Compositions and methods for detection of venereal disease lymphogranuloma (LGV) chlamydia trachomatis serovar

Abstract

The present disclosure relates generally to methods for rapidly detecting the presence or absence of chlamydia trachomatis serovariant causing venereal disease lymphogranuloma (LGV) in a biological or non-biological sample. The method may include performing an amplification step, a hybridization step, and a detection step. In addition, oligonucleotide primers and probes as well as kits are provided that are designed to detect pmpH genes of targeted Chlamydia trachomatis L serovariant of L serovariant.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 357,179, filed on June 30, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on June 29, 2023 is named "P37525-WO_Sequence_Listing" and is 26.813 bytes in size. Background Art

[0005] The present disclosure relates to the field of molecular diagnostics, and more particularly to the detection of lymphogranuloma venereum (LGV)-causing serotypes (L serotypes) of Chlamydia trachomatis (C. trachomatis) by polymerase chain reaction (PCR) assays.

[0006] Infection from Chlamydia trachomatis is the leading bacterial cause of sexually transmitted diseases worldwide, with approximately 89.1 million cases occurring each year (Bebear C., de Barbeyrac B., Clinical Microbial Infect. 2009; 15: 4-10). In the United States, Chlamydia trachomatis is the most frequently reported bacterial sexually transmitted disease (STD), and has the highest prevalence in people aged ≤24 years. In 2013, the Centers for Disease Control and Prevention (CDC) reported a total of 1,401,906 cases of Chlamydia trachomatis infection, equivalent to 446.6 cases per 100,000 people (CDC, Sexually Transmitted Disease Surveillance 2013).

[0007] The microorganism Chlamydia trachomatis is a Gram-negative, non-motile, obligate intracellular bacterium with a unique biphasic life cycle. A variety of infections are caused by Chlamydia trachomatis, including urethritis, cervicitis, proctitis, conjunctivitis, endometritis, and salpingitis; if left untreated, these infections may ascend to the uterus, fallopian tubes, and ovaries, leading to pelvic inflammatory syndrome, ectopic pregnancy, and tubal factor infertility. Reiter's syndrome (urethritis, conjunctivitis, arthritis, and mucocutaneous lesions) has also been associated with genital Chlamydia trachomatis infection. Many infections remain asymptomatic, and a large number of infected patients may not seek care. Patients often become reinfected if their sexual partners are not treated. Infants born to infected mothers may develop conjunctivitis, pharyngitis, and pneumonia. The main symptoms in both males and females are increased excretion and difficulty urinating; females may also experience irregular uterine bleeding.

[0008] The serotypes of C. trachomatis can be divided into three groups: serotypes A, B, and C (AC), serotypes D, E, F, G, I, J, and K (DK), and serotype L, each of which is associated with a different clinical presentation of chlamydial disease. Serotype AC presents with ocular infections, serotype DK presents with anogenital infections, and serotype L presents with lymphogranuloma venereum or LGV (genital ulcers / lesions). Notably, serotype L includes a variety of different strains, including L1, L2, L2b, and L3. Differentiating between sexually transmitted serotypes (DK vs. L) is particularly important because different treatments are prescribed for each serotype—typically 100 mg doxycycline / oral / twice daily / 7 days for most chlamydial infections and 100 mg doxycycline / oral / twice daily / 21 days for LGV (Sexually Transmitted Infections Treatment Guidelines, 2021. “Lymphogranuloma Venereum.” Centers for Disease Control and Prevention, https: / / www.cdc.gov / std / treatment-guidelines / lgv.htm, September 3, 2021). The most accurate diagnosis ensures the most appropriate antibiotic therapy.

[0009] The nucleic acid sequences of the polymorphic membrane protein H (pmpH) genes of 19 serotypes / serovariants have been previously 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).

[0010] Current approaches to diagnosing LGV include clinical diagnosis, epidemiological information, and LGV-specific molecular testing (ie, nucleic acid amplification testing or NAAT); molecular testing is the only option that can definitively diagnose LGV. In current molecular diagnosis of LGV, targeting the polymorphic membrane protein H (pmpH) gene is dominant because all LGV serotypes have a unique gap of 36 nucleotides that is not present in other serotypes (see Figure 1 ). For at least the foregoing reasons, there is a need for improved methods for specifically and selectively detecting the L serotype of Chlamydia trachomatis in nucleic acid samples. Summary of the invention

[0011] The present invention overcomes the above disadvantages by providing a method for specifically and selectively detecting the L serotype of Chlamydia trachomatis in a nucleic acid sample.

[0012] In the present disclosure, PCR is provided to measure the pmpH gene of the L serotype of Chlamydia trachomatis specifically to detect biological samples and non-biological samples. Although all Chlamydia trachomatis serotypes have the pmpH gene, the pmpH gene of the L serotype includes at least two regions, relative to the pmpH gene of non-L serotype (for example, serotype A, B, C, D, E, F, G, I, J and K), these two regions can be realized by PCR specificity and selective detection. In one aspect, the first region of the pmpH gene of the L serotype has unique 36 base pairs (bp) deletions that can be used for molecular diagnosis. In another aspect, the second region of the 3' end of the pmpH gene comprising the L serotype can be used for molecular diagnosis additionally or alternatively.

[0013] The present disclosure relates to a method for rapidly detecting the presence or absence of L serotype in biological samples or non-biological samples by real-time PCR (RT-PCR) in a single test tube. In this article, the method for detecting L serotype comprises carrying out at least one cycle step, which may include an amplification step and a hybridization step. In addition, primers, probes and test kits designed to detect L serotype in a single test tube are provided. Detection method is designed to the pmpH gene for targeting L serotype, which allows people to detect L serotype in a single test.

