sequences. In one embodiment, the internal control sequence is added to each sample preparation reaction. The internal control is then processed through the entire sample preparation and amplification procedure, together with the test sample and calibrator (if present), to confirm correct sample processing and assay validity. In one embodiment, the internal control sequence is added directly to the PCR reaction to test RT-PCR inhibition and confirm assay validity. The internal control can be any suitable non-CT, non-NG, non-TV or non-MG nucleic acid sequence (also referred to as an "exogenous" sequence), and desirably comprises, consists essentially of, or consists of: an armored RNA target sequence. As used herein, the term "armored RNA" refers to an RNase-resistant RNA that is a complex of MS2 bacteriophage capsid protein and RNA produced in Escherichia coli by inducing an expression plasmid encoding the capsid protein and RNA standard sequence (see, e.g., Pasloske et al., J. Clin. Microbiol., 36(12):3590-359 (1998); and U.S. Pat. Nos. 5,677,124, 5,919,625, and 5,939,262). In one embodiment, for example, the internal control may comprise an RNA sequence derived from or obtained from a hydroxypyruvate reductase gene of the squash plant Curcurbita pepo. In this regard, the oligonucleotide set described herein may further comprise an internal control forward primer oligonucleotide sequence comprising SEQ ID NO: 13, an internal control reverse primer oligonucleotide sequence comprising SEQ ID NO: 14, and an internal control probe oligonucleotide sequence comprising SEQ ID NO: 15.

[0038] The oligonucleotide sets described herein for detecting CT, NG, TV, and MG may further comprise primers and probe oligonucleotide sequences for amplifying and detecting cell controls, which serve as controls for sample input and preparation. In this regard, the cell control primer and probe sets may amplify and detect any suitable human gene, such as a human housekeeping gene. In one embodiment, the cell control primer and probe set amplifies and detects the human β-globin gene and includes a cell control forward primer oligonucleotide sequence comprising SEQ ID NO: 16, a cell control reverse primer oligonucleotide sequence comprising SEQ ID NO: 17, and a cell control probe oligonucleotide sequence comprising SEQ ID NO: 18 and a detectable label.

[0039] Both the internal control probe and the cell control probe desirably comprise a detectable label, such as any of those described herein. In one embodiment, the internal control probe and the cell control probe can be labeled with different fluorophores that are different from the probes used to detect CT, NG, TV, and MG, allowing for simultaneous detection and differentiation of internal controls, cell controls, and amplified target sequences in the same reaction. The internal control and cell control probes can also comprise a quencher moiety, such as any of those described herein.

[0040] Method for amplifying and detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) The present disclosure provides methods for detecting CT, NG, TV, and MG in a sample suspected of containing CT, NG, TV, and MG. The method comprises: (a) contacting a sample from a human with a set of oligonucleotide sequences described herein and a reagent for amplifying and detecting nucleic acid sequences, (b) amplifying a portion of a CT 23S rRNA sequence, a portion of a NG opa gene, a portion of a TV 18S rRNA, and a portion of a MG 23S rRNA present in the sample, (c) hybridizing a first oligonucleotide probe, a second oligonucleotide probe, a third oligonucleotide probe, and a fourth oligonucleotide probe to the amplified portions of the CT 23S rRNA, the NG opa gene, the TV 18S rRNA, and the MG 23S rRNA, respectively, and (d) detecting hybridization of the first oligonucleotide probe, the second oligonucleotide probe, the third oligonucleotide probe, and the fourth oligonucleotide probe to the amplified portions of the CT 23S rRNA, the NG opa gene, the TV 18S rRNA, and the MG 23S rRNA, respectively, by evaluating a signal from each detectable label, whereby (i) the presence of a signal indicates that the first oligonucleotide probe, the second oligonucleotide probe, the third oligonucleotide probe, and the fourth oligonucleotide probe hybridize to the CT 23S rRNA, the NG opa gene, the TV 18S rRNA, and the MG 23S rRNA, respectively. Hybridization of an amplified portion of 23S rRNA, and the presence of CT, NG, TV, and MG in the sample; and (ii) the absence of a signal indicates the absence of CT, NG, TV, and MG in the sample. The description of the primers and probe oligonucleotides set forth herein with respect to the aforementioned oligonucleotide sets also applies to those same aspects of the methods described herein. The methods described herein can be performed to detect any one or combination of CT, NG, TV, and / or MG simultaneously or sequentially. For example, a sample can be analyzed for the presence of: only CT, CT and NG, CT and TV, only NG, NG and TV, only TV, TV and MG, only MG, NG and MG, CT, NG and TV, and CT, NG, TV, and MG, etc. Analysis of only one or a subset of CT, NG, TV, and MG results in "masking" of unselected pathogens, which may be useful in low prevalence areas where the positive predictive value of a particular assay is very low.

