Compositions and Methods for Detecting Lymphogranuloma Venereum (LGV) Serotypes of Chlamydia trachomatis

Abstract

The present disclosure generally relates to a method for rapidly detecting the presence or absence of serotypes that cause lymphogranuloma venereum (LGV) of Chlamydia trachomatis in biological or non-biological samples. The method may include performing an amplification step, a hybridization step, and a detection step. Further provided are oligonucleotide primers and probes targeting the pmpH gene of the L serotype of Chlamydia trachomatis, as well as a kit designed to detect the L serotype.

Description

Technical Field

[0001] Cross - reference to related applications This application claims the priority and benefit of U.S. Provisional Application No. 63 / 357,179, filed on June 30, 2022, the content of which is incorporated herein by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing that was electronically submitted in ASCII format and is incorporated herein by reference in its entirety. The name of the ASCII copy created on June 29, 2023 is "P37525 - WO_Sequence_Listing", and the size is 26.813 bytes.

Background Art

[0003] The present disclosure relates to the field of molecular diagnostics, and more particularly, to the detection of serotypes (L serotypes) of Chlamydia trachomatis (C. trachomatis) that cause lymphogranuloma venereum (LGV) by polymerase chain reaction (PCR) assays.

[0004] Infection from C. trachomatis is a major cause of sexually transmitted diseases (STDs) by bacteria worldwide, with approximately 89.1 million cases occurring annually (Bebear C., de Barbeyrac B., Clinical Microbial Infect. 2009;15:4 - 10). In the United States, C. trachomatis is the most frequently reported bacterial STD, and the prevalence is highest in people under 24 years of age. In 2013, a total of 1,401,906 cases of C. trachomatis infection were reported to the Centers for Disease Control and Prevention (CDC) in the United States, corresponding to a rate of 446.6 cases per 100,000 population (CDC, Sexually Transmitted Disease Surveillance 2013).

[0005] Chlamydia trachomatis is a Gram-negative, non-motile, obligate intracellular bacterium with a characteristic biphasic life cycle. It causes various infections including urethritis, cervicitis, proctitis, conjunctivitis, endometritis, and salpingitis. If left untreated, these infections can ascend to the uterus, fallopian tubes, and ovaries, causing pelvic inflammatory syndrome, ectopic pregnancy, and tubal factor infertility. Reiter's syndrome (urethritis, conjunctivitis, arthritis, and mucocutaneous lesions) is also associated with genital C. trachomatis infection. Many infections are asymptomatic, and a large number of infected patients may not seek treatment. Patients are often reinfected if their sexual partners are not treated. Infants born to infected mothers can develop conjunctivitis, pharyngitis, and pneumonia. The main symptoms in both men and women are increased secretions and urination disorders, and women may also present with irregular uterine bleeding.

[0006] The serotypes of C. trachomatis can be separated into three groups: serotypes A, B, and C (A–C), serotypes D, E, F, G, H, I, J, and K (D–K), and L serotypes, which are each associated with distinct clinical manifestations of chlamydial disease. Serotypes A–C present as eye infections, serotypes D–K as anogenital infections, and L serotypes as lymphogranuloma venereum or LGV (genital ulcers / lesions). Notably, the L serotypes include multiple different strains, including L1, L2, L2b, and L3. Usually, in most chlamydial infections, doxycycline 100 mg / oral / twice daily / for 7 days, and in LGV, doxycycline 100 mg / oral / twice daily / for 21 days, are prescribed different treatment regimens, respectively. Therefore, differentiating the serotypes of sexual infection (D–K vs. L) is particularly important (Sexual Infections Treatment Guidelines, 2021. "Lymphogranuloma Venereum" Disease Management Prevention Center https: / / www.cdc.gov / std / treatment-guidelines / lgv.htm, 3 September 2021). The most accurate diagnosis ensures the most appropriate antibiotic therapy.

[0007] The nucleic acid sequences of the polymorphic membrane protein H (pmpH) gene of serotype / serovar 19 are described, for example, in Verweij et al., Clin Microbiol Infect (2011); 17:1717-1726 - NCBI accession numbers: A (AY184155), B (AY184156), Ba (AY184157), C (AY184158), D (AY184159), Da (AY967759), E (AY184160), Swedish variant E (SW-E; FN652779), F (AY184161), G (AY184162), H (AY184163), I (AY184164), Ia (AY967760), J (AY184165), K (AY184166), L1 (AY184167), L2 (AY184168) and L3 (AY184169).

[0008] Current methods for diagnosing LGV include clinical diagnosis, epidemiological information, and LGV-specific molecular tests (i.e., nucleic acid amplification tests or NAATs), and the molecular test is the only option that can provide a definitive diagnosis of LGV. In current molecular diagnosis of LGV, since all LGV serotypes have a unique 36-nucleotide gap, it is advantageous to target the polymorphic membrane protein H (pmpH) gene, which is not present in other serotypes (see Figure 1). For at least the foregoing reasons, there is a need for an improved method for specifically and selectively detecting serotype L of C. trachomatis in a nucleic acid sample. SUMMARY OF THE INVENTION

[0009] The present invention overcomes the foregoing drawbacks by providing a method for specifically and selectively detecting serotype L of C. trachomatis in a nucleic acid sample.

[0010] The present disclosure provides a PCR assay for specifically detecting the pmpH gene of the L serotype of C. trachomatis in biological and non-biological samples. All C. trachomatis serotypes have the pmpH gene, but the pmpH gene of the L serotype contains at least two regions that enable specific and selective detection by PCR as compared to the pmpH gene of non-L serotypes (e.g., serotypes A, B, C, D, E, F, G, I, J, and K). In one aspect, the first region of the pmpH gene of the L serotype has a unique 36 base pair (bp) deletion that can be utilized for molecular diagnosis. In another aspect, the second region, including the 3' end of the pmpH gene of the L serotype, can be additionally or alternatively utilized for molecular diagnosis.

[0011] The present disclosure relates to a method for rapidly detecting the presence or absence of the L serotype in a biological or non-biological sample by real-time PCR (RT-PCR) in a single test tube. As used herein, a method for detecting the L serotype includes performing at least one cycling step that may include an amplification step and a hybridization step. Further provided are primers, probes, and kits designed to detect the L serotype in a single tube. The detection method is designed to target the pmpH gene of the L serotype, thereby enabling detection of the L serotype in a single test.

[0012] In one aspect, a method for detecting the L serotype in a sample is provided, the method comprising providing a sample, and, when the L serotype is present in the sample, performing an amplification step that includes contacting the sample with at least one set of primers designed to target the polymorphic membrane protein H (pmpH) gene of the L serotype to produce an amplification product; performing a hybridization step that includes contacting the amplification product with at least one detectable probe that targets the pmpH gene of the L serotype when the L serotype is present in the sample; and performing a detection step that includes detecting the presence or absence of the amplification product (wherein the presence of the amplification product indicates the presence of the L serotype in the sample and the absence of the amplification product indicates the absence of the L serotype in the sample). As used herein, the at least one set of primers and the at least one detectable probe include or consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 6, and 7, or a combination thereof, a forward primer that includes or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 8, and 9, or a combination thereof, or a reverse primer that includes or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, and 11, or a probe that includes or consists of the complement thereof. In some embodiments, the forward primer includes or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2, or a combination thereof, the reverse primer includes or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 4, or a combination thereof, and the probe includes or consists of the nucleic acid sequence of SEQ ID NO: 5 or the complement thereof.In some embodiments, the forward primer comprises or consists of the nucleic acid sequences of SEQ ID NOs: 1 and 2, or a combination thereof, the reverse primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 3, the probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 5 or its complement, or the forward primer comprises or consists of the nucleic acid sequences of SEQ ID NOs: 1 and 2, or a combination thereof, the reverse primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 4, and the probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 5 or its complement. In some embodiments, the forward primer comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7, or a combination thereof, the reverse primer comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9, or a combination thereof, and the probe comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11 or their complements. In certain embodiments, the forward primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 7, the reverse primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 9, and the probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 11 or its complement. In some embodiments, the oligonucleotide primer and / or oligonucleotide probe has 50 or fewer nucleotides. In certain embodiments, the oligonucleotide primer and / or oligonucleotide probe has 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides). In some embodiments, the hybridization step comprises contacting an amplicon with a detectable probe labeled with a donor fluorophore and a corresponding acceptor moiety, and the detection step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorophore and the acceptor moiety of the probe, and the presence or absence of fluorescence indicates the presence or absence of the L serotype in the sample.In some embodiments, a polymerase enzyme having nuclease activity from 5' to 3' is used in the amplification step. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is a vaginal swab specimen, a vaginal swab specimen collected by a clinician, a cervical swab specimen, a mid-pharyngeal (throat) swab specimen, or an anorectal swab specimen. In some embodiments, the amplification step and the hybridization step are repeated. As used herein, the number of repetitions depends, for example, on the nature of the sample. If the sample is a complex mixture of nucleic acids, more amplification steps and hybridization steps are required to amplify a sufficient amount of the target sequence for detection. In some embodiments, the amplification step and the hybridization step are repeated at least about 20 times, but may be repeated at least 25, 30, 40, 50, 60, or even 100 times. Further, the detection of the presence or absence of the amplification product may be performed during or after each amplification step and hybridization step, every other amplification step and hybridization step, during or after a specific amplification step and hybridization step, or during or after a specific set of amplification steps and hybridization steps, and if present, an amplification product sufficient for detection is expected. In some embodiments, the donor fluorescent moiety and the acceptor moiety, such as a quencher, can be within 5 to 20 nucleotides (e.g., 8 or 10) of each other along the length of the probe. In some embodiments, the donor fluorescent moiety and the corresponding acceptor moiety are within 8 to 20 nucleotides of each other on the probe. In some embodiments, the acceptor moiety is a quencher. In another embodiment, the detectable probe further comprises a nucleic acid sequence that allows for the formation of a secondary structure. Such formation of the secondary structure generally results in a spatial proximity between the first fluorescent moiety and the second fluorescent moiety. According to this method, the second fluorescent moiety on the probe can be a quencher. In some embodiments, the detectable probe can be labeled with a fluorescent dye that acts as a reporter. The probe can also have a second dye that acts as a quencher.The reporter dye is measured at a defined wavelength and thus enables the detection and discrimination of the amplified pmpH gene of the L serotype. The fluorescence signal of the intact probe is suppressed by the quencher dye. During the amplification step of PCR, hybridization of the probe to a specific single-stranded DNA template results in cleavage by the 5'-3' nuclease activity of DNA polymerase, leading to separation of the reporter and quencher dyes and generation of a fluorescence signal. With each PCR cycle, the amount of cleaved probe increases, and the cumulative signal of the reporter dye increases accordingly. Optionally, one or more additional probes (e.g., internal control or other targeted probes such as other viral nucleic acids) can also be labeled with a unique reporter fluorescent dye different from the fluorescent dye label associated with the target gene probe. In such cases, since a specific reporter dye is measured at a defined wavelength, simultaneous detection and discrimination of the amplified target and one or more additional probes are possible.