[0014] In one aspect, a method for detecting L serotype in a sample is provided, comprising providing a sample, performing an amplification step, the amplification step comprising 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, if the L serotype is present in the sample, performing a hybridization step, the hybridization step comprising contacting the amplification product (if the L serotype is present in the sample) with at least one detectable probe to target the pmpH gene of the L serotype, and performing a detection step, the 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 wherein the absence of the amplification product indicates the absence of the L serotype in the sample. Herein, the at least one set of primers and the at least one detectable probe include a forward primer comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 6, and 7 or a combination thereof; a reverse primer comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 8, and 9 or a combination thereof; and a probe comprising or consisting of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, and 11 or a complementary sequence 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; 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; and the probe comprises or consists of a nucleic acid sequence of SEQ ID NO: 5 or a complementary sequence thereof. In some embodiments, the forward primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 1 and 2 or a combination thereof; the reverse primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 3; and the probe comprises or consists of a nucleic acid sequence of SEQ ID NO: 5 or its complement, or the forward primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 1 and 2 or a combination thereof; the reverse primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 4; and the probe comprises or consists of a 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 NO: 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 NO: 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 NO: 10 and 11 or its complement. 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 less nucleotides. In certain embodiments, the oligonucleotide primer and / or oligonucleotide probe have 40 or less nucleotides (e.g., 35 or less nucleotides, 30 or less nucleotides, 25 or less nucleotides). In some embodiments, the hybridization step includes contacting the amplification product with a detectable probe, which is 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, wherein the presence or absence of fluorescence indicates the presence or absence of L serotype in the sample. In some embodiments, the amplification step uses a polymerase with 5' to 3' nuclease activity. In some embodiments, the sample is a biological sample. In a specific embodiment, the biological sample is a vaginal swab sample, a vaginal swab sample collected by a clinician, an intracervical swab sample, an oropharyngeal (throat) swab sample, or an anorectal swab sample. In some embodiments, the amplification and hybridization steps are repeated. In this article, the number of repetitions depends on, for example, the nature of the sample. If the sample is a complex mixture of nucleic acid, more amplification steps and hybridization steps will be needed to amplify the target sequence that is enough for detection. In certain embodiments, repeat amplification and hybridization steps at least about 20 times, but can repeat up to at least 25 times, 30 times, 40 times, 50 times, 60 times or even 100 times. In addition, can during or after each amplification and hybridization step, during or after every other amplification and hybridization step, during or after a specific amplification and hybridization step, or during or after a specific amplification and hybridization step, detect the presence or absence of amplified product, wherein if present, expect to have enough amplified products for detection. In certain embodiments, donor fluorescent moiety and acceptor moiety (for example, quencher) can be no more than 5 to 20 nucleotides (for example, 8 or 10) apart from each other along the length of probe. In certain embodiments, donor fluorescent moiety and corresponding acceptor moiety are no more than 8-20 nucleotides apart from each other on probe. In certain embodiments, acceptor moiety is quencher. In another embodiment, detectable probe further includes the nucleic acid sequence that allows secondary structure to form. This secondary structure formation usually causes the space between the first fluorescent moiety and the second fluorescent moiety to approach. According to the method, the second fluorescent part on the probe can be a quencher. In certain embodiments, the detectable probe can be labeled with a fluorescent dye that serves as a reporter. The probe may also have a second dye that serves as a quencher. The reporter dye is measured at a defined wavelength, thereby allowing detection and differentiation of the amplified pmpH gene of the L serotype. The fluorescent signal of the complete probe is suppressed by the quencher dye. During the PCR amplification step, the hybridization of the probe with the specific single-stranded DNA template results in the cutting of the 5' to 3' nuclease activity of the DNA polymerase, thereby causing the reporter dye and the quencher dye to separate and generate a fluorescent signal.With each PCR cycle, an increasing number of cleaved probes are generated, and the cumulative signal of the reporter dye increases. Optionally, one or more additional probes (e.g., such as an internal reference control or other targeted probes (e.g., other viral nucleic acids) can also be labeled with a reporter fluorescent dye that is unique and different from the fluorescent dye label associated with the target gene probe. In this case, because the specific reporter dye is measured at a defined wavelength, the amplified target and one or more additional probes can be detected and distinguished simultaneously.