[0041] A sample as defined herein is "suspected" of containing CT, NG, TV and MG if the sample is obtained from a subject (preferably a human) suspected of being infected by CT, NG, TV and MG. A subject is suspected of being infected by CT, NG, TV and MG if the subject has an increased risk of contracting a sexually transmitted infection or disease (STI or STD). Factors that may increase the risk of STIs include, for example, having unprotected sexual intercourse, sexual activity with multiple partners, a history of STIs, rape, injection drug use, age (half of STIs occur in people aged 15 to 24 years), male prescription drug use for erectile dysfunction, and transmission from mother to baby during pregnancy.

[0042] Sample can be any suitable sample obtained from any suitable subject, and the subject is usually a mammal, such as a person. Sample can be derived from any biological source, such as cervical tissue or fluid, vaginal tissue or fluid, intracervical tissue or fluid or anal swab or brush or physiological fluid, including but not limited to urine, whole blood, serum, blood plasma, interstitial fluid, saliva, lens fluid, cerebrospinal fluid, sweat, emulsion, ascites, mucus, nasal fluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menstrual fluid, amniotic fluid, semen, etc. Sample can be obtained from a subject using conventional techniques known to those skilled in the art, and sample can be used directly or after pretreatment to change the characteristics of sample as obtained from biological source. Such pretreatment can include, for example, preparing blood plasma, diluting viscous fluid, filtering, precipitation, diluting, distilling, mixing, concentrating, interfering with the inactivation of components, the addition of reagent, cracking, etc. from blood.

[0043] After obtaining a sample from a subject, the sample can be contacted with an oligonucleotide set as described herein to form a reaction mixture. The reaction mixture is then placed under amplification conditions. The primers hybridize with a portion of the CT 23S rRNA sequence, a portion of the NG opa gene, a portion of the TV 18S rRNA, and / or a portion of the MG 23S rRNA (if present in the sample), and amplify the portion of the CT 23S rRNA sequence, the NG opa gene, the TV 18S rRNA, and / or the MG 23S rRNA present in the sample.

[0044] Amplifying the CT, NG, TV and / or MG nucleic acid sequences in a sample can be performed using any suitable nucleic acid sequence amplification method known in the art, including but not limited to polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), real-time PCR, transcription-mediated amplification (TMA), rolling circle amplification, nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA) and ligase chain reaction (LCR).

[0045] In one embodiment, RT-PCR is performed, such as real-time RT-PCR, to amplify Chlamydia trachomatis ribosomal RNA sequences, Neisseria gonorrhoeae genomic DNA sequences, Trichomonas vaginalis ribosomal RNA sequences, Mycoplasma genitalium ribosomal RNA sequences, exogenous internal control RNA sequences, and human genomic DNA sequences. As used herein, "RT-PCR" refers to an enzymatic reaction in which complementary DNA (cDNA) fragments are synthesized from a substrate RNA template. The reaction generally involves the use of a synthetic oligonucleotide primer and the use of a reverse transcriptase, and the synthetic oligonucleotide primer is complementary to the nucleotide sequence in the substrate RNA. The reaction consists of a cycle, in which the oligonucleotide primer present in a large amount of excess hybridizes with the substrate RNA to form a double-stranded structure along the complementary nucleotide sequence. Primer-substrate DNA: RNA complex will then serve as the starting site of the cDNA synthesis reaction catalyzed by reverse transcriptase, resulting in the synthesis of a cDNA chain complementary to the RNA chain. RNA can be messenger RNA (mRNA), transfer RNA (tRNA), genomic RNA (gRNA), ribosomal RNA (rRNA) or small nuclear RNA (snRNA). Methods and reagents for RT-PCR are well known in the art and are commercially available from a variety of sources (see, e.g., Freeman et al., Biotechniques, 26(1):112-122, 142-125 (1999); Joyce, C., Methods Mol. Biol., 193:83-92 (2002); and O'Connell, J. (ed.), RT-PCR Protocols, 1st ed., Springer-Verlag, New York, NY (2010)). Reverse transcription can be performed using one-step or two-step techniques known in the art, such as by using reverse transcription kits available from Thermo Fisher Scientific (Waltham, MA), Qiagen (Hilden, Germany), and Promega Corp. (Madison, WI).

[0046] As used herein, "real-time PCR" refers to a PCR method in which the accumulation of amplified products is measured in real time as the reaction progresses, accompanied by product quantification after each cycle, in contrast to conventional PCR in which the amplified DNA products are detected in endpoint analysis. Real-time PCR is also referred to as "quantitative PCR (qPCR)" in the art. Real-time detection of PCR products generally involves the use of nonspecific fluorescent dyes that are embedded in any double-stranded DNA and sequence-specific fluorescently labeled DNA probes. Real-time PCR techniques and systems are known in the art (see, e.g., Dorak, M. Tevfik, ed., Real-time PCR, Taylor & Francis (2007); and Fraga et al., "Real-time PCR," Current protocols essential laboratory techniques: 10-3 (2008)), and are commercially available from various sources (e.g., m2000rt REALTIME TM PCR Systems (Abbott Molecular, Inc., Des Plaines, IL); CFX Real-Time PCR Detection Systems (Bio-Rad Laboratories, Inc., Hercules, CA); and TAQMAN TM Real-Time PCR System (ThermoFisher Scientific, Waltham, MA)), any of which can be employed in the methods described herein.