[0013] In another aspect, there is provided a method for detecting the serotype (L serotype) that causes lymphogranuloma venereum of Chlamydia trachomatis in a sample, the method comprising: (a) when the L serotype is present in the sample, performing an amplification step comprising contacting the sample with at least one set of primers designed to target the 3′ end of the polymorphic membrane protein H (pmpH) gene of the L serotype to produce an amplification product; (b) when the L serotype is present in the sample, performing a hybridization step comprising contacting the amplification product with at least one detectable probe that targets the pmpH gene of the L serotype; and (c) performing a detection step comprising detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of the L serotype in the sample and the absence of the amplification product indicates the absence of the L serotype in the sample, and the at least one set of primers is designed to target a region comprising at least a part of the last 1000 nucleotides at the 3′ end of the pmpH gene of the L serotype. In some embodiments, the at least one set of primers is designed to target a region comprising at least a part of the last 500 nucleotides at the 3′ end of the pmpH gene of the L serotype. In some embodiments, the at least one set of primers is designed to target a region comprising at least a part of nucleotides 2600 to 2900 of the pmpH gene of the L1 serotype or the corresponding region of a different L serotype. In some embodiments, the at least one set of primers is designed to target a region comprising at least a part of nucleotides 2600 to 2800 of the pmpH gene of the L1 serotype or the corresponding region of a different L serotype. In some embodiments, the at least one set of primers is designed to target a region comprising at least a part of nucleotides 2635 to 2766 of the pmpH gene of the L1 serotype or the corresponding region of a different L serotype. As used herein, the sequence of the pmpH gene of the L1 serotype can be the nucleic acid shown in Genbank accession number HE601950 (gene: L1440_00934; CDS: CCP62925.1; position: 1032228..1035245, length: 3,018 nt) and further provided as SEQ ID NO: 18.In this specification, the oligonucleotide primers and / or oligonucleotide probes are selected to hybridize efficiently to the L serotype but not to other serotypes. In some embodiments, the oligonucleotide probes are selected to hybridize only to the L serotype and not to any other serotypes. In certain embodiments, the oligonucleotide probes are selected to hybridize to at least a portion of nucleotides 2660 to 2740 (SEQ ID NO: 18) of the pmpH gene of the L1 serotype or the corresponding region of a different L serotype, more specifically at least a portion of nucleotides 2670 to 2730 (SEQ ID NO: 18) of the pmpH gene of the L1 serotype or the corresponding region of a different L serotype, or even more specifically at least a portion of nucleotides 2680 to 2720 (SEQ ID NO: 18) of the pmpH gene of the L1 serotype or the corresponding region of a different L serotype. In some embodiments, the oligonucleotide primers and / or oligonucleotide probes have 50 or fewer nucleotides. In certain specific embodiments, the oligonucleotide primers and / or oligonucleotide probes have 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides). In some embodiments, the hybridization step includes contacting an amplicon with a detectable probe labeled with a donor fluorescent moiety and a corresponding acceptor moiety, and the detection step includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, and the presence or absence of fluorescence indicates the presence or absence of the L serotype in the sample. In some embodiments, a polymerase enzyme having 5'-to-3' nuclease activity is used in the amplification step. In some embodiments, the sample is a biological sample. In certain embodiments, the biological sample is a vaginal swab specimen, a vaginal swab specimen collected by a clinician, a cervical swab specimen, a mid-pharyngeal (throat) swab specimen, or an anorectal swab specimen. In some embodiments, the amplification step and the hybridization step are repeated. In this specification, the number of repetitions depends, for example, on the nature of the sample.When the sample is a complex mixture of nucleic acids, more amplification steps and hybridization steps are required to amplify a target sequence sufficient for detection. In some embodiments, the amplification and hybridization steps are repeated at least about 20 times, but may be repeated at least 25, 30, 40, 50, 60, or even 100 times. Further, detection of the presence or absence of the amplification product may be performed during or after each amplification and hybridization step, every other amplification and hybridization step, during or after a specific amplification and hybridization step, or during or after a specific set of amplification and hybridization steps, and when present, an amplification product sufficient for detection is expected. In some embodiments, the donor fluorescent moiety and the acceptor moiety, e.g., quencher, can be within 5 to 20 nucleotides (e.g., 8 or 10) of each other along the length of the probe. In some embodiments, the donor fluorescent moiety and the corresponding acceptor moiety are within 8 to 20 nucleotides of each other on the probe. In some embodiments, the acceptor moiety is a quencher. In another embodiment, the detectable probe further comprises a nucleic acid sequence that allows for the formation of a secondary structure. Such formation of the secondary structure generally results in a spatial proximity between the first fluorescent moiety and the second fluorescent moiety. According to this method, the second fluorescent moiety on the probe can be a quencher. In some embodiments, the detectable probe can be labeled with a fluorescent dye that acts as a reporter. The probe can also have a second dye that acts as a quencher. The reporter dye is measured at a defined wavelength and thus allows for the detection and discrimination of the amplified pmpH gene of the L serotype. The fluorescent signal of the intact probe is suppressed by the quencher dye. During the amplification step of PCR, hybridization of the probe to a specific single-stranded DNA template results in cleavage by the 5'-3' nuclease activity of DNA polymerase, leading to separation of the reporter and quencher dyes and generation of a fluorescent signal.With each PCR cycle, the amount of cleaved probe increases, and the cumulative signal of the reporter dye increases accordingly. Optionally, one or more additional probes (e.g., internal reference controls or other targeted probes such as (e.g., other viral nucleic acids) can also be labeled with a unique reporter fluorescent dye different from the fluorescent dye label associated with the target gene probe. In such cases, since a specific reporter dye is measured at a defined wavelength, simultaneous detection and discrimination of the amplified target and one or more additional probes are possible.

[0014] In another aspect, a kit for detecting the L serotype is provided. A kit for detecting the pmpH gene of the lymphogranuloma venereum serotype (L serotype) of Chlamydia trachomatis in a sample is provided, the kit comprising: (a) a DNA polymerase having 5'-3' nuclease activity; (b) nucleotide monomers; and (c) (i) a forward primer comprising the nucleic acid sequences of SEQ ID NOs: 1, 2, 6, and 7 or any combination thereof, (ii) a reverse primer comprising the nucleic acid sequences of SEQ ID NOs: 3, 4, 8, and 9 or a combination thereof, and (iii) a detectable probe comprising the nucleic acid sequences of SEQ ID NOs: 5, 10, and 11 or their complements, comprising at least one pair of primers and at least one detectable probe. In some embodiments, the forward primer comprises, consists of, or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2, or a combination thereof; the reverse primer comprises, consists of, or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 4, or a combination thereof; and the probe comprises, consists of, or consists of the nucleic acid sequence of SEQ ID NO: 5 or its complement. In some embodiments, the forward primer comprises, consists of, or consists of the nucleic acid sequences of SEQ ID NOs: 1 and 2, or a combination thereof; the reverse primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 3; and the probe comprises, consists of, or consists of the nucleic acid sequence of SEQ ID NO: 5 or its complement, or the forward primer comprises, consists of, or consists of the nucleic acid sequences of SEQ ID NOs: 1 and 2, or a combination thereof; the reverse primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 4; and the probe comprises, consists of, or consists of the nucleic acid sequence of SEQ ID NO: 5 or its complement. In some embodiments, the forward primer comprises, consists of, or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7, or a combination thereof; the reverse primer comprises, consists of, or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9, or a combination thereof; and the probe comprises, consists of, or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11 or their complements, or a combination thereof.In certain embodiments, the forward primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 7, the reverse primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 9, and the probe comprises or consists of the nucleic acid sequence of SEQ ID NO: 11 or its complement. In some embodiments, the oligonucleotide primer and / or oligonucleotide probe have 50 or fewer nucleotides. In certain embodiments, the oligonucleotide primer and / or oligonucleotide probe have 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides). In some embodiments, the kit can include a probe already labeled with a donor and a corresponding acceptor fluorophore, or can include a fluorogenic moiety for labeling the probe. In some embodiments, the detectable probe is labeled with a donor fluorophore and a corresponding acceptor moiety. In some embodiments, the kit can also include nucleoside triphosphates, a nucleic acid polymerase, and a buffer necessary for the function of the nucleic acid polymerase. In some embodiments, the kit can also include an instruction sheet and instructions for using the primers, probes, and fluorescent dye moieties to detect the presence or absence of L serotype in a sample.