[0015] In another aspect, a method for detecting a serotype of Chlamydia trachomatis that causes lymphogranuloma venereum (L serotype) in a sample is provided, the method comprising: (a) performing an amplification step, the 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, if the L serotype is present in the sample; (b) performing a hybridization step, comprising contacting the amplification product (if the L serotype is present in the sample) with at least one detectable probe to target 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 wherein the absence of the amplification product indicates the absence of the L serotype in the sample, wherein the at least one set of primers is designed to target a region comprising at least a portion of the last 1000 nucleotides of 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 portion of the last 500 nucleotides of 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 portion of nucleotides 2600 to 2900 of the pmpH gene of the L1 serotype or a 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 portion of nucleotides 2600 to 2800 of the pmpH gene of the L1 serotype or a 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 portion of nucleotides 2635 to 2766 of the pmpH gene of the L1 serotype or a corresponding region of a different L serotype. Herein, the sequence of the pmpH gene of the L1 serotype can be the nucleic acid shown in Genbank Accession No. HE601950 (Gene: L1440_00934; CDS: CCP62925.1; Position: 1032228..1035245, Length: 3,018 nt) and is further provided as SEQ ID NO: 18. Herein, the oligonucleotide primers and / or oligonucleotide probes are selected to effectively hybridize with the L serotype, but not with other serotypes. In some embodiments, the oligonucleotide probe is selected to hybridize only with the L serotype, but not with all other serotypes.In certain embodiments, the oligonucleotide probe is selected to hybridize to at least a portion of nucleotides 2660 to 2740 of the pmpH gene of the L1 serotype (SEQ ID NO: 18) or a corresponding region of a different L serotype, more specifically to at least a portion of nucleotides 2670 to 2730 of the pmpH gene of the L1 serotype (SEQ ID NO: 18) or a corresponding region of a different L serotype, or even more specifically to at least a portion of nucleotides 2680 to 2720 of the pmpH gene of the L1 serotype (SEQ ID NO: 18) or a corresponding region of a different L serotype. 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 includes contacting the amplification product with a detectable probe, which is 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, wherein the presence or absence of fluorescence indicates the presence or absence of L serotype in the sample. In some embodiments, the amplification step employs a polymerase with 5' to 3' nuclease activity. In some embodiments, the sample is a biological sample. In a specific embodiment, the biological sample is a vaginal swab sample, a vaginal swab sample collected by a clinician, an endocervical swab sample, an oropharyngeal (throat) swab sample, or an anorectal swab sample. In some embodiments, the amplification and hybridization steps are repeated. In this article, the number of repetitions depends on, for example, the nature of the sample. If the sample is a complex mixture of nucleic acids, more amplification steps and hybridization steps will be required to amplify target sequences sufficient for detection. In some embodiments, the amplification and hybridization steps are repeated at least about 20 times, but can be repeated up to at least 25 times, 30 times, 40 times, 50 times, 60 times or even 100 times. In addition, can during or after each amplification and hybridization step, during or after every other amplification and hybridization step, during or after a specific amplification and hybridization step, or during or after a specific amplification and hybridization step, detect the presence or absence of amplified product, wherein if present, it is expected that there are enough amplified products for detection. In certain embodiments, donor fluorescent moiety and acceptor moiety (for example, quencher) can be no more than 5 to 20 nucleotides (for example, 8 or 10) apart from each other along the length of probe. In certain embodiments, donor fluorescent moiety and corresponding acceptor moiety are no more than 8-20 nucleotides apart from each other on probe. In certain embodiments, acceptor moiety is a quencher. In another embodiment, detectable probe further includes a nucleic acid sequence that allows secondary structure to form. This secondary structure formation usually causes the space between the first fluorescent moiety and the second fluorescent moiety to approach.According to the method, the second fluorescent part on the probe can be a quencher. In certain embodiments, the detectable probe can be labeled with a fluorescent dye that serves as a reporter. The probe may also have a second dye that serves as a quencher. The reporter dye is measured at a defined wavelength, thereby allowing detection and differentiation of the amplified pmpH gene of the L serotype. The fluorescent signal of the complete probe is suppressed by a quencher dye. During the PCR amplification step, the hybridization of the probe with the specific single-stranded DNA template causes the cutting of the 5' to 3' nuclease activity of the DNA polymerase, thereby causing the reporter dye and the quencher dye to separate, and generate a fluorescent signal. With each PCR cycle, an increasing number of cut probes are generated, and the cumulative signal of the reporter dye also increases therewith. Optionally, one or more other probes (for example, such as an internal reference control or other targeting probes (for example, other viral nucleic acids) may also be labeled with a reporter fluorescent dye, which is unique and different from the fluorescent dye marker associated with the target gene probe. In this case, because the specific reporter dye is measured at a defined wavelength, it is possible to simultaneously detect and differentiate the amplified target and one or more other probes.

[0016] In another aspect, a kit for detecting L serotype is provided. Specifically, a kit for detecting the pmpH gene of the serotype (L serotype) of Chlamydia trachomatis that causes lymphogranuloma venereum in a sample is provided, the kit comprising an amplification reagent, the amplification reagent comprising: (a) a DNA polymerase having 5′ to 3′ nuclease activity; (b) nucleotide monomers; (c) at least one pair of primers and at least one detectable probe, comprising: (i) a forward primer comprising a nucleic acid sequence of SEQ ID NO: 1, 2, 6 and 7 or any combination thereof; (ii) a reverse primer comprising a nucleic acid sequence of SEQ ID NO: 3, 4, 8 and 9 or a combination thereof; and (iii) a detectable probe comprising a nucleic acid sequence of SEQ ID NO: 5, 10 and 11 or a complementary sequence 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; 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; and the probe comprises or consists of a 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 of SEQ ID NOs: 1 and 2 or a combination thereof; the reverse primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 3; and the probe comprises or consists of a nucleic acid sequence of SEQ ID NO: 5 or its complement, or the forward primer comprises or consists of a nucleic acid sequence of SEQ ID NOs: 1 and 2 or a combination thereof; the reverse primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 4; and the probe comprises or consists of a 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 a nucleic acid sequence of SEQ ID NO: 7; the reverse primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 9; and the probe comprises or consists of a 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 kit may include probes that have been labeled with donor and corresponding acceptor fluorescent moieties, or may include fluorescent moieties for labeling probes.In some embodiments, the detectable probe is labeled with a donor fluorescent moiety and a corresponding acceptor moiety. In some embodiments, the kit may 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 may also include a package insert and instructions for using the primers, probes, and fluorescent moieties to detect the presence or absence of L serotypes in a sample.

[0017] In another aspect, an oligonucleotide is provided, comprising or consisting of a nucleic acid sequence selected from SEQ ID NO: 1-11 or its complementary sequence. In some embodiments, the oligonucleotide may have 50 or fewer nucleotides. In certain embodiments, the oligonucleotide may have 40 or fewer nucleotides (e.g., 35 or fewer nucleotides, 30 or fewer nucleotides, 25 or fewer nucleotides). In some embodiments, the oligonucleotide may 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 change nucleic acid hybridization stability relative to unmodified nucleotides. In some embodiments, the at least one modified nucleotide is selected from the group consisting of N6-benzyl-dA, N4-benzyl-dC, N6-p-tert-butyl-benzyl-dA and N4-p-tert-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. "Conservatively modified variations" or simply "conservative variations" of a particular nucleic acid sequence refers to those nucleic acids that encode identical or substantially identical amino acid sequences, or, where the nucleic acid does not encode an amino acid sequence, to substantially identical sequences. One skilled in the art will recognize that individual substitutions, deletions or additions that change, add or delete a single amino acid or a small portion of amino acids (usually less than 5%, more usually less than 4%, 2% or 1%) in the encoded sequence are "conservatively modified variations", wherein these changes result in the deletion of an amino acid, the addition of an amino acid, or the substitution of an amino acid with a chemically similar amino acid. In some embodiments, the oligonucleotides are provided in the form of a set of oligonucleotides. Herein, the set of oligonucleotides may include: (i) a forward primer comprising or consisting of a nucleic acid sequence of SEQ ID NOs: 1, 2, 6 and 7 or any combination thereof; (ii) a reverse primer comprising or consisting of a nucleic acid sequence 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 a nucleic acid sequence of SEQ ID NOs: 5, 10 and 11 or a complementary sequence 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. Herein, in certain embodiments, the probe may comprise or consist of a nucleic acid sequence of SEQ ID NO: 5 or its complementary sequence. In some embodiments, the forward primer comprises or consists of a nucleic acid sequence of SEQ ID NOs: 1 and 2 or a combination thereof; and the reverse primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 3.Herein, in certain embodiments, the probe may comprise or consist of a nucleic acid sequence of SEQ ID NO: 5 or its complementary sequence. In certain embodiments, the forward primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 1 and 2 or a combination thereof; and the reverse primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 4. Herein, in certain embodiments, the probe may comprise or consist of a nucleic acid sequence of SEQ ID NO: 5 or its complementary sequence. In certain embodiments, the forward primer comprises or consists of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 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 NO: 8 and 9 or a combination thereof. Herein, in certain embodiments, the probe may comprise or consist of a nucleic acid sequence selected from the group consisting of SEQ ID NO: 10 and 11 or its complementary sequence. In certain embodiments, the forward primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 7; and the reverse primer comprises or consists of a nucleic acid sequence of SEQ ID NO: 9. Herein, in certain embodiments, the probe may comprise or consist of a nucleic acid sequence of SEQ ID NO: 11 or its complementary sequence. In some embodiments, at least one primer and / or detectable probe targeting the pmpH gene of the L serotype comprises at least one modified nucleotide as detailed above.