[0047] After amplification of a portion of the CT 23S rRNA sequence, NG opa gene, TV 18S rRNA and / or MG 23S rRNA (if present in the sample), the method described herein further comprises hybridizing the first, second, third, and fourth oligonucleotide probes described herein to the amplified portions of the CT 23S rRNA, NG opa gene, TV 18S rRNA, and MG 23S rRNA, respectively. In one embodiment, a reaction mixture comprising a CT 23S rRNA amplicon, a NG opa gene amplicon, a TV 18S rRNA amplicon, and a MG 23S rRNA amplicon may be contacted with the first, second, third, and fourth oligonucleotide probes as described herein (which preferentially hybridize to the target nucleic acid sequence of the amplicon or its complement, thereby forming a hybrid duplex that is stable for detection) under stringent hybridization and washing conditions. "Hybridization" as used herein refers to the formation of a duplex structure by two single-stranded nucleic acids due to complementary base pairing. Hybridization can occur between completely complementary nucleic acid chains, or between "substantially complementary" nucleic acid chains containing minor mismatch regions. As used herein, "stringent hybridization conditions" means conditions under which hybridization of completely complementary nucleic acid chains is strongly preferred. Under stringent hybridization conditions, for example, in a complex nucleic acid mixture, a first nucleic acid sequence (e.g., a primer) will hybridize with a second nucleic acid sequence (e.g., a target sequence). Stringent conditions are sequence-dependent and will be different in different situations. Stringent conditions can be selected to be higher than the thermal melting point (T) of the specific sequence at a defined ionic strength pH. m ) is about 5-10℃ lower. m It can be the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the oligonucleotides complementary to the target hybridize to the target sequence at equilibrium (since the target sequence is present in excess, at T mStringent conditions may be those in which the salt concentration is less than about 1.0 M sodium ion, such as about 0.01-1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., about 10-50 nucleotides) and at least about 60° C. for long probes (e.g., greater than about 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least 2 to 10 times the background hybridization. Exemplary stringent hybridization conditions include the following: 50% formamide, 5x SSC, and 1% SDS, incubated at 42° C., or 5x SSC, 1% SDS, incubated at 65° C., with washes in 0.2x SSC and 0.1% SDS at 65° C. Any suitable methods and conditions known in the art for hybridizing oligonucleotide probes to target nucleic acid sequences may be used in the methods disclosed herein.

[0048] After hybridizing the first, second, third, and fourth oligonucleotide probes described herein to the amplified portions of CT 23S rRNA, NG opa gene, TV 18S rRNA, and MG 23S rRNA, respectively, the method comprises detecting hybridization of the probe oligonucleotide sequences to the portions of CT 23S rRNA, NG opa gene, TV 18S rRNA, and MG 23S rRNA by evaluating the signal from each detectable label, whereby (i) the presence of the signal indicates hybridization of the first, second, third, and fourth oligonucleotide probes to the amplified portions of CT 23S rRNA, NG opa gene, TV 18S rRNA, and MG 23S rRNA, respectively, and the presence of CT, NG, TV, and MG in the sample; and (ii) the absence of the signal indicates the absence of CT, NG, TV, and MG in the sample. Detection of the signal from the probe oligonucleotide sequences can be performed using various well-known methods, including, for example, homogeneous or heterogeneous techniques.

[0049] Homogeneous detection methods involve detecting the products of an amplification reaction as they are formed, i.e., in real time. As a result, when the reaction mixture is subjected to amplification conditions, amplification product / probe hybrids are formed and detected. Homogeneous detection methods include, but are not limited to, the use of FRET labels that are attached to the probe and emit a signal in the presence of the target sequence, Molecular Beacons (see Tyagi et al., Nature Biotechnol., 14:303-308 (1996); Tyagi et al., Nature Biotechnol., 16:49-53 (1998); Kostrikis et al., Science, 279:1228-1229 (1998); Sokol et al., Proc. Natl. Acad. Sci. USA, 95:11538-11543 (1998); Marras et al., Genet. Anal., 14:151-156 (1999); and U.S. Patents 5,846,726, 5,925,517, 6,277,581, and 6,235,504), Assays (see, e.g., U.S. Pat. Nos. 5,210,015; 5,804,375; 5,487,792 and 6,214,979, and International Patent Application Publication No. WO 01 / 86001), and hybridization protection assays (HPA) utilizing probes labeled with an acridinium ester (AE) (see, e.g., Weeks et al., Clin. Chem., 29:1474-1479 (1983); Berry et al., Clin. Chem., 34:2087-2090 (1988)).