[0015] In another aspect, an oligonucleotide is provided that comprises or consists of a nucleic acid sequence selected from SEQ ID NOs: 1-11 or their complements. In some embodiments, the oligonucleotide can have 50 or fewer nucleotides. In certain embodiments, the oligonucleotide can have 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides). In some embodiments, the oligonucleotide can be used as an oligonucleotide primer and / or an oligonucleotide probe. In some embodiments, the oligonucleotide comprises at least one modified nucleotide, for example, to alter nucleic acid hybridization stability as compared to unmodified nucleotides. In some embodiments, at least one modified nucleotide is selected from the group consisting of N6-benzyl-dA, N4-benzyl-dC, N6-parater-butyl-benzyl-dA, and N4-parater-butyl-benzyl-dC. Optionally, the oligonucleotide comprises at least one label and / or at least one quencher moiety. In some embodiments, the oligonucleotide comprises at least one conservatively modified variation. A "conservatively modified variation" or simply "conservative variation" of a particular nucleic acid sequence refers to a nucleic acid that encodes the same or substantially the same amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, to a substantially the same sequence. One of ordinary skill in the art will recognize that individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a low percentage of amino acids (typically less than 5%, more typically less than 4%, 2%, or 1%) in the encoded sequence are "conservatively modified variations" where the alteration results in the deletion, addition, or substitution of an amino acid with a chemically similar amino acid. In some embodiments, the oligonucleotide is provided as a set of oligonucleotides.In this specification, a set of oligonucleotides may include (i) a forward primer comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 1, 2, 6, and 7, or any combination thereof, and (ii) a reverse primer comprising or consisting of the nucleic acid sequences of SEQ ID NOs: 3, 4, 8, and 9, or a combination thereof. In certain embodiments, the set of oligonucleotides further includes (iii) a detectable probe comprising the nucleic acid sequences of SEQ ID NOs: 5, 10, and 11, or a complement thereof. In some embodiments, the forward primer comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2, or a combination thereof, and the reverse primer comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 4, or a combination thereof. As used herein, in certain embodiments, the probe may comprise or consist of the nucleic acid sequence of SEQ ID NO: 5, or a complement thereof. In some embodiments, the forward primer comprises or consists of the nucleic acid sequences of SEQ ID NOs: 1 and 2, or a combination thereof, and the reverse primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 3. As used herein, in certain embodiments, the probe may comprise or consist of the nucleic acid sequence of SEQ ID NO: 5, or a complement thereof. In some embodiments, the forward primer comprises or consists of the nucleic acid sequences of SEQ ID NOs: 1 and 2, or a combination thereof, and the reverse primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 4. As used herein, in certain embodiments, the probe may comprise or consist of the nucleic acid sequence of SEQ ID NO: 5, or a complement thereof. In some embodiments, the forward primer comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7, or a combination thereof, and the reverse primer comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9, or a combination thereof. As used herein, in certain embodiments, the probe may comprise or consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11, or a complement thereof.In certain embodiments, the forward primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 7, and the reverse primer comprises or consists of the nucleic acid sequence of SEQ ID NO: 9. As used herein, in certain embodiments, the probe may comprise or consist of the nucleic acid sequence of SEQ ID NO: 11, or its complement. In some embodiments, at least one of the primers and / or detectable probes targeting the pmpH gene of the L serotype comprises at least one of the modified nucleotides detailed above.

[0016] In another aspect, determination of the presence of the L serotype nucleic acid of Chlamydia trachomatis in the methods and kits provided above can be combined with determination of the presence of all serotype nucleic acids of Chlamydia trachomatis. As used herein, the oligonucleotide sets disclosed above for use in the methods and kits above can be combined with an oligonucleotide set comprising at least forward and reverse oligonucleotide primers that amplify and detect all serotypes of Chlamydia trachomatis (CT) and at least one oligonucleotide probe. In some embodiments, the oligonucleotide set comprises at least one pan-CT forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13, or a combination thereof, at least one pan-CT reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14 and 15, or a combination thereof, and at least one pan-CT probe comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 17, or a complement thereof. In certain embodiments, the amplification, hybridization, and detection steps using the set of pan-CT oligonucleotides are performed in a parallel reaction using a second portion of the sample. In an alternative embodiment, the amplification, hybridization, and detection steps using the set of pan-CT oligonucleotides are performed in a multiplex reaction within the same vial as the reaction for determining the L serotype. As used herein, the fluorophore (or donor-acceptor pair) for labeling the pan-CT oligonucleotide probe is different from the fluorophore (or donor-acceptor pair) for labeling the L serotype-specific oligonucleotide probe.More specifically, the hybridization step may include contacting the amplification product with two or more probes labeled with different donor fluorescent moieties (e.g., a first and a second donor fluorescent moieties) and corresponding acceptor moieties, and the detection step includes detecting, for both the different donor fluorescent moieties and the corresponding acceptor moieties, the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety of the probe (e.g., the first and second donor fluorescent moieties) and the acceptor moiety, and the presence or absence of fluorescence indicates the presence or absence of Chlamydia trachomatis (CT) serovar L and / or the presence or absence of any Chlamydia trachomatis (CT) serovar in the sample.

[0017] The present disclosure also provides methods for detecting the presence or absence of L serotype (i.e., pmpH gene having a 36-base deletion) nucleic acid in a biological sample derived from an individual. Using these methods, the presence or absence of L serotype or L serotype nucleic acid in a biological sample such as a vaginal swab specimen, a vaginal swab specimen collected by a clinician, a cervical swab specimen, a mid-pharyngeal (throat) swab specimen, and an anorectal swab specimen, or other biological materials where L serotype is thought to be present, can be detected for use in diagnostic tests. Further, the same tests may be used by those skilled in the art to evaluate other sample types for detecting L serotype nucleic acid. Such methods generally involve performing at least one cycling step that includes an amplification step and either a detectable probe hybridization step or a dye binding step. Typically, the amplification step includes contacting the sample with at least one pair of oligonucleotide primers to generate one or more amplification products if nucleic acid molecules are present in the sample, the probe hybridization step includes contacting the amplification products with one or more detectable probes specific for the amplification products, and the dye binding step includes contacting the amplification products with a double-stranded DNA binding dye. Such methods also include detecting the presence or absence of binding of the double-stranded DNA binding dye to the amplification products, the presence of binding indicating the presence of L serotype nucleic acid in the sample, and the absence of hybridization indicating the absence of L serotype or L serotype nucleic acid in the sample. A representative double-stranded DNA binding dye is ethidium bromide. Other nucleic acid binding dyes include DAPI, Hoechst dyes, PicoGreen®, RiboGreen®, OliGreen®, and cyanine dyes such as YO-YO® and SYBR® Green. Further, such methods may also include determining the melting temperature between the amplification products and the double-stranded DNA binding dye, and the melting temperature is used to confirm the presence or absence of L serotype or L serotype nucleic acid.

[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the subject matter, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0019] Details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the drawings and the detailed description of the embodiments for carrying out the invention, as well as from the claims.

Brief Description of the Drawings

[0020]

Figure 1

Figure 2

BEST MODE FOR CARRYING OUT THE INVENTION

[0021] DETAILED DESCRIPTION I. Definitions In this application, unless otherwise clear from the context: (i) the term "a" can be understood to mean "at least one"; (ii) the term "or" can be understood to mean "and / or"; (iii) the terms "comprising" and "including" can be understood to include the listed components or steps by themselves or together with one or more additional components or steps, regardless of whether or not they are present; (iv) the terms "about" and "approximately" can be understood to allow for standard variations as would be understood by one of ordinary skill in the art; and (v) when ranges are provided, endpoints are included.

[0022] As used herein, the term "amplifying" or "amplification" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid molecule (e.g., the pmpH gene from the L serotype of C. trachomatis). Amplifying a nucleic acid molecule typically involves denaturing the template nucleic acid, annealing primers to the template nucleic acid at a temperature below the melting temperature of the primers, and enzymatically extending from the primers to generate an amplification product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase enzyme (e.g., Platinum® Taq), and an appropriate buffer and / or cofactor for optimal activity of the polymerase enzyme (e.g., MgCl2 and / or KCl).

[0023] Approximately: As used herein, the term "about" or "approximately" when applied to one or more values of interest refers to a value similar to the recited reference value. In certain embodiments, the term "about" or "approximately" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater than or less than) of the recited reference value, unless otherwise specified or otherwise apparent from the context (except where such numbers would exceed 100% of the possible values).