[0018] In another aspect, determining the presence of nucleic acid of the L serotype of Chlamydia trachomatis in the methods and kits provided above can be combined with determining the presence of nucleic acid of all serotypes of Chlamydia trachomatis. Herein, the oligonucleotide sets disclosed above for use in the above methods and kits can be combined with an oligonucleotide set comprising at least forward and reverse oligonucleotide primers and at least one oligonucleotide probe for amplifying and detecting all serotypes of Chlamydia trachomatis (CT). 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 their complements. In certain embodiments, the amplification, hybridization, and detection steps using a set of pan-CT oligonucleotides are performed in parallel reactions using a second portion of the sample. In alternative embodiments, the amplification, hybridization, and detection steps using a set of pan-CT oligonucleotides are performed in a multiplex reaction in the same vial as the reaction used to determine the L serotype. Herein, the fluorophore (or donor-acceptor pair) used to label the pan-CT oligonucleotide probe is different from the fluorophore (or donor-acceptor pair) used to label the L serotype-specific oligonucleotide probe. More specifically, the hybridization step may include contacting the amplification product with two or more probes, each of which is labeled with a different donor fluorescent moiety (e.g., a first donor fluorescent moiety and a second 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 moieties (e.g., the first donor fluorescent moiety and the second donor fluorescent moiety) and the acceptor moiety of the probes for both the different donor fluorescent moieties and the corresponding acceptor moieties, wherein 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 Chlamydia trachomatis (CT) serotype in the sample.

[0019] The present disclosure also provides a method for detecting the presence or absence of L serotype (i.e., pmpH gene with 36 base deletions) nucleic acid in a biological sample from an individual. These methods can be used to detect the presence or absence of L serotype or L serotype nucleic acid in a biological sample for diagnostic testing, and the biological sample is such as a vaginal swab specimen, a vaginal swab specimen collected by a clinician, a cervical swab specimen, an oropharyngeal (throat) swab specimen, and anorectal swab specimens or other biological materials that are considered to have L serotypes. In addition, experienced people in the art can use the same test to evaluate other sample types to detect L serotype nucleic acids. Such methods generally include performing at least one cycle step, and the at least one cycle step includes an amplification step and 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 produce one or more amplification products in the case where nucleic acid molecules are present in the sample, the probe hybridization step includes contacting the amplification product with one or more detectable probes that are specific to the amplification product, and the dye binding step includes contacting the amplification product with a double-stranded DNA binding dye. Such methods also include detecting the presence or absence of a double-stranded DNA binding dye bound to the amplified product, wherein the presence of binding indicates the presence of L serotype in the sample, and wherein the absence of hybridization indicates 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 dye, and cyanine dyes, e.g. and In addition, such methods may further comprise determining the melting temperature between the amplified product and the double-stranded DNA binding dye, wherein the melting temperature confirms the presence or absence of the L serotype or the L serotype nucleic acid.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although in the practice or testing of this theme, methods and materials similar or equivalent to those described herein can be used, suitable methods and materials are described below. In addition, materials, methods and examples are only illustrative and are not intended to be limiting. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification (including definitions) shall prevail.

[0021] The 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 detailed description, and from the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1is a sequence alignment of Chlamydia trachomatis serotypes AC / K / J compared to serotypes L1 / L2 / L3, showing a unique 36-bp deletion present only in serotypes L1 / L2 / L3. An example of how a primer / probe detection set can be designed to specifically detect the L serotype.

[0023] Figure 2 1 is a sequence alignment of the pmpH genes of Chlamydia trachomatis serotypes A, B, C, D, E, F, G, Ia, J and K compared to serotypes L1, L2, L2b and L3, showing a first region (denoted as R1) containing a unique 36-bp deletion that exists only in the L serotype and a second region (denoted as R2) at the 3' end of the pmpH gene. The x-axis indicates the distance of the pmpH gene in nucleotides along the 5' to 3' direction. The y-axis shows a chart from top to bottom, illustrating the consensus identity (CID) of the listed serotypes, followed by serotypes L2, L2b, L3, L1, A, B, C, F, G, E, D, Ia, K and J. The triangle indicates the binding position of the primers and probes of the present disclosure (see Table 1). DETAILED DESCRIPTION

[0024] I. Definitions

[0025] In this application, unless the context clearly indicates otherwise, (i) the term "a" (a) may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the term "comprising" (and including) may be understood to cover the components or steps listed individually, whether they are presented alone or together with one or more other components or steps; (iv) the terms "about" and "approximately" may be understood to allow for standard variations, as would be understood by a person of ordinary skill in the art; and (v) where a range is provided, the endpoints are included.

[0026] As used herein, the term "amplifying" or "amplification" refers to the process of synthesizing a nucleic acid molecule that is complementary to one or both strands of a template nucleic acid molecule (e.g., the pmpH gene from serotype L of Chlamydia 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 enzymatic extension from the primers to produce an amplified product. Amplification typically requires the presence of deoxyribonucleoside triphosphates, a DNA polymerase (e.g., Taq) and appropriate buffers and / or cofactors (e.g., MgCl2 and / or KCl) for optimizing polymerase activity.