[0050] Heterogeneous detection systems generally use capture reagents to separate the sequence of amplification from other materials in the reaction mixture. Capture reagents generally include solid support materials (e.g., microtiter wells, beads, chips, etc.) coated with one or more specific binding sequences. The binding sequence can be complementary to the tail sequence of the oligonucleotide probe of the present invention. Alternatively, the binding sequence can be complementary to the sequence of the capture oligonucleotide, and the sequence of the capture oligonucleotide itself includes a sequence complementary to the tail sequence of the probe. After the amplified product / probe hybrid combined with the capture reagent is separated from the remaining reaction mixture, any suitable detection method known in the art or described herein can be used to detect the amplified product / probe hybrid.

[0051] Kits and compositions for amplifying and detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) The present invention also provides a kit for amplifying and detecting CT, NG, TV and / or MG in a sample. The kit comprises a primer and a probe set, which amplifies and detects a portion of Chlamydia trachomatis 23S rRNA, a portion of Neisseria gonorrhoeae opa gene, a portion of Trichomonas vaginalis 18S rRNA, and / or a portion of Mycoplasma genitalium 23S rRNA, as well as reagents and instructions for amplifying and detecting CT, NG, TV and / or MG. The description of the primer oligonucleotides and probe oligonucleotides described herein for the aforementioned method also applies to the same aspects of the kit described herein. Examples of suitable reagents for inclusion in the kit (in addition to the oligonucleotide primers and probes described herein) include conventional reagents used in nucleic acid amplification reactions, such as one or more enzymes with polymerase activity, enzyme cofactors (e.g., magnesium or nicotinamide adenine dinucleotide (NAD)), salts, buffers, deoxyribonucleotides or ribonucleotide triphosphates (dNTP / rNTP; e.g., deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytidine triphosphate and deoxythymidine triphosphate) blocking reagents, labeling reagents, and the like. Many such agents are described herein or are otherwise known in the art and are commercially available.

[0052] In one embodiment, the kit may comprise, consist essentially of, or consist of: (a) a primer and probe set for amplifying and detecting a portion of Chlamydia trachomatis 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a first probe oligonucleotide sequence comprising SEQ ID NO: 3; (b) a primer and probe set for amplifying and detecting a portion of Neisseria gonorrhoeae opa gene, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 4, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 5, and a second probe oligonucleotide sequence comprising SEQ ID NO: 6; (c) a primer and probe set for amplifying and detecting a portion of Trichomonas vaginalis 18S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 7, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 8, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; (d) a primer and probe set for amplifying and detecting Mycoplasma genitalium 23S A primer and probe set for a portion of rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 10, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 11, and a fourth probe oligonucleotide sequence comprising SEQ ID NO: 12, (e) reagents for amplifying and detecting nucleic acid sequences; and (f) instructions for use, wherein the probe oligonucleotide sequences each comprise a detectable label.

[0053] The kit may include instructions for using the amplification reagents and primers and probe oligonucleotides described herein, such as for processing test samples, extracting nucleic acid molecules and / or performing tests; and for interpreting the results obtained, and notifications in a form prescribed by a government agency. Such instructions may optionally be in printed form, or on a CD, DVD or other recording medium.

[0054] The present disclosure also provides compositions for amplifying and detecting CT, NG, TV and / or MG in a sample. The composition comprises, consists essentially of, or consists of: (a) a primer and probe set for amplifying and detecting a portion of Chlamydia trachomatis 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a first probe oligonucleotide sequence comprising SEQ ID NO: 3; (b) a primer and probe set for amplifying and detecting a portion of Neisseria gonorrhoeae opa gene, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 4, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 5, and a second probe oligonucleotide sequence comprising SEQ ID NO: 6; (c) a primer and probe set for amplifying and detecting a portion of Trichomonas vaginalis 18S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 7, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 8, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; and (d) a primer and probe set for amplifying and detecting a portion of Mycoplasma genitalium 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9. NO: 10 forward primer oligonucleotide sequence, comprising SEQ ID NO: 11 reverse primer oligonucleotide sequence, and comprising SEQ ID NO: 12 fourth probe oligonucleotide sequence; wherein each of the probe oligonucleotide sequences comprises a detectable label. The description of the primer oligonucleotide and probe oligonucleotide set forth herein for the aforementioned methods and kits also applies to those same aspects of the compositions described herein. In some embodiments, the composition comprises a carrier, preferably a pharmaceutically (e.g., physiologically acceptable) carrier. Any suitable carrier can be used within the context of the present invention, and such carriers are well known in the art. The composition can optionally be sterile or sterile in addition to the oligonucleotides described herein.