[0024] Associated with: As used herein, two events or entities are "associated with" each other if the presence, level, and / or form of one event or entity correlates with the presence, level, and / or form of the other. For example, a particular entity (e.g., a polypeptide, gene signature, metabolite, etc.) is considered to be associated with a particular disease, disorder, or condition if its presence, level, and / or form correlates with (e.g., throughout a relevant population) the incidence and / or susceptibility to such disease, disorder, or condition. In some embodiments, two or more entities are physically "associated with" each other if they interact directly or indirectly, such that they are physically proximate to and / or remain proximate to each other. In some embodiments, two or more entities that are physically associated with each other are covalently bonded to each other, and in some embodiments, two or more entities that are physically associated with each other are not covalently bonded to each other but are non-covalently bonded, e.g., by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0025] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained or derived from a biological source of interest (e.g., tissue or organism or cell culture) as described herein. In some embodiments, the source of interest includes or consists of an organism, such as an animal or a human. In some embodiments, the biological sample comprises or consists of a biological tissue or a body fluid. In some embodiments, the biological sample may be or may include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy sample; cell-containing body fluid; floating nucleic acid; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph fluid; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; lavage or wash fluid such as ductal lavage or bronchoalveolar lavage; aspirate; scrape; bone marrow material; tissue biopsy material; surgical material; other body fluids, secretions and / or excretions; and / or cells derived from or including these, etc. In some embodiments, the biological sample comprises or consists of cells obtained from an individual. In some embodiments, the obtained cells are cells derived from or including cells of the individual from whom the sample was obtained. In some embodiments, the sample is a "primary sample" obtained directly from the source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid (e.g., blood, lymph fluid, feces, etc.). In some embodiments, as apparent from the context, the term "sample" refers to a preparation obtained by processing a primary sample (e.g., by removing one or more components and / or by adding one or more agents). For example, filtration using a semipermeable membrane. Such a "processed sample" may contain, for example, nucleic acids or proteins obtained by subjecting the primary sample to techniques such as extraction from the sample or amplification or reverse transcription of mRNA, isolation and / or purification of certain components.

[0026] The term "its complement" refers to a nucleic acid that is the same length as a given nucleic acid and is exactly complementary.

[0027] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open-ended and means that while the named element or step is essential, other elements or steps may be added within the scope of the composition or method. It is understood that a composition or method described as "including" (or "comprising") one or more named elements or steps also describes a corresponding more limited composition or method "consisting essentially of" (or "consisting essentially of") the same named elements or steps, meaning that the composition or method includes the named essential element or step and may also include additional elements or steps that do not substantially affect the basic and novel features(s) of the composition or method. Any composition or method described herein as "including" or "consisting essentially of" one or more named elements or steps also describes a corresponding, more limited, and closed-ended composition or method "consisting of" (or "consisting of") the named elements or steps, excluding other unnamed elements or steps. In any composition or method disclosed herein, a known or disclosed equivalent of any named essential element or step can be used in place of that element or step.

[0028] Designed: As used herein, the term "designed" refers to (i) an agent whose structure has been selected by human hand, or a selected agent, (ii) an agent produced by a process that requires human hand, and / or (iii) an agent that is different from natural substances and other known agents.

[0029] To determine: One of ordinary skill in the art reading this specification will understand that "to determine" can be accomplished by utilizing any of a variety of techniques available to the skilled artisan, including, for example, the specific techniques expressly recited in this specification. In some embodiments, to determine includes the manipulation of physical samples. In some embodiments, to determine includes the consideration and / or manipulation of data or information, e.g., utilizing a computer or other processing device adapted to perform relevant analysis. In some embodiments, to determine includes receiving relevant information and / or materials from an information source. In some embodiments, to determine includes comparing one or more characteristics of a sample or entity to a comparable reference.

[0030] The terms "extension" or "elongation," when used with respect to a nucleic acid, refer to the incorporation of additional nucleotides (or other similar molecules) into the nucleic acid. For example, a nucleic acid is optionally extended by nucleotides that incorporate a biocatalyst, such as a polymerase, that adds nucleotides to the 3' end of the nucleic acid.

[0031] The terms "hybridize" or "hybridization" refer to the annealing of one or more probes to an amplification product. Hybridization conditions typically include a temperature that is lower than the melting temperature of the probe, but that avoids non-specific hybridization of the probe.

[0032] Identity: As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules), and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. For example, the calculation of percent identity between two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment. Non-identical sequences can be ignored for comparison). In certain embodiments, the length of the aligned sequences for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. Next, the nucleotides at the corresponding positions are compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecule is identical at that position. The percent identity between two sequences is a function of the number of gaps and the number of identical positions shared by the sequences taking into account the length of each gap required to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4:11-17) incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the comparison of nucleic acid sequences performed using the ALIGN program uses the PAM120 weight residue table, a 12 gap length penalty, and a 4 gap penalty.Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program of the GCG software package with the NWSgapdna.CMP matrix.

[0033] "Modified nucleotide" in the context of an oligonucleotide refers to a change in which at least one nucleotide of the oligonucleotide sequence is replaced by a different nucleotide that provides a desired property to the oligonucleotide. Exemplary modified nucleotides that can be substituted in the oligonucleotides described herein include, for example, C5-methyl-dC, C5-ethyl-dC, C5-methyl-dU, C5-ethyl-dU, 2,6-diaminopurine, C5-propynyl-dC, C5-propynyl-dU, C7-propynyl-dA, C7-propynyl-dG, C5-propargylamino-dC, C5-propargylamino-dU, C7-propargylamino-dA, C7-propargylamino-dG, 7-deaza-2-deoxyxanthosine, pyrazolopyrimidine analogs, pseudodU, nitropyrrole, nitroindole, 2'-O-methylriboU, 2'-O-methylriboC, N4-ethyl-dC, N6-methyl-dA, and the like. Many other modified nucleotides that can be substituted in an oligonucleotide are mentioned herein or are known in the art. In certain embodiments, the modified nucleotide substitution modifies the melting temperature (Tm) of the oligonucleotide as compared to the melting temperature of the corresponding unmodified oligonucleotide. Further, some modified nucleotide substitutions can, in some embodiments, reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation, etc.) and increase the yield of the intended target amplicon. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is hereby incorporated by reference herein. Examples of these types of nucleic acid modifications are described, for example, in U.S. Patent No. 6,001,611, which is hereby incorporated by reference herein.

[0034] As used herein, the term "primer" is known to those skilled in the art and refers to an oligomeric compound, primarily an oligonucleotide, as well as a modified oligonucleotide that can "prime" DNA synthesis by a template-dependent DNA polymerase, i.e., for example, the 3' end of the oligonucleotide provides a free 3'-OH group to which "nucleotides" are further attached by a template-dependent DNA polymerase to establish a 3'-to-5' phosphodiester bond, whereby deoxynucleoside triphosphates are used and pyrophosphate is released. Thus, there is probably no fundamental difference between a "primer", an "oligonucleotide", or a "probe" except perhaps for the intended function.

[0035] Sample: As used herein, the term "sample" refers to a substance that is or contains a composition of interest for qualitative and / or quantitative evaluation. In some embodiments, the sample is a biological sample (i.e., derived from a living organism (e.g., a cell or an organism)). In some embodiments, the sample is derived from a geological resource, a water resource, an astronomical resource, or an agricultural resource. In some embodiments, the source of interest comprises or consists of a living organism, such as an animal or a human. In some embodiments, a sample for forensic analysis is or comprises a biological tissue, a body fluid, an organic substance, or a non-organic substance such as clothing, dirt, plastic, water, etc. In some embodiments, an agricultural sample comprises or consists of an organic substance such as a leaf, a petal, a bark, a wood, a seed, a plant, a fruit, etc.

[0036] Specificity: As used herein, the term "specificity" means the preference of an enzyme for a particular substrate.

[0037] Selectivity: As used herein, the term "selectivity" means the preference of an enzyme for a particular substrate over another substrate.

[0038] Substantially: As used herein, the term "substantially" refers to a qualitative condition presenting an overall or nearly overall degree or extent of a feature or characteristic of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, complete and / or proceed to completion, or achieve or avoid absolute results. Therefore, the term "substantially" is used herein to capture the possibility of lack of completion inherent in many biological and chemical phenomena.

[0039] Synthetic: As used herein, the word "synthetic" means being in a form that does not exist in nature, being produced by human hand and thus having a structure that does not exist in nature, or being associated with one or more other components that do not exist in nature or are not associated in nature, or not being associated with one or more other components that are associated in nature.

[0040] The term "thermostable polymerase" refers to a polymerase enzyme that is thermostable, i.e., the enzyme does not irreversibly denature when exposed to high temperatures for the time required to effect the formation of primer extension products complementary to the template and to cause denaturation of double-stranded template nucleic acids. Generally, synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases are isolated from, for example, Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nevertheless, non-thermostable polymerases can be used in PCR assays if the enzyme is replenished.

[0041] The term "5'-to-3' nuclease activity" typically refers to the activity of a nucleic acid polymerase that is associated with nucleic acid strand synthesis and, thereby, removes nucleotides from the 5' end of a nucleic acid strand.

[0042] II. Detailed Description of Certain Embodiments Diagnosis of the serotype (L serotype) of Chlamydia trachomatis that causes lymphogranuloma venereum (LGV) by nucleic acid amplification provides a method for rapidly and accurately detecting bacterial infections. An RT-PCR assay for detecting the L serotype in a sample is described herein. Primers and probes for detecting the pmpH gene of the L serotype of C. trachomatis are provided, and products or kits containing such primers and probes are also provided. The increased sensitivity of RT-PCR for detecting the L serotype, as well as the improved features of RT-PCR including sample containment and real-time detection of amplification products, compared to other methods, enable the implementation of this technology for the routine diagnosis of infections caused by the L serotype of C. trachomatis that causes LGV in clinical laboratories.