[0027] Approximately: As used herein, the term "approximately" or "about", as applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​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 or less than) of the reference value, unless otherwise stated or obvious from the context (unless the number exceeds 100% of the possible value).

[0028] Correlation: Two events or entities are "correlated" with each other, as the term is used herein, if the presence, level, and / or form of one event or entity is related to the presence, level, and / or form of another event or entity. For example, a particular entity (e.g., a polypeptide, genetic marker, metabolite, etc.) is considered to be associated with a particular disease, disorder, or condition (e.g., in a relevant population) if the presence, level, and / or form of the entity is associated with the incidence and / or susceptibility of the 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 close to each other and / or remain in physical proximity to each other. In some embodiments, two or more entities that are physically bound to each other are covalently attached to each other; in some embodiments, two or more entities that are physically bound to each other are not covalently attached to each other, but are non-covalently attached, such as by hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0029] Biological sample: As used herein, the term "biological sample" typically refers to a sample obtained or derived from a target biological source (e.g., a tissue or organism or cell culture), as described herein. In some embodiments, the target source includes an organism, such as an animal or a human, or is composed thereof. In some embodiments, the biological sample includes a biological tissue or fluid, or is composed thereof. In some embodiments, the biological sample can be or include bone marrow; blood; blood cells; ascites; tissue or fine needle biopsy samples; body fluids containing cells; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid; peritoneal fluid; pleural fluid; feces; lymph; gynecological fluid; skin swab; vaginal swab; oral swab; nasal swab; washing or lavage fluid, such as catheter lavage fluid or bronchoalveolar lavage fluid; aspirate; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; other body fluids, secretions and / or excretions; and / or cells therefrom, etc. In some embodiments, the biological sample comprises or is composed of cells obtained from an individual. In certain embodiments, the cell obtained is or includes a cell from an individual from which a sample is obtained. In certain embodiments, a sample is a "primary sample" obtained directly from a target source by any appropriate means. For example, in certain embodiments, an original biological sample is obtained by a method selected from the group consisting of the following items: biopsy (e.g., fine needle aspiration or tissue biopsy), surgery, body fluid collection (e.g., blood, lymph, feces, etc.). In certain embodiments, as will be clear from the context, the term "sample" refers to a preparation obtained by processing the original sample (e.g., by removing one or more components thereof and / or by adding one or more reagents thereto). For example, a semipermeable membrane is used for filtration. Such "processed sample" may include, for example, nucleic acid or protein extracted from a sample, or nucleic acid or protein obtained by subjecting the primary sample to techniques such as amplification or reverse transcription of mRNA, separation and / or purification of certain components.

[0030] The term "its complementary sequence" refers to a nucleic acid that has the same length as a given nucleic acid and is completely complementary thereto.

[0031] Comprising: A composition or method described herein as "comprising" one or more named elements or steps is open ended, meaning that the named elements or steps are essential, but other elements or steps may be added within the scope of the composition or method. It should be understood that a composition or method described as "comprising" or "comprises" one or more named elements or steps also describes a corresponding, more limited composition or method "consisting essentially of" or "consists essentially of" the same named elements or steps, meaning that the composition or method includes the named basic elements or steps and may also include additional elements or steps that do not substantially affect the basic and novel features of the composition or method. It should also be understood that any composition or method described herein as "comprising" or "consisting essentially of" one or more named elements or steps also describes a corresponding, more limited, closed composition or method "consisting of" or "consists of" the named elements or steps to exclude any other unnamed elements or steps. In any composition or method disclosed herein, a known or published equivalent of any named essential element or step may be substituted for that element or step.

[0032] Designed: As used herein, the term "designed" refers to an agent that: (i) has a structure that is selected by human effort; (ii) is produced by a process that requires human effort; and / or (iii) is different from natural substances and other known agents.

[0033] Determining: Those of ordinary skill in the art reading this specification will understand that "determining" can be accomplished using or by using any of a variety of techniques available to those skilled in the art, including, for example, specific techniques explicitly mentioned herein. In some embodiments, determining involves manipulation of a physical sample. In some embodiments, determining involves consideration and / or manipulation of data or information, such as using a computer or other processing unit suitable for performing the relevant analysis. In some embodiments, determining involves receiving relevant information and / or material from a source. In some embodiments, determining involves comparing one or more features of a sample or entity to a comparable reference.

[0034] When applied to nucleic acids, the term "extension" or "elongation" refers to when additional nucleotides (or other similar molecules) are incorporated into the nucleic acid. For example, the nucleic acid is optionally extended by a biological catalyst that incorporates nucleotides, such as a polymerase that typically adds nucleotides at the 3' end of the nucleic acid.

[0035] The term "hybridizing" or "hybridization" refers to the annealing of one or more probes to an amplification product. Hybridization conditions generally include a temperature that is below the melting temperature of the probes but avoids non-specific hybridization of the probes.

[0036] Identity: As used herein, the term "identity" refers to the overall correlation between polymer molecules, for example, between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, if the sequence of the polymer molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical, then they are considered to be "substantially identical" to each other. For example, for the purpose of optimal comparison, the percentage of identity of two nucleic acid or polypeptide sequences can be calculated by comparing the two sequences (for example, a gap can be introduced in one or both of the first sequence and the second sequence for optimal comparison, and for the purpose of comparison, non-identical sequences can be ignored). In certain embodiments, the length of the sequence compared 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. Then compare the nucleotides at the corresponding positions. When the 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. Considering the number of each gap and the length of the gap, the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, which needs to be introduced to achieve the best comparison of the two sequences. The comparison of sequences and the determination of the percent identity between the two sequences can be completed using a mathematical algorithm. For example, the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17) can be used to determine the percent identity between two nucleotide sequences, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the nucleic acid sequence comparison performed with the ALIGN program uses a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the NWSgapdna.CMP matrix can be used to determine the percent identity between two nucleotide sequences using the GAP program in the GCG software package.