[0055] The aforementioned kits and compositions may further comprise primers and probe sets for amplifying and detecting exogenous internal control nucleic acid sequences (e.g., zucchini hydroxypyruvate reductase gene), and primers and probe sets for amplifying and detecting cell control nucleic acid sequences (e.g., human β-globin gene), as described herein. In this regard, the kits and / or compositions may include an internal control forward primer oligonucleotide sequence comprising SEQ ID NO: 13, an internal control reverse primer oligonucleotide sequence comprising SEQ ID NO: 14, and an internal control probe oligonucleotide sequence comprising SEQ ID NO: 15 and a detectable label. In another embodiment, the kits and / or compositions may include a cell control forward primer oligonucleotide sequence comprising SEQ ID NO: 16, a cell control reverse primer oligonucleotide sequence comprising SEQ ID NO: 17, and a cell control probe oligonucleotide sequence comprising SEQ ID NO: 18 and a detectable label.

[0056] Test kit and / or composition can be supplied in solid (e.g., lyophilized) or liquid form. In one embodiment, primer oligonucleotide, probe oligonucleotide and other reagents are lyophilized (i.e., freeze-dried). Many nucleic acid detection systems known in the art provide PCR reagents in liquid form, which require frozen storage and batch testing. However, the freeze-drying of the various components of the test kit and composition described herein eliminates the need for frozen storage, and allows the determination component to be delivered in unit dose form, so that the user can run the determination of the required exact number, thereby minimizing reagent waste. For each individual component (e.g., primer oligonucleotide, probe oligonucleotide or buffer), the various components of the test kit of the present invention and composition can be optionally included in different containers (e.g., vials, ampoules, test tubes, flasks or bottles). Each component is generally appropriately divided in its respective containers or provided in concentrated form. Other containers suitable for carrying out certain steps of amplification / detection determination can also be provided. Each container is preferably maintained in a closed constraint for commercial sale.

[0057] The invention is further illustrated by the following examples which, however, should not, of course, be construed in any way as limiting its scope. Example

[0058] This example demonstrates methods for amplifying and detecting Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium in a sample according to the present disclosure.

[0059] Assays that utilize real-time RT-PCR to amplify and detect genomic sequences of Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium extracted from human specimens have been commercialized by Abbott Molecular, Inc. (Des Plaines, IL) under the trade name ALINITY TM m STI developed under ALINITY TM The m STI assay utilizes real-time RT-PCR to amplify and detect Chlamydia trachomatis ribosomal RNA sequences, Neisseria gonorrhoeae genomic DNA sequences, Trichomonas vaginalis ribosomal RNA sequences, Mycoplasma genitalium ribosomal RNA sequences, and human genomic DNA sequences, which have been isolated from endocervical swab specimens, vaginal swab specimens, male and female urine specimens, and ALINITY ELISA was performed on gynecological specimens stored in ELISA solution (Hologic, Inc., Marlborough, MA). Endocervical swabs, vaginal swabs, and urine specimens were analyzed using ALINITY TM m multi-Collect SpecimenTransport Kit for collection. The sample is transferred to a transport tube for use in ALINITY TM m processing on the system.

[0060] ALINITY TM The steps of mSTI assay consist of the following: sample preparation, RT-PCR assembly, amplification / detection, and result calculation and reporting. TM All stages of the m STI determination procedure are performed by ALINITY TM mThe system performs this automatically. The user does not perform any intermediate processing or transfer steps. TM The m system is designed as a random access analyzer that can be used with other ALINITY TM mAssays performed in parallel ALINITY TM mSTI determination.

[0061] Using ALINITY TM mDNA Sample Prep Kit, ALINITY TM m Lysis Solution and ALINITY TM m Diluent Solution in ALINITY TM mThe instrument automatically extracts nucleic acids from samples on board. TMThe instrument uses magnetic particle technology to facilitate nucleic acid capture, washing, and elution. Specifically, at the beginning of the sample preparation protocol, the sample (specimen or control) and magnetic particles are drawn through the instrument into a liquid containing ALINITY TM m LysisSolution and ethanol into the wells of the sample preparation cartridge. The conditions of the lysis step promote lysis of cells, denaturation of proteins, and dissociation of bacterial phage capsids from armored RNA (RNA targets in controls). The lysis conditions also promote binding of nucleic acids to magnetic particles. At the end of the lysis step, the magnetic particles with bound sample nucleic acids are captured by a magnetic plunger wrapped by a disposable plastic sleeve. The magnetic particles are then transferred sequentially to the wells in the sample preparation cartridge containing a series of wash solutions. After the wash steps are completed, the magnetic particles are captured by the plunger magnet and transferred to the elution wells in the sample preparation cartridge, where the purified nucleic acids are eluted from the particles into the ALINITY TM m DNAElution Buffer.