[0043] The L serotype of C. trachomatis is a pathogen that has emerged in the industry for men who have sex with men, often in association with HIV coinfection. The L serotype causes acute disease and can persist for long periods, and if untreated, may promote the spread of HIV. Clinical care, surveillance, and research are significantly hampered by the lack of a widely available, rapid, and standardized test for the diagnosis of LGV.

[0044] Recently, a number of PCR-based test systems have been published that facilitate the identification of the L serotype associated with LGV. Some of these have addressed the detection and typing of almost all serotypes of C. trachomatis in a given sample. Distinguishing the L serotype from other serotypes of C. trachomatis is primarily clinically important because different antibiotic treatment regimens are required. Detection is often based on amplification of the pmpH gene, and unique deletions in the L serotype facilitate the design of L serotype-specific PCR.

[0045] The present disclosure provides a method for detecting the L serotype of C. trachomatis, for example, by amplifying the first region or the second region of the pmpH nucleic acid sequence derived from the L serotype (see FIG. 2). Nucleic acid sequences of the pmpH gene from various serotypes of C. trachomatis are available (e.g., GenBank accession number AY184167 from serotype L1, AY184168 from serotype L2, AY184169 for serotype L3). Further, as described above, the sequence of the pmpH gene of the L1 serotype can be the nucleic acid shown in Genbank accession number HE601950 (gene: L1440_00934, CDS: CCP62925.1, position: 1032228..1035245, length: 3,018 nt). Specifically, primers and probes for amplifying and detecting the pmpH gene from an L serotype nucleic acid molecular target are provided by embodiments of the present disclosure.

[0046] The present disclosure is based, at least in part, on the unexpected discovery that the pmpH gene provides at least two regions that are useful for selectively and specifically detecting the L serotype of C. trachomatis as compared to other serotypes that cause non-LGV. As shown in FIG. 2 herein, region R1, which is the region from 400 to 600 nucleotides from the 5' end of the pmpH gene, contains a 36 bp deletion in the L serotype (marked as a gray box in the L serotype), while all other serotypes lack this deletion. Thus, in a first aspect, a set of oligonucleotide primers is arranged to hybridize to the R1 region to amplify this target region (the gray triangle in FIG. 2), while an oligonucleotide probe that hybridizes across the deletion site is selected. Using this setup, the oligonucleotide probe that spans the deletion site hybridizes exclusively to the deletion site that is present only in the L serotype, and thus, a signal is detected only for the L serotype (L1, L2, L2b, L3).

[0047] In a second aspect, unexpectedly, a second region, region R2 shown in FIG. 2, which is in the region close to 2600 to 2900 nucleotides from the 3′ end of the pmpH gene and the 5′ end of the pmpH gene, was identified (in some aspects, 2600 to 2800 nucleotides from the 5′ end of the pmpH gene). As used herein, the identification of the L serotype does not depend on the determination of specific deletions that are only present in the L serotype. Rather, region R2 provides various nucleotides that differ between the sequence of the L serotype and the sequences of all other serotypes. As used herein, oligonucleotide primers and / or oligonucleotide probes are selected to hybridize efficiently to the L serotype but not to other serotypes. In particular, the oligonucleotide probe can be selected to hybridize only to the L serotype and not to any of the other serotypes (see the triangles in FIG. 2 indicating useful hybridization sites for oligonucleotide primer sets and / or oligonucleotide probes). As used herein, the oligonucleotide probe can be selected to hybridize to at least a portion of nucleotides 2660 to 2740 of the pmpH gene of the L1 serotype or the corresponding region of a different L serotype, more specifically at least a portion of nucleotides 2670 to 2730 of the pmpH gene of the L1 serotype or the corresponding region of a different L serotype, or even more specifically at least a portion of nucleotides 2680 to 2720 of the pmpH gene of the L1 serotype or the corresponding region of a different L serotype.

[0048]

Table 1

[0049] More specifically, as shown in Table 1, suitable primer and probe oligonucleotides include nucleic acids having sequences selected from SEQ ID NOs: 1 to 11, substantially identical variants thereof having at least, for example, 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 1 to 11, or the complements of SEQ ID NOs: 1 to 11, and variants, respectively. In the present specification, primer and probe oligonucleotides that hybridize to region R1 are provided, each including a nucleic acid having a sequence selected from SEQ ID NOs: 1 to 5, substantially identical variants thereof having at least, for example, 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 1 to 5, or the complements of SEQ ID NOs: 1 to 5, and variants. Further, primer and probe oligonucleotides that hybridize to region R2 are also provided, each including a nucleic acid having a sequence selected from SEQ ID NOs: 6 to 11, substantially identical variants thereof having at least, for example, 80%, 90%, or 95% sequence identity to one of SEQ ID NOs: 6 to 11, or the complements of SEQ ID NOs: 6 to 11, and variants.

[0050] In one embodiment, the set of primers and probes described in Table 1 is used to provide detection of one or more L serotypes of C. trachomatis in a biological sample suspected of containing the one or more L serotypes. The set of primers and probes can include or consist of primers and probes specific for the pmpH gene derived from the L serotype nucleic acid sequence that includes or consists of the nucleic acid sequences of SEQ ID NOs: 1 to 11.

[0051] Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1 to 11 can be identified by using the primers and / or probes in the disclosed methods. Functionally active variants of any of the primers and / or probes of SEQ ID NOs: 1 to 11 relate to primers and / or probes that provide similar or higher specificity and sensitivity compared to the respective sequences of SEQ ID NOs: 1 to 11 in the described methods or kits.

[0052] The variant can vary from the sequences of SEQ ID NOs: 1 to 11 by, for example, addition, deletion, or substitution of one or more nucleotides at the 5'-end and / or 3'-end of each of the sequences of SEQ ID NOs: 1 to 11. As detailed above, the primer (and / or probe) may be chemically modified, i.e., the primer and / or probe may contain a modified nucleotide or a non-nucleotide compound. Thus, the probe (or primer) is a modified oligonucleotide. A "modified nucleotide" (or "nucleotide analog") differs from a natural "nucleotide" by several modifications, but still consists of a base or base-like compound, a pentofuranosyl sugar or pentofuranosyl sugar-like compound, a phosphate moiety or phosphate-like moiety, or a combination thereof. For example, a "label" can be attached to the base portion of a "nucleotide" to thereby obtain a "modified nucleotide". The natural base in a "nucleotide" may also be replaced, for example, by 7-deazapurine, thereby also obtaining a "modified nucleotide". The terms "modified nucleotide" or "nucleotide analog" are used interchangeably in the present application. A "modified nucleoside" (or "nucleoside analog") differs from a natural nucleoside by several modifications as outlined above for "modified nucleotides" (or "nucleotide analogs").

[0053] Furthermore, in some applications, it may be beneficial to combine the determination of the presence of nucleic acids of the L serotype of Chlamydia trachomatis with the determination of the presence of nucleic acids of all serotypes of Chlamydia trachomatis. This approach may be particularly beneficial when using the setup of FIG. 2 above, either by detecting the deletion of the L serotype in region R1 using a probe spanning the deletion, or by detecting the L serotype using oligonucleotide primers and / or oligonucleotide probes that hybridize within region R2 of the L serotype but not to other serotypes. As used herein, a signal is detected only for the presence of the L serotype and no signal is detected for the presence of all other serotypes. However, the absence of a signal may also be caused by a failure of the amplification / detection run and thus may result in false negative results. For this reason, oligonucleotide primers and probes that enable the determination of all serotypes of Chlamydia trachomatis (CT) are also provided herein. As shown in Table 1, at least one pan-CT forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 12 and 13, or a combination thereof, at least one pan-CT reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 14 and 15, or a combination thereof, and at least one pan-CT probe comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 17, or a complement thereof, are provided. It is disclosed herein that the amplification and detection reactions using the pan-CT oligonucleotides can be carried out using a second portion of the sample in a parallel reaction. However, it is also possible to carry out the amplification and detection reactions using the pan-CT oligonucleotides in a multiplex reaction within the same vial as the reaction for determining the L serotype. As used herein, the fluorophore (or donor-acceptor pair) for labeling the pan-CT oligonucleotide probe is different from the fluorophore (or donor-acceptor pair) for labeling the L serotype-specific oligonucleotide probe.More specifically, the hybridization step may include contacting the amplification product with two or more probes labeled with different donor fluorescent moieties (e.g., the first and second donor fluorescent moieties) and corresponding acceptor moieties, and the detection step includes, for both the different donor fluorescent moieties and the corresponding acceptor moieties, detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety (e.g., the first and second donor fluorescent moieties) of the probe and the acceptor moiety, and the presence or absence of fluorescence indicates the presence or absence of Chlamydia trachomatis (CT) L serotype and / or the presence or absence of any of the Chlamydia trachomatis (CT) serotypes in the sample.

[0054] Oligonucleotides comprising modified oligonucleotides and oligonucleotide analogs that amplify nucleic acid molecules derived from the pmpH gene of the L serotype nucleic acid sequence can be designed using a computer program such as OLIGO (Molecular Biology Insights Inc., Cascade, Colorado), for example. Important features in designing oligonucleotides for use as amplification primers include an appropriately sized amplification product to facilitate detection (e.g., by electrophoresis), a similar melting temperature for members of a pair of primers, and the length of each primer (i.e., the primer needs to be long enough to anneal with sequence specificity and initiate synthesis, but not so long that the fidelity decreases during oligonucleotide synthesis.), but are not limited thereto. Typically, the oligonucleotide primer is 8 to 50 nucleotides in length (e.g., 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, or 50 nucleotides in length).