[0037] "Modified nucleotides" in the context of an oligonucleotide refers to changes wherein at least one nucleotide of the oligonucleotide sequence is replaced with a different nucleotide, thereby providing the oligonucleotide with desired properties. In the oligonucleotides described herein, exemplary modified nucleotides that can be substituted 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-deoxyxanthine nucleoside, pyrazolopyrimidine, pseudo-dU, nitropyrrole, nitroindole, 2′-O-methylribose-U, 2′-O-methylribose-C, N4-ethyl-dC, N6-methyl-dA, etc. Many other modified nucleotides that can be substituted in oligonucleotides are mentioned herein or are otherwise known in the art. In certain embodiments, the modified nucleotide substitution modifies the melting temperature (Tm) of the oligonucleotide relative to the melting temperature of the corresponding unmodified oligonucleotide. To further illustrate, in some embodiments, certain modified nucleotide substitutions can reduce non-specific nucleic acid amplification (e.g., minimize primer dimer formation, etc.), increase the yield of the expected target amplicon, etc. Examples of these types of nucleic acid modifications are described in, for example, U.S. Patent No. 6,001,611 (incorporated herein by reference). Examples of these types of nucleic acid modifications are described in, for example, U.S. Patent No. 6,001,611 (incorporated herein by reference).

[0038] The term "primer" as used herein is known to those skilled in the art and refers to an oligomeric compound capable of "priming" DNA synthesis by a template-dependent DNA polymerase, primarily to oligonucleotides, but also to modified oligonucleotides, i.e., for example, the 3'-end of the oligonucleotide provides a free 3'-OH group to which further "nucleotides" can be attached by the template-dependent DNA polymerase, thereby establishing a 3' to 5' phosphodiester bond, wherein deoxynucleoside triphosphates are used and pyrophosphate is thereby released. Therefore, there is no fundamental difference between "primer", "oligonucleotide" or "probe", except for the possible intended function.

[0039] Sample: As used herein, the term "sample" refers to a substance that is or contains a target composition for qualitative and / or quantitative evaluation. In some embodiments, the sample is a biological sample (i.e., from a living thing (e.g., a cell or organism)). In some embodiments, the sample is from a geological, aquatic, astronomical, or agricultural source. In some embodiments, the target source includes an organism, such as an animal or a human, or consists of it. In some embodiments, the sample used for forensic analysis is or contains biological tissue, biological fluid, organic or inorganic matter, such as, for example, clothing, dirt, plastic, water. In some embodiments, agricultural samples contain or consist of organic matter, such as leaves, petals, bark, wood, seeds, plants, fruits, etc.

[0040] Specificity: As used herein, the term "specificity" refers to the preference of an enzyme for a particular substrate.

[0041] Selectivity: As used herein, the term "selectivity" means the preference of an enzyme for one particular substrate over another.

[0042] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full range or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will appreciate that biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completion or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0043] Synthetic: As used herein, the term "synthetic" means produced by the hand of man, and therefore produced in a form that does not occur in nature, either because it has a structure that does not occur in nature, or because it is combined with one or more other components with which it is not combined in nature, or is not combined with one or more other components with which it is combined in nature.

[0044] The term "thermostable polymerase" refers to a thermostable polymerase, i.e., an enzyme that catalyzes the formation of a primer extension product complementary to the template and does not irreversibly denature when subjected to elevated temperatures for the time required to achieve denaturation of a double-stranded template nucleic acid. Typically, synthesis is initiated at the 3' end of each primer and proceeds in a 5' to 3' direction along the template strand. Thermostable polymerases have been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. However, polymerases that are not thermostable may also be used in PCR assays provided that the enzyme is supplemented.

[0045] The term "5' to 3' nuclease activity" refers to the activity of a nucleic acid polymerase, typically associated with nucleic acid strand synthesis, thereby removing nucleotides from the 5' end of the nucleic acid strand.

[0046] II. Detailed Description of Certain Embodiments

[0047] Diagnosing infection with a serotype (L serotype) of Chlamydia trachomatis that causes lymphogranuloma venereum (LGV) by nucleic acid amplification provides a method for quickly and accurately detecting bacterial infection. This article describes an RT-PCR assay for detecting the L serotype in a sample. Primers and probes for detecting the pmpH gene of the L serotype of Chlamydia trachomatis, as well as products or kits containing such primers and probes, are provided. Compared with other methods, RT-PCR is used to detect the increased sensitivity of the L serotype, and the improved features of RT-PCR, including real-time detection of sample accommodation and amplified products, make it feasible to implement this technology in clinical laboratories for routine diagnosis of infection with the serotype of LGV caused by Chlamydia trachomatis.

[0048] The L serotypes of Chlamydia trachomatis are an emerging pathogen of men who have sex with men in the industrial setting, often associated with HIV co-infection. The L serotypes cause acute illness that may persist for a prolonged period of time and, if untreated, may facilitate HIV transmission. Clinical care, surveillance, and research are severely hampered by the lack of a widely available, rapid, standardized test for the diagnosis of LGV.

[0049] Recently, a number of PCR-based test systems have been published that aid in the identification of LGV-associated L serotypes. Some of these involve the detection and typing of nearly all serotypes of C. trachomatis in a given sample. Differentiating the L serotypes of C. trachomatis from other serotypes has important clinical implications as different antibiotic treatment regimens are required. Detection is usually based on the amplification of the pmpH gene, in which a unique deletion in the L serotypes facilitates the design of an L serotype-specific PCR.

[0050] The present disclosure provides a method for detecting the L serotype of Chlamydia trachomatis by amplifying a first region or a second region of a pmpH nucleic acid sequence, for example, from the L serotype (see Figure 2 ). Nucleic acid sequences of pmpH genes from various serotypes of Chlamydia trachomatis are available (e.g., GenBank Accession No. AY184167 from serotype L1, AY184168 from serotype L2, AY184169 from serotype L3). In addition, as described above, the sequence of the pmpH gene of the L1 serotype can be a nucleic acid as shown in Genbank Accession No. HE601950 (gene: L1440_00934; CDS: CCP62925.1; position: 1032228..1035245, length: 3,018nt). Specifically, primers and probes for amplifying and detecting the pmpH gene from an L serotype nucleic acid molecule target are provided by the embodiments in the present disclosure.