[0062] The resulting purified nucleic acid is then mixed with a liquid unit dose activator reagent, a lyophilized unit dose ALINITY TM mSTI Amplification Reagents and ALINITY TM The m Vapor Barrier Solution is combined and transferred to the amplification / detection module through the instrument for reverse transcription, PCR amplification, and real-time fluorescence detection.

[0063] Test assay controls at or above the established minimum frequency to help ensure that instrument and reagent performance remains satisfactory. During each control event, process negative and positive controls by sample preparation and RT-PCR procedures equivalent to those used for the specimens. Assay controls are used to confirm proper sample handling and assay validity. ALINITY TM The mSTI amplification reagents include primers and probes that amplify and detect the single copy human gene β-globin. Amplification and detection of the β-globin gene confirms proper sample processing and sufficient sample input. An armored RNA internal control containing the sequence of the hydroxypyruvate reductase gene from the squash plant Cucurbita pepo is included in each PCR reaction. Both the β-globin control and the internal control are used to confirm assay validity.

[0064] The primer and probe oligonucleotide sequences used for amplification and detection of CT, NG, TV, MG, and internal and cell controls are described in Table 1 below. TM The mSTI assay is designed to target a region of the 23S ribosomal RNA. This design is different from the Abbott REALTIME assay that targets CT-cryptic plasmid DNA.TM CT / NG Assay. Targeting ribosomal RNA increases CT analytical sensitivity because ribosomal RNA copy numbers far exceed the number of cryptic plasmid copies per organism. CT primer / probe sets are designed to target sequences of 23S ribosomal RNA that are highly conserved among all CT serovars but do not cross-react with RNA from commensal and closely related bacterial species. For the qualitative detection of Neisseria gonorrhoeae (NG), ALINITY TM The mSTI assay is designed to target the multicopy opa gene. For the qualitative detection of Trichomonas vaginalis (TV), ALINITY TM mSTI assays are designed to target a region of the 18S ribosomal RNA. For the qualitative detection of Mycoplasma genitalium (MG), ALINITY TM The mSTI assay was designed to target a region of the 23S ribosomal RNA.

[0065] Table 1. Primer and probe sequences F = fluorophore Q = quencher.

[0066] The assay utilizes two multi-well plates for nucleic acid amplification. One plate (the master mix plate) contains the above oligonucleotide primers and probes, PCR buffer components, reverse transcriptase, DNA polymerase, dNTP mix, armored RNA internal control, and excipients, and is lyophilized (see Table 2). The second plate (activation plate) contains MgCl2 and any monovalent salts required for the reaction (see Table 3) and is stored as a liquid.

[0067] Table 2. Main mixing well components &Indicates the master mix excipient component.

[0068] Table 3. Activated pore components Components concentration <![CDATA[MgCl2]]> 5.94mM Tetramethylammonium chloride (TMAC) 104.03mM KCl 37.77mM ProClin950 0.03％ .

[0069] For each sample, the sample eluate and a defined volume of liquid activator are automatically transferred to the freeze-dried master mix well to form the PCR reaction. The complete PCR reaction is then transferred to the thermal cycler for amplification and detection without additional operator intervention.

[0070] At Abbott ALINITY TMDuring the first step of RT-PCR on the m system, the target RNA is converted to cDNA by the activity of reverse transcriptase. The CT, TV, MG and IC reverse primers anneal to their respective RNA targets and are extended during a short incubation period at a temperature that allows reverse transcription activity.

[0071] At the end of the reverse transcription step, the reaction temperature rises to a temperature that dissociates the double-stranded cDNA / RNA products (from CT, TV, MG and IC) generated during the reverse transcriptase step, and the double-stranded DNA targets (from NG and humans). This unwinding step also promotes the inactivation of reverse transcriptase and the hot start activation of DNA polymerase. As the reaction temperature is subsequently reduced, the CT, TV, MG and IC forward primers anneal to their respective cDNA chains and are extended by DNA polymerase. At the same time, the forward and reverse primers of NG and BG anneal to their respective genomic DNA chains and are extended by DNA polymerase. During successive rounds of thermal cycling, the amplified product dissociates into single strands at high temperatures, followed by primer annealing and extension as the temperature decreases. Exponential amplification is achieved by repeated cycles between high and low temperatures. Amplification of all six assay targets (CT, NG, TV, MG, BG and IC) is performed simultaneously in the same reaction. Table 4 illustrates the results of the ALINITY TM m RT-PCR cycling conditions used for STI assay.

[0072] Table 4: PCR Cycling Protocol

[0073] During the primer annealing / extension step, a reading step is performed to allow for real-time fluorescence detection of amplified products as the probes anneal to their respective targets. In the absence of a target, fluorescence is quenched. In the presence of a target, hybridization of the probe with a complementary sequence separates the fluorophore and the quencher, allowing fluorescence emission and detection. The number of amplification cycles (CN) at which the fluorescence signal exceeds the threshold value is used to detect the presence of the target nucleic acid. Samples with CN earlier than the defined CN cutoff value are reported as positive, while samples without CN or CN later than the cutoff value are reported as negative.