[0055] In addition to the primer set, the method may use one or more probes to detect the presence or absence of the L serotype. The term "probe" refers to a synthetic or biologically produced nucleic acid (DNA or RNA), which, by design or selection, contains a specific nucleotide sequence that enables it to hybridize specifically (i.e., preferentially) under defined stringency conditions to, in this case, the pmpH gene derived from the L serotype nucleic acid. The "probe" may be referred to as a "detection probe" which means detecting the target nucleic acid.

[0056] In some embodiments, the probe can be labeled with at least one fluorescent label. In one embodiment, the probe for the pmpH gene derived from the L serotype can be labeled with a donor fluorescent moiety, such as a fluorescent dye, and a corresponding acceptor fluorescent moiety, such as a quencher. In one embodiment, the probe comprises or consists of a fluorescent moiety and the nucleic acid sequence comprises or consists of SEQ ID NO: 5. In another embodiment, the probe comprises or consists of a fluorescent moiety and the nucleic acid sequence comprises or consists of SEQ ID NO: 11.

[0057] The design of the oligonucleotide used as a probe can be carried out in the same manner as the design of the primer. In embodiments, a single probe or a pair of probes may be used for the detection of the amplification product. Depending on the embodiment, the probe(s) used may contain at least one label and / or at least one quencher moiety. Similar to the primer, the probe usually has a similar melting temperature and the length of each probe should be long enough for sequence-specific hybridization to occur but not so long as to reduce fidelity during synthesis. Oligonucleotide probes generally have a length of 15 to 30 (e.g., 16, 18, 20, 21, 22, 23, 24, or 25) nucleotides.

[0058] A construct containing a target gene nucleic acid molecule can be propagated in a host cell. As used herein, the term host cell means including prokaryotes and eukaryotes, such as yeast, plant, and animal cells. Prokaryotic hosts can include Escherichia coli (E. coli), Salmonella typhimurium, Serratia marcescens, and Bacillus subtilis. Eukaryotic hosts include yeasts such as S. cerevisiae, S. pombe, Pichia pastoris, mammalian cells such as COS cells or Chinese hamster ovary (CHO) cells, insect cells, as well as plant cells such as Arabidopsis thaliana and Nicotiana tabacum. The construct can be introduced into the host cell using any of the techniques generally known to those skilled in the art. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and virus-mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells. In addition, naked DNA can be directly delivered into cells (see, for example, U.S. Patent Nos. 5,580,859 and 5,589,466).

[0059] U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188 disclose conventional PCR techniques. PCR typically uses two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides (e.g., SEQ ID NOs: 1-4) that can act as starting points for nucleic acid synthesis within the described target nucleic acid sequences. Primers can be purified from restriction digests by conventional methods or can be generated synthetically. Primers are preferably single-stranded for maximum efficiency in amplification, although primers can be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double-stranded nucleic acids is by heating.

[0060] If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be achieved by any suitable denaturation method, including physical, chemical, or enzymatic means. One method of separating nucleic acid strands involves heating until the nucleic acid is predominantly denatured (e.g., denatured by more than 50%, 60%, 70%, 80%, 90%, or 95%). The heating conditions required to denature the template nucleic acid will depend, for example, on the buffer salt concentration and the length and nucleotide composition of the nucleic acid being denatured, but will typically be in the range of about 90°C to about 105°C, depending on the characteristics of the reaction, such as temperature and nucleic acid length. Denaturation is typically carried out for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0061] When the double-stranded template nucleic acid is denatured by heat, the reaction mixture is cooled to a temperature that promotes annealing of each primer to its target sequence on the disclosed target gene nucleic acid molecule. The annealing temperature is typically about 35°C to about 65°C (e.g., about 40°C to about 60°C, about 45°C to about 50°C). The annealing time can be about 10 seconds to about 1 minute (e.g., about 20 seconds to about 50 seconds, about 30 seconds to about 40 seconds). The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature sufficient for extension to occur from the annealed primers to produce a product complementary to the template nucleic acid. The temperature must be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template but should not be so high as to denature the extension product from its complementary template (e.g., the temperature for extension is generally in the range of about 40°C to about 80°C (e.g., about 50°C to about 70°C, about 60°C)). The extension time can be about 10 seconds to about 5 minutes (e.g., about 30 seconds to about 4 minutes, about 1 minute to about 3 minutes, about 1 minute 30 seconds to about 2 minutes).

[0062] PCR assays can use template nucleic acids such as RNA or DNA (cDNA). The template nucleic acid need not be purified and can be a minor fraction of a complex mixture such as C. trachomatis nucleic acid contained in human cells. C. trachomatis nucleic acid molecules can be extracted from biological samples by routine techniques such as those described in Diagnostic Molecular Microbiology: Principles and Applications (Persing et al., (eds.), 1993, American Society for Microbiology, Washington D.C.). Nucleic acids can be obtained from any number of sources, e.g., plasmids, or natural sources including bacteria, yeast, viruses, organelles, or higher organisms such as plants or animals.

[0063] Oligonucleotide primers (e.g., SEQ ID NOs: 1-4 and 6-9) are combined with PCR reagents under reaction conditions that induce primer extension. For example, a chain extension reaction generally contains 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5-1.0 μg of denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U of Taq polymerase, and 10% DMSO. The reaction generally contains 150-320 μM each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0064] The newly synthesized strands form double-stranded molecules that can be used in subsequent steps of the reaction. To generate the desired amount of amplification product corresponding to the target gene nucleic acid molecule, the steps of strand separation, annealing, and extension can be repeated as many times as necessary. The limiting factors of the reaction are the amounts of primer, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling step (i.e., denaturation, annealing, and extension) is preferably repeated at least once. The number of cycling steps depends, for example, on the nature of the sample for detection. If the sample is a complex mixture of nucleic acids, more cycling steps are required to amplify a sufficient amount of the target sequence for detection. Generally, the cycling step is repeated at least about 20 times, but may be repeated 40, 60, or even 100 times.

[0065] FRET technology (e.g., U.S. Patent Nos. 4,996,143, 5,565,322, 5,849,489, 6,162,603) is based on the concept that energy transfer occurs between two fluorescent moieties such that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are placed within a certain distance of each other, they can be visualized or otherwise detected and / or quantified. Typically, when the donor is excited by light radiation of a suitable wavelength, it transfers energy to the acceptor. Typically, the acceptor re-emits the transferred energy in the form of light radiation of a different wavelength. In certain systems, non-fluorescent energy can be transferred between the donor and acceptor moieties through a biomolecule that includes a substantially non-fluorescent donor moiety (see, e.g., U.S. Patent No. 7,741,467).

[0066] In one example, an oligonucleotide probe can contain a donor fluorescent moiety and a corresponding quencher that may or may not be fluorescent and that dissipates energy transferred in a form other than light. When the probe is intact, energy transfer typically occurs between two fluorescent moieties, resulting in quenching of fluorescence emission from the donor fluorescent moiety. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved, for example, by the 5' to 3' nuclease activity of Taq polymerase, so that the fluorescence emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described, for example, in U.S. Patent Nos. 5,210,015, 5,994,056, and 6,171,785. Commonly used donor-acceptor pairs include the FAM-TAMRA pair. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers™ (BHQ) (Biosearch Technologies, Inc., Novato, CA), Iowa Black™ (Integrated DNA Tech., Inc., Coralville, IA), and BlackBerry® Quencher 650 (BBQ-650) (Berry & Assoc, Dexter, MI).

[0067] In another example, two oligonucleotide probes each containing a fluorescent moiety can hybridize to the amplification product at specific positions determined by the complementarity of the oligonucleotide probe to the C. trachomatis target nucleic acid sequence. When the oligonucleotide probe hybridizes to the nucleic acid of the amplification product at an appropriate position, a FRET signal is generated. The hybridization temperature can range from about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0068] Fluorescence analysis can be performed using, for example, a photon counting epi-fluorescence microscope system (equipped with an appropriate dichroic mirror and a filter for monitoring fluorescence emission in a specific range), a photon counting photomultiplier tube system, or a fluorometer. Excitation to initiate energy transfer or to enable direct detection of the phosphor can be performed using an argon ion laser, a high-intensity mercury (Hg) arc lamp, an optical fiber light source, or other high-intensity light sources appropriately filtered for excitation in the desired range.

[0069] As used herein with respect to "corresponding" for a donor fluorescent moiety and a corresponding acceptor fluorescent moiety, it refers to an acceptor fluorescent moiety having an absorbance spectrum that overlaps with the emission spectrum of the donor fluorescent moiety. The wavelength maximum of the emission spectrum of the acceptor fluorescent moiety must be at least 100 nm greater than the wavelength maximum of the excitation spectrum of the donor fluorescent moiety. Thereby, efficient non-irradiative energy transfer can occur between them.

[0070] The fluorescent donor moiety and the corresponding acceptor moiety are generally selected for (a) high-efficiency Forster energy transfer, (b) a large final Stokes shift (>100 nm), (c) a shift of emission to the red portion of the visible spectrum (>600 nm) as much as possible; and (d) a shift of emission to a higher wavelength than the Raman water fluorescence emission caused by excitation at the donor excitation wavelength. For example, a donor fluorescent moiety having its maximum excitation wavelength near the laser line (e.g., helium-cadmium 442 nm or argon 488 nm), a high extinction coefficient, a high quantum yield, and good overlap with the excitation spectrum of the acceptor fluorescent moiety corresponding to its fluorescence emission can be selected. A corresponding acceptor fluorescent moiety having a high extinction coefficient, a high quantum yield, good overlap of its excitation with the emission of the donor fluorescent moiety, and emission in the red portion of the visible spectrum (>600 nm) can be selected.