[0051] The present disclosure is based, at least in part, on the surprising discovery that the pmpH gene provides at least two regions that can be used to selectively and specifically detect the L serotype of Chlamydia trachomatis relative to other non-LGV causing serotypes. Figure 2 As shown, region R1, which is a region of 400-600 nucleotides from the 5' end of the pmpH gene, contains a 36 bp deletion in the L serotype (marked as a grey box in the L serotype), while all other serotypes lack this deletion. Therefore, in a first aspect, a set of oligonucleotide primers are positioned to hybridize to the R1 region in order to amplify this target region ( Figure 2 ), while selecting oligonucleotide probes that hybridize across the deletion site. Using this setting, the oligonucleotide probes that hybridize across the deletion site specifically hybridize to the deletion site that is only present in the L serotype, and therefore, signals are detected only for the L serotypes (L1, L2, L2b, L3).

[0052] In a second aspect, a second region was surprisingly identified, such as Figure 2Region R2 shown, which is a region near the 3' end of the pmpH gene and 2600 to 2900 nucleotides from the 5' end of the pmpH gene (in some embodiments, 2600 to 2800 nucleotides from the 5' end of the pmpH gene). Herein, identification of the L serotype does not rely on the specific determination of deletions that are present only in the L serotype. Instead, region R2 provides a variety of nucleotides that are different between the sequences of the L serotype and all other serotypes. Herein, oligonucleotide primers and / or oligonucleotide probes are selected to effectively hybridize with the L serotype and not with other serotypes. In particular, oligonucleotide probes can be selected to hybridize only with the L serotype and not with all other serotypes (see Figure 2 Triangles indicate useful hybridization sites of oligonucleotide primer sets and / or oligonucleotide probes). Herein, the oligonucleotide probe can be selected to hybridize with at least a portion of nucleotides 2660 to 2740 of the pmpH gene of the L1 serotype or corresponding regions of different L serotypes, more specifically, to hybridize with at least a portion of nucleotides 2670 to 2730 of the pmpH gene of the L1 serotype or corresponding regions of different L serotypes, or even more specifically, to hybridize with at least a portion of nucleotides 2680 to 2720 of the pmpH gene of the L1 serotype or corresponding regions of different L serotypes.

[0053] Table 1: Primers and probes for the pmpH gene of Chlamydia trachomatis serotype L

[0054]

[0055]

[0056] As shown in Table 1, F primer = forward primer; R primer = reverse primer; F / FAM / HEX = dye; Q / ZEN / BHQ2 = quencher; 3IABkF′Q = blocker; t_BB_dA = tert-butylbenzyl A; Spc_C3 = 3′C3 spacer.

[0057] More specifically, as shown in Table 1, suitable primers and probe oligonucleotides are provided, each comprising a nucleic acid having a sequence selected from SEQ ID NOs: 1-11, substantially identical variants thereof (wherein the variant has at least, for example, 80%, 90% or 95% sequence identity with one of SEQ ID NOs: 1-11 or a complementary sequence to SEQ ID NOs: 1-11), and variants. Herein, primers and probe oligonucleotides that hybridize to region R1 are provided, each comprising a nucleic acid having a sequence selected from SEQ ID NOs: 1-5, substantially identical variants thereof (wherein the variant has at least, for example, 80%, 90% or 95% sequence identity with one of SEQ ID NOs: 1-5 or a complementary sequence to SEQ ID NOs: 1-5), and variants. In addition, primers and probe oligonucleotides that hybridize to region R2 are also provided, each comprising a nucleic acid having a sequence selected from SEQ ID NOs: 6-11, substantially identical variants thereof (wherein the variant has at least, for example, 80%, 90% or 95% sequence identity with one of SEQ ID NOs: 6-11 or the complementary sequence of SEQ ID NOs: 6-11), and variants.

[0058] In one embodiment, the primer and probe sets described in Table 1 are used to provide detection of one or more L serotypes of Chlamydia trachomatis in a biological sample suspected of containing the one or more L serotypes. The primer and probe sets may include or consist of primers and probes specific for the pmpH gene from an L serotype nucleic acid sequence, which may contain or consist of the nucleic acid sequences of SEQ ID NOs: 1-11.

[0059] Functionally active variants of any primer and / or probe of SEQ ID NO: 1-11 can be identified by using the primer and / or probe in the disclosed methods. Functionally active variants of primers and / or probes of any of SEQ ID NO: 1-11 relate to primers and / or probes that provide similar or higher specificity and sensitivity in the described methods or kits compared to the corresponding sequences of SEQ ID NO: 1-11.

[0060] The variant may differ from the sequence of SEQ ID NOs: 1-11, for example, by one or more nucleotide additions, deletions or substitutions, such as one or more nucleotide additions, deletions or substitutions at the 5' end and / or 3' end of the corresponding sequence of SEQ ID NOs: 1-11. As described above, the primer (and / or probe) may be chemically modified; that is, the primer and / or probe may include a modified nucleotide or a non-nucleotide compound. The probe (or primer) is then a modified oligonucleotide. "Modified nucleotides" (or "nucleotide analogs") differ from natural "nucleotides" in some modifications, but still consist of a base or base-like compound, a pentofuranosyl sugar or a pentofuranosyl sugar-like compound, a phosphate moiety or a phosphate-like moiety, or a combination thereof. For example, a "label" may be attached to the base moiety of a "nucleotide", thereby obtaining a "modified nucleotide". The natural base in a "nucleotide" may also be replaced by, for example, 7-deazapurine, thereby also obtaining a "modified nucleotide". The terms "modified nucleotide" or "nucleotide analogs" are used interchangeably in this application. "Modified nucleosides" (or "nucleoside analogs") differ from natural nucleosides by certain modifications in the manner as outlined above for "modified nucleotides" (or "nucleotide analogs").