[0074] When in ALINITY m TM When selecting an STI assay for a given sample on the system, the user will be able to select one or more pathogens at the start of the run. For example, if the user only wants to evaluate CT or only wants to evaluate CT and NG, the user can select only those pathogens, and the results for the unselected pathogens will be masked. This can be useful for hiding pathogen results in low prevalence areas where the positive predictive value is very low.

[0075] The PCR formulation and cycling conditions described above can be further modified to optimize the assay.

[0076] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0077] In the context of describing the present invention (especially in the context of the following claims), the use of the terms "one" and "a kind of" and "the / said" and "at least one / kind" and similar indicators should be interpreted as covering both the singular and the plural, unless otherwise specified herein or clearly contradicted by the context. The use of the term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be understood to mean an item selected from the listed items (A or B), or any combination of two or more listed items (A and B), unless otherwise specified herein or clearly contradicted by the context. Unless otherwise specified, the terms "comprising", "having", "including" and "containing" should be interpreted as open terms (i.e., meaning "including but not limited to"). The phrase "essentially consisting of..." should also be interpreted as an open phrase, intended to include steps or materials that do not substantially affect the basic and novel features of the described product or method. The phrase "consisting of..." should be interpreted as a closed phrase, which excludes any element, step or component that is not clearly specified in the specification or claims. Unless otherwise specified herein, the narration of the value range in this article is only expected to serve as a shorthand method for individually referring to each separate value falling within the range, and each separate value is introduced into the specification as it is individually narrated in this article. Unless otherwise specified herein or obviously contradictory with the context, all methods described herein can be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (such as, "for example") provided herein is only expected to better illustrate the present invention, and does not limit the scope of the present invention. Any language in the specification should not be interpreted as indicating that any unclaimed element is necessary for the practice of the present invention.

[0078] Preferred embodiments of the present invention are described herein, including the best mode known to the inventor for carrying out the present invention. After reading the foregoing description, the changes of those preferred embodiments may become apparent to those of ordinary skill in the art. The inventor expects that a skilled person will appropriately adopt such changes, and the inventor expects that the present invention will be practiced in a manner different from that specifically described herein. Accordingly, the present invention includes all modifications and equivalents of the subject matter described in the claims attached thereto as permitted by applicable law. In addition, unless otherwise specified herein or clearly contradictory to the context, any combination of the above-mentioned elements with all possible variations thereof is encompassed by the present invention.

Claims

1. A set of oligonucleotide sequences for amplifying and detecting nucleic acid sequences of Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) in a sample, comprising: (a) a primer and probe set for amplifying and detecting at least a portion of Chlamydia trachomatis 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a first probe oligonucleotide sequence comprising SEQ ID NO: 3; (b) a primer and probe set for amplifying and detecting at least a portion of the opa gene of Neisseria gonorrhoeae, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 4, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 5, and a second probe oligonucleotide sequence comprising SEQ ID NO: 6; (c) a primer and probe set for amplifying and detecting at least a portion of Trichomonas vaginalis 18S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 7, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 8, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; and (d) A primer and probe set for amplifying and detecting at least a portion of Mycoplasma genitalium 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 10, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 11, and a fourth probe oligonucleotide sequence comprising SEQ ID NO: 12, wherein each of the probe oligonucleotide sequences comprises a detectable label.

2. The set of claim 1, further comprising an internal control primer and probe set.

3. The set of claim 2, wherein the internal control primer and probe set comprises: (e) an internal control forward primer oligonucleotide sequence comprising SEQ ID NO: 13, (f) an internal control reverse primer oligonucleotide sequence comprising SEQ ID NO: 14, and (g) An internal control probe oligonucleotide sequence comprising SEQ ID NO: 15 and a detectable label.

4. The set of any one of claims 1-3, further comprising a cellular control primer and probe set.

5. The panel of claim 4, wherein the cell control primer and probe set amplifies and detects the human β-globin gene.

6. The set of claim 4 or claim 5, wherein the cell control primer and probe set comprises: (h) a cell control forward primer oligonucleotide sequence comprising SEQ ID NO: 16, (i) a cell control reverse primer oligonucleotide sequence comprising SEQ ID NO: 17, and (j) A cellular control probe oligonucleotide sequence comprising SEQ ID NO: 18 and a detectable label.