[0071] In FRET technology, representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties include fluorescein, lucifer yellow, B-phycoerythrin, 9-acridine isothiocyanate, lucifer yellow VS, 4-acetamido-4'-isothio-cyanatostilbene-2,2'-disulfonic acid, 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrenebutyrate, and 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid derivatives. Representative acceptor fluorescent moieties include LC Red640, LC Red705, Cy5, Cy5.5, Lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosin isothiocyanate, fluorescein, diethylenetriaminepentaacetate, or other chelates of lanthanide ions (e.g., europium, or terbium), depending on the donor fluorescent moiety used. The donor fluorescent moiety and the acceptor fluorescent moiety can be obtained, for example, from Molecular Probes (Eugene, Oregon) or Sigma Chemical Co. (St. Louis, Missouri).

[0072] The donor fluorescent moiety and the acceptor fluorescent moiety can be attached to appropriate probe oligonucleotides via a linker arm. Since the linker arm affects the distance between the donor fluorescent moiety and the acceptor fluorescent moiety, the length of each linker arm is important. The length of the linker arm can be the distance in angstroms (Å) from the nucleotide base to the fluorescent moiety. Generally, the linker arm is about 10 Å to about 25 Å. The linker arm can be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching the linker arm to specific nucleotide bases and methods for attaching the fluorescent moiety to the linker arm.

[0073] An acceptor fluorescent moiety such as LC Red640 can be combined with an oligonucleotide containing an amino linker (e.g., a C6 - aminophosphoramidite available from ABI (Foster City, Calif.) or Glen Research (Sterling, Va.)) to generate, for example, an LC Red640 - labeled oligonucleotide. Linkers frequently used to couple a donor fluorescent moiety such as fluorescein to an oligonucleotide include a thiourea linker (derived from FITC, e.g., fluorescein - CPG manufactured by Glen Research or ChemGene (Ashland, Mass.)), an amide - linker (derived from fluorescein - NHS - ester, e.g., CX - fluorescein - CPG manufactured by BioGenex (San Ramon, Calif.)), or a 3’ - amino - CPG that requires coupling of fluorescein - NHS - ester after oligonucleotide synthesis.

[0074] Detection of L serotype of C. trachomatis The present disclosure provides a method for detecting the presence or absence of the L serotype of C. trachomatis in a biological or non - biological sample. The provided method avoids problems of sample contamination, false negatives, and false positives. The method includes at least one cycling step of amplifying a portion of the pmpH gene from an L serotype - target nucleic acid molecule from the sample using at least one pair of primer pairs, and performing a FRET detection step. The plurality of cycling steps are preferably performed in a thermocycler. The method can be carried out using gene primers and probes for detecting the presence of the pmpH gene from the L serotype, indicating the presence of one or more L serotypes in the sample.

[0075] As described herein, amplification products can be detected using labeled hybridization probes that utilize FRET technology. One FRET format utilizes TaqMan® technology to detect the presence or absence of amplification products, and thus the presence or absence of one or more L serotypes. TaqMan® technology utilizes one single-stranded hybridization probe labeled with one fluorescent dye and one quencher, which may or may not be fluorescent, for example. When the first fluorescent moiety is excited with light of an appropriate wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is generally a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplification product) and is then degraded, for example, by the 5' to 3' nuclease activity of Taq polymerase during the subsequent extension phase. As a result, the fluorescent moiety and the quencher moiety are spatially separated from each other. Consequently, when the first fluorescent moiety is excited in the absence of the quencher, fluorescence emission from the first fluorescent moiety can be detected. By way of example, the ABI PRISM® 7700 Sequence Detection System (Applied Biosystems) uses TaqMan® technology and is suitable for performing the methods described herein for detecting the presence or absence of L serotypes in a sample.

[0076] The presence of amplification products can also be detected using real-time PCR with molecular beacons in combination with FRET. Molecular beacon technology uses hybridization probes labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and the fluorescent labels are typically placed at each end of the probe. Molecular beacon technology uses probe oligonucleotides having sequences that allow secondary structure formation (e.g., hairpins). As a result of secondary structure formation within the probe, when the probe is in solution, both fluorescent moieties are spatially proximate. After hybridization to the target nucleic acid (i.e., amplification product), the secondary structure of the probe is disrupted and the fluorescent moieties are separated from each other, whereby emission from the first fluorescent moiety can be detected after excitation with light of a suitable wavelength.

[0077] Another common form of FRET technology utilizes two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is generally designed to hybridize in close proximity to each other within a target DNA molecule (e.g., amplification product). A donor fluorescent moiety, such as fluorescein, is excited at 470 nm by the light source of a LightCycler® instrument. During FRET, fluorescein transfers its energy to an acceptor fluorescent moiety, such as LightCycler®-Red640 (LC Red640) or LightCycler®-Red705 (LC Red705). The acceptor fluorescent moiety then emits light at a longer wavelength, which is detected by the optical detection system of the LightCycler® instrument. Efficient FRET can occur only when the fluorescent moieties are directly locally proximate and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be correlated with the number of original target DNA molecules. When amplification of the target nucleic acid occurs and the amplification product is purified, the hybridization step results in a FRET-based detectable signal between members of the probe pair.

[0078] Generally, the presence of FRET indicates the presence of one or more L serotypes in the sample, and the absence of FRET indicates the absence of L serotypes in the sample. However, insufficient specimen collection, transport delays, inappropriate transport conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that can affect the success and / or accuracy of the test results. Using the methods disclosed herein, for example, detection of FRET within a 45-cycle process indicates infection with the L serotype of C. trachomatis.

[0079] Representative biological samples that can be used in the practice of this method include, but are not limited to, blood, plasma, serum, liver samples, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Methods for collecting and preserving biological samples are known to those skilled in the art. Biological samples can be processed (e.g., by nucleic acid extraction methods and / or kits known in the art) to release nucleic acids, or in some cases, biological samples can be brought into direct contact with PCR reaction components and appropriate oligonucleotides.

[0080] Melting curve analysis is an additional step that can be included in the cycling profile. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called the melting temperature (Tm), which is defined as the temperature at which half of the DNA double strands separate into single strands. The melting temperature of DNA depends mainly on its nucleotide composition. Thus, DNA molecules rich in G and C nucleotides have a higher Tm than DNA molecules rich in A and T nucleotides. By detecting the temperature at which the signal is lost, the melting temperature of the probe can be determined. Similarly, the annealing temperature of the probe can be determined by detecting the temperature at which the signal is generated. The melting temperature of the probe from each amplification product can confirm the presence or absence of the target nucleic acid in the sample.

[0081] While each thermocycler is operating, cycling of a control sample can be performed similarly. A positive control sample can amplify a target nucleic acid control template (other than the amplification product of the described target gene), for example, using control primers and a control probe. A positive control sample can also amplify, for example, a plasmid construct containing the target nucleic acid molecule. Such plasmid controls can be amplified internally (e.g., within the sample) or in a separate sample run in parallel with the patient sample using the same primers and probes as those used for detection of the intended target. Such controls are indicators of the success or failure of amplification, hybridization, and / or FRET reactions. Each thermocycler run can also include, for example, a negative control lacking the target template DNA. The negative control can measure contamination. This ensures that the system and reagents do not produce false positive signals. Thus, control reactions can readily determine, for example, the ability of primers to anneal by sequence specificity and initiate extension, and the ability of probes to hybridize by sequence specificity and generate FRET.

[0082] In one embodiment, the method includes a step of avoiding contamination. For example, enzymatic methods utilizing uracil-DNA glycosylase are described in U.S. Patent Nos. 5,035,996, 5,683,896, and 5,945,313 to reduce or eliminate contamination between one thermocycler run and the next.

[0083] The method can be practiced using a conventional PCR method combined with FRET technology. In one embodiment, a LightCycler® instrument is used. The following patent applications describe real-time PCR used in LightCycler® technology: International Publication Nos. WO 97 / 46707, WO 97 / 46714, and WO 97 / 46712.

[0084] LightCycler® can be operated using a PC workstation and utilize the Windows® NT operating system. Signals from the samples are obtained when the machine sequentially positions capillaries over the optical unit. The software can display the fluorescence signal in real time immediately after each measurement. The fluorescence acquisition time is 10 to 100 milliseconds (msec). After each cycling step, the quantitative display of fluorescence versus the number of cycles can be continuously updated for all samples. The generated data can be saved for further analysis.

[0085] As an alternative to FRET, amplification products can be detected using a double-stranded DNA-binding dye such as a fluorescent DNA-binding dye (e.g., SYBR® Green or SYBR® Gold (Molecular Probes)). When interacting with double-stranded nucleic acids, such fluorescent DNA-binding dyes emit a fluorescence signal upon excitation with light of a suitable wavelength. Also, double-stranded DNA-binding dyes such as nucleic acid intercalating dyes can be used. When using a double-stranded DNA-binding dye, melting curve analysis is typically performed to confirm the presence of the amplification product.

[0086] It is understood that the embodiments of the present disclosure are not limited by the configurations of one or more commercially available devices.