7. The panel of any one of claims 1 to 6, wherein the detectable label is a fluorophore.

8. The set of any one of claims 1 to 7, wherein each of the probe oligonucleotides further comprises a quencher moiety.

9. A method for detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) in a sample suspected of containing Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis and Mycoplasma genitalium, the method comprising: (a) contacting a sample from a human with the oligonucleotide sequence set according to any one of claims 1 to 8 and a reagent for amplifying and detecting nucleic acid sequences, (b) amplifying at least a portion of the CT 23S rRNA sequence, at least a portion of the NG opa gene, at least a portion of the TV 18S rRNA, and at least a portion of the MG 23S rRNA present in the sample, (c) hybridizing the first oligonucleotide probe, the second oligonucleotide probe, the third oligonucleotide probe, and the fourth oligonucleotide probe to the amplified portions of CT 23S rRNA, NG opa gene, TV 18S rRNA, and MG 23S rRNA, respectively, (d) detecting hybridization of the first oligonucleotide probe, the second oligonucleotide probe, the third oligonucleotide probe, and the fourth oligonucleotide probe with the amplified portions of CT 23S rRNA, NG opa gene, TV 18S rRNA, and MG 23S rRNA by evaluating the signal from each detectable label, thereby (i) the presence of the signal indicates hybridization of the first oligonucleotide probe, the second oligonucleotide probe, the third oligonucleotide probe, and the fourth oligonucleotide probe to the amplified portions of CT 23S rRNA, NG opa gene, TV 18S rRNA, and MG 23S rRNA, and the presence of CT, NG, TV, and MG in the sample, and (ii) The absence of a signal indicates the absence of CT, NG, TV and MG in the sample.

10. The method of claim 9, wherein the sample comprises endocervical tissue or fluid, vaginal tissue or fluid, urine, or cervical tissue or fluid.

11. A kit for detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) in a sample, the kit comprising (a) a primer and probe set for amplifying and detecting at least a portion of Chlamydia trachomatis 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a first probe oligonucleotide sequence comprising SEQ ID NO: 3; (b) a primer and probe set for amplifying and detecting at least a portion of the opa gene of Neisseria gonorrhoeae, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 4, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 5, and a second probe oligonucleotide sequence comprising SEQ ID NO: 6; (c) a primer and probe set for amplifying and detecting at least a portion of Trichomonas vaginalis 18S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 7, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 8, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; (d) a primer and probe set for amplifying and detecting at least a portion of Mycoplasma genitalium 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 10, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 11, and a fourth probe oligonucleotide sequence comprising SEQ ID NO: 12; (e) reagents for amplifying and detecting nucleic acid sequences; and (f) instructions for use, wherein the probe oligonucleotide sequences each comprise a detectable label.

12. The kit of claim 11, further comprising an internal control primer and probe set.

13. The kit of claim 12, wherein the internal control primer and probe set comprises: (e) an internal control forward primer oligonucleotide sequence comprising SEQ ID NO: 13, (f) an internal control reverse primer oligonucleotide sequence comprising SEQ ID NO: 14, and (g) An internal control probe oligonucleotide sequence comprising SEQ ID NO: 15 and a detectable label.

14. The kit of any one of claims 11-13, further comprising a cell control primer and probe set.

15. The kit of claim 14, wherein the cell control primer and probe set amplifies and detects human β-globin gene.

16. The kit of claim 14 or claim 15, wherein the cell control primer and probe set comprises: (h) a cell control forward primer oligonucleotide sequence comprising SEQ ID NO: 16, (i) a cell control reverse primer oligonucleotide sequence comprising SEQ ID NO: 17, and (j) A cellular control probe oligonucleotide sequence comprising SEQ ID NO: 18 and a detectable label.

17. The kit of any one of claims 11-16, wherein the primers, probes and reagents are lyophilized.

18. A composition for amplifying and detecting Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), Trichomonas vaginalis (TV) and Mycoplasma genitalium (MG) in a sample, comprising: (a) a primer and probe set for amplifying and detecting at least a portion of Chlamydia trachomatis 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 1, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 2, and a first probe oligonucleotide sequence comprising SEQ ID NO: 3; (b) a primer and probe set for amplifying and detecting at least a portion of the opa gene of Neisseria gonorrhoeae, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 4, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 5, and a second probe oligonucleotide sequence comprising SEQ ID NO: 6; (c) a primer and probe set for amplifying and detecting at least a portion of Trichomonas vaginalis 18S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 7, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 8, and a third probe oligonucleotide sequence comprising SEQ ID NO: 9; and (d) A primer and probe set for amplifying and detecting at least a portion of Mycoplasma genitalium 23S rRNA, comprising a forward primer oligonucleotide sequence comprising SEQ ID NO: 10, a reverse primer oligonucleotide sequence comprising SEQ ID NO: 11, and a fourth probe oligonucleotide sequence comprising SEQ ID NO: 12; wherein each of the probe oligonucleotide sequences comprises a detectable label.

19. The composition of claim 18, wherein the primer oligonucleotides, probe oligonucleotides and reagents are lyophilized.

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