[0087] Manufactured Product / Kit Embodiments of the present disclosure further provide a product, composition, or kit for detecting the L serotype. The product may include primers and probes used for detecting the L serotype, along with a suitable packaging material. Representative primers and probes for detecting the pmpH gene of the L serotype can hybridize to a target nucleic acid molecule. Further, the kit may also include appropriately packaged reagents and materials necessary for DNA immobilization, hybridization, and detection, such as a solid support, buffer, enzyme, and DNA standard. Methods for designing primers and probes are disclosed herein, and representative examples of primers and probes that amplify and hybridize to a target nucleic acid molecule are provided.

[0088] The product may also include one or more fluorescent moieties for labeling the probe, or the probe supplied with the kit may be labeled. For example, the product may include donor and / or acceptor fluorescent moieties for labeling the target gene probe. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0089] The product can also include an accompanying document or packaging label having instructions for using the primers and probes for the pmpH gene from the L serotype to detect the L serotype in a sample. The product may further include reagents (e.g., buffer, polymerase enzyme, cofactor, or agent for preventing contamination) for implementing the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0090] Embodiments of the present disclosure are further described in the following examples, which do not limit the scope of the invention described in the claims.

[0091] In particular, embodiments of the method according to the present disclosure may include one or more additional steps or omit one or more of the described steps of the method. Generally, the method can be modified by any suitable method that still enables the selective and specific detection of one or more L serotypes of C. trachomatis in a sample. Further variations of the method within the scope of the present disclosure will be apparent from the additional examples and descriptions included herein.

Examples

[0092] The following examples are intended to be illustrative and are in no way intended to be limiting.

[0093] Example 1: This example describes the PCR experimental conditions for the RT-PCR detection of the pmpH gene target. Detection of the pmpH gene target by RT-PCR was performed using the cobas® 6800 / 8800 system (Roche Molecular Systems, Inc., Pleasanton, CA). The final concentrations of the amplification reagents and the thermal profile used for the PCR amplification reaction are shown in Table 2.

[0094]

Table 2

[0095] The pre-PCR program included incubations at 55°C, 60°C, and 65°C for initial denaturation and reverse transcription of the RNA template. Incubation at the three temperatures synthesizes the beneficial effect that even slightly mismatched target sequences (such as genetic variants of organisms) are transcribed at lower temperatures, while the formation of RNA secondary structures is suppressed at higher temperatures, thus resulting in more efficient transcription. The PCR cycling was divided into two measurements, and a one-step setting (combining annealing and extension) was applied to both measurements. The first 5 cycles at 55°C allow for an inclusive increase by pre-amplifying slightly mismatched target sequences, while the 45 cycles of the second measurement enhance the specificity by using an annealing / extension temperature of 58°C.

[0096] Example 2: Table 3 shows the results of a PCR assay using a set of primers and probes for detecting the L serotype of C. trachomatis. Master Mix (MMx) 1 used the forward primer of SEQ ID NO: 1, the reverse primer of SEQ ID NO: 3, and the probe of SEQ ID NO: 5. Master Mix (MMx) 2 used the forward primer of SEQ ID NO: 2, the reverse primer of SEQ ID NO: 4, and the probe of SEQ ID NO: 5. Master Mix (MMx) 3 used the forward primer of SEQ ID NO: 7, the reverse primer of SEQ ID NO: 9, and the probe of SEQ ID NO: 11. Template plasmids having a consensus nucleic acid sequence of any of serotypes L1, L2, and L3 or serotypes A, B, and C, serotypes D, E, F, G, H, and I, and serotypes J and K were tested at concentrations from 1.00E+01 copies per reaction to 1.00E+07 copies per reaction. The initial tests demonstrated that these primer / probe pairings yielded good results regarding the sensitivity and specificity for the L serotype (i.e., serotypes L1, L2, and L3).

[0097]

Table 3

[0098] The present invention is presented in several various embodiments in the following description with reference to the drawings, and like numbers represent the same or similar elements. Throughout this specification, references to "one embodiment", "an embodiment", or similar language mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment", "in an embodiment", and similar language throughout this specification are not necessarily all referring to the same embodiment.

[0099] The described features, structures, or characteristics of the present invention may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are set forth in order to provide a thorough understanding of embodiments of the system. However, one of ordinary skill in the art will recognize that the system and method may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the present invention. Accordingly, the foregoing description is intended to be illustrative and not limiting of the scope of the concepts of the present invention.

[0100] Each reference identified in this application is hereby incorporated by reference in its entirety into this specification.

Claims

1. A method for detecting the serotype (L serotype) of Chlamydia trachomatis in a sample that causes lymphogranulomatosis of the inguinal region, (a) If the L serotype is present in the sample, an amplification step is carried out, which includes contacting the sample with at least one set of primers designed to target the polymorphic membrane protein H(pmpH) gene of the L serotype to produce an amplification product. (b) If the L serotype is present in the sample, a hybridization step is performed which includes contacting the amplification product with at least one detectable probe that targets the pmpH gene of the L serotype. (c) Performing a detection step that includes detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of the L serotype in the sample, and the absence of the amplification product indicates the absence of the L serotype in the sample. Includes, The at least one set of primers and the at least one detectable probe are A forward primer comprising nucleic acid sequences selected from the group consisting of SEQ ID NOs: 6 and 7, or a combination thereof, A reverse primer containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9, or a combination thereof, A probe comprising nucleic acid sequences selected from the group consisting of SEQ ID NOs. 10 and 11, or their complements. Methods that include...

2. The method according to claim 1, wherein the hybridization step includes contacting a detectable probe labeled with a donor fluorescent portion and a corresponding acceptor portion with the amplification product, and the detection step includes detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent portion and the acceptor portion of the probe, the presence or absence of fluorescence indicating the presence or absence of the L serotype in the sample.

3. The method according to claim 1, wherein the amplification step uses a polymerase enzyme having nuclease activity from 5' to 3'.

4. The method according to claim 1, wherein the sample is a biological sample.

5. The method according to claim 4, wherein the biological sample is a vaginal swab sample, a vaginal swab sample collected by a clinician, a cervical swab sample, an oropharyngeal (throat) swab sample, or an anorectal swab sample.

6. A method according to any one of claims 1 to 5, further comprising a second set of primers and at least a second detectable probe, wherein the second set of primers comprises at least a forward primer and at least a reverse primer, the forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2, or a combination thereof, the reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 4, or a combination thereof, and the second detectable probe comprising the nucleic acid sequence of SEQ ID NO: 5 or a complement thereof.

7. The method according to any one of claims 1 to 5, wherein the forward primer comprises the nucleic acid sequence of SEQ ID NO: 7, the reverse primer comprises the nucleic acid sequence of SEQ ID NO: 9, and the probe comprises the nucleic acid sequence of SEQ ID NO: 11 or a complement thereof.

8. A method for detecting the serotype (L serotype) of Chlamydia trachomatis in a sample that causes lymphogranulomatosis of the inguinal region, (a) If the L serotype is present in the sample, an amplification step is carried out, which includes contacting the sample with at least one set of primers designed to target the 3' end of the polymorphic membrane protein H(pmpH) gene of the L serotype, as shown in SEQ ID NO: 18, to produce an amplification product. (b) If the L serotype is present in the sample, a hybridization step is performed which includes contacting the amplification product with at least one detectable probe that targets the pmpH gene of the L serotype. (c) Performing a detection step that includes detecting the presence or absence of the amplification product, wherein the presence of the amplification product indicates the presence of the L serotype in the sample, and the absence of the amplification product indicates the absence of the L serotype in the sample. A method including, A method wherein the at least one set of primers is designed to target a region consisting of at least a portion of nucleotides 2600 to 2800 of the pmpH gene of the L serotype having SEQ ID NO: 18, or a corresponding region of a different L serotype.

9. A kit for detecting the pmpH gene of the Chlamydia trachomatis serotype (L serotype) that causes lymphogranulomatosis of the inguinal region in a sample, wherein the kit includes an amplification reagent, and the amplification reagent is as follows: (a) DNA polymerase having nuclease activity from 5' to 3', (b) Nucleotide monomers and (c) at least one pair of primers and at least one detectable probe, i. A forward primer containing a nucleic acid sequence selected from the group consisting of Sequence IDs 6 and 7, or any combination thereof. ii. A reverse primer containing a nucleic acid sequence selected from the group consisting of Sequence IDs 8 and 9, or a combination thereof, and iii. A detectable probe comprising at least one pair of primers and at least one detectable probe, each containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs. 10 and 11, or their complements, A kit that includes this.

10. The kit according to claim 9, wherein the detectable probe is labeled with a donor fluorescence portion and a corresponding acceptor portion.

11. A kit according to any one of claims 9 to 10, further comprising a set of second primers and at least a second detectable probe, wherein the set of second primers comprises at least a forward primer and at least a reverse primer, the forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2, or a combination thereof, the reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 4, or a combination thereof, and the second detectable probe comprising the nucleic acid sequence of SEQ ID NO: 5 or a complement thereof.

12. The kit according to any one of claims 9 to 10, wherein the forward primer comprises the nucleic acid sequence of SEQ ID NO: 7, the reverse primer comprises the nucleic acid sequence of SEQ ID NO: 9, and the probe comprises the nucleic acid sequence of SEQ ID NO: 11 or its complement.

13. An oligonucleotide comprising or consisting of a nucleotide sequence selected from SEQ ID NOs. 6-11 or its complement.

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