[0061] Furthermore, in some applications, it may be beneficial to combine the determination of the presence of nucleic acid of the L serotype of Chlamydia trachomatis with the determination of the presence of nucleic acid of all serotypes of Chlamydia trachomatis. Figure 2When the method is set up, the method may be particularly beneficial by detecting the absence of L serotype in region R1 using a probe spanning the absence or detecting L serotype using an oligonucleotide primer and / or an oligonucleotide probe that hybridizes in region R2 of L serotype but does not hybridize to other serotypes. In this article, a signal is detected only for the presence of L serotype, while no signal is detected for the presence of all other serotypes. However, the lack of signal may also be caused by a failure of the amplification / detection run, which may lead to a false negative result. To this end, oligonucleotide primers and probes are also provided herein, which allow all serotypes of Chlamydia trachomatis (CT) to be determined. As shown in Table 1, an exemplary oligonucleotide set is provided, which includes at least one pan-CT forward primer, which includes 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, which includes 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, which includes a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16 and 17 or their complements. It is disclosed herein that amplification and detection reactions using pan-CT oligonucleotides can be performed in parallel reactions using a second portion of the sample. However, the pan-CT oligonucleotides may also be used in a multiplex reaction in the same vial as the reaction for determining the L serotype for amplification and detection reactions. Herein, the fluorophore (or donor-acceptor pair) used to label the pan-CT oligonucleotide probe is different from the fluorophore (or donor-acceptor pair) used to label the L serotype-specific oligonucleotide probe. More specifically, the hybridization step may include contacting the amplification product with two or more probes each labeled with a different donor fluorescent moiety (e.g., a first donor fluorescent moiety and a second 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 (e.g., a first donor fluorescent moiety and a second donor fluorescent moiety) and the acceptor moiety of the probe for both the different donor fluorescent moieties and the corresponding acceptor moieties, wherein 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 Chlamydia trachomatis (CT) serotype in the sample.

[0062] For example, computer programs such as OLIGO (Molecular Biology Insights Inc., Cascade, Colo.) can be used to design the oligonucleotides of the nucleic acid molecules of the pmpH gene of the amplification from the L serotype nucleic acid sequence, including modified oligonucleotides and oligonucleotide analogs. When designing the oligonucleotide to be used as an amplification primer, important features include but are not limited to the amplification product of appropriate size for the convenience of detection (for example, by electrophoresis), the similar melting temperature of the members of a pair of primers and the length of each primer (that is, the primer needs to be long enough to anneal with sequence specificity and start synthesis, but it cannot be too long that the fidelity is reduced during the synthesis of the oligonucleotide). Typically, the length of the oligonucleotide primer is 8 to 50 nucleotides (for example, a length of 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48 or 50 nucleotides).

[0063] In addition to a set of primers, these methods may also use one or more probes in order to detect the presence or absence of serotype L. The term "probe" refers to synthetic or biologically produced nucleic acids (DNA or RNA) that contain specific nucleotide sequences by design or selection that allow them to specifically (i.e., preferentially) hybridize to a "target nucleic acid," in this case, the pmpH gene from a serotype L nucleic acid, under a defined predetermined stringency. A "probe" may be referred to as a "detection probe," meaning that it detects a target nucleic acid.

[0064] In some embodiments, the probe can be labeled with at least one fluorescent marker. In one embodiment, the probe for the pmpH gene from the L serotype can be labeled with a donor fluorescent moiety (e.g., a fluorescent dye) and a corresponding acceptor fluorescent moiety (e.g., 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.

[0065] Designing oligonucleotides used as probes can be carried out in a manner similar to primer design. Embodiments can use a single probe or a pair of probes to detect amplified products. According to an embodiment, the probe used may include at least one label and / or at least one quencher moiety. Like primers, probes generally have similar melting temperatures, and the length of each probe must be sufficient for sequence-specific hybridization to occur, but not too long so that fidelity is reduced during synthesis. The length of the oligonucleotide probe is generally 15 to 30 (e.g., 16, 18, 20, 21, 22, 23, 24 or 25) nucleotides.

[0066] The construct containing the target gene nucleic acid molecule can be propagated in a host cell. As used herein, the term host cell is intended to include prokaryotes and eukaryotes, such as yeast, plants and animal cells. Prokaryotic hosts may include Escherichia coli (E. coli), Salmonella typhimurium (Salmonella typhimurium), Serratia marcescens (Serratiamarcescens) and Bacillus subtilis (Bacillus subtilis). Eukaryotic hosts include yeast (such as Saccharomyces cerevisiae (S. cerevisiae), Schizosaccharomyces pombe (S. pombe), Pichia pastoris (Pichia pastoris)), mammalian cells (such as COS cells or Chinese hamster ovary (CHO) cells), insect cells and plant cells (such as Arabidopsis thaliana (Arabidopsis thaliana) and tobacco (Nicotiana tabacum)). Any technique known to those of ordinary skill in the art can be used to introduce the construct into a host cell. For example, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection and virus-mediated nucleic acid transfer are common methods for introducing nucleic acid into host cells. In addition, naked DNA can be delivered directly to cells (see, e.g., U.S. Patent Nos. 5,580,859 and 5,589,466).

[0067] U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159 and 4,965,188 disclose conventional PCR techniques. PCR typically employs two oligonucleotide primers that bind to a selected nucleic acid template (e.g., DNA or RNA). Primers useful in some embodiments include oligonucleotides that can serve as starting points for nucleic acid synthesis within the described target nucleic acid sequence (e.g., SEQ ID NOs: 1-4). The primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically. For maximum efficiency in amplification, the primer is preferably single-stranded, but the primer can be double-stranded. The double-stranded primer is first denatured (i.e., treated) to separate the strands. One method of denaturing a double-stranded nucleic acid is by heating.

[0068] If the template nucleic acid is double-stranded, the two strands must be separated before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturation method, including physical, chemical or enzymatic methods. One method of separating nucleic acid strands involves heating the nucleic acid until it is mostly denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). 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 are typically in the range of about 90°C to about 105°C for a period of time, depending on the characteristics of the reaction such as temperature and nucleic acid length. Denaturation is typically performed for about 30 seconds to 4 minutes (e.g., 1 minute to 2 minutes 30 seconds, or 1.5 minutes).

[0069] If the double-stranded template nucleic acid is denatured by heating, the reaction mixture is cooled to a temperature that promotes annealing of each primer to the target sequence on the described target gene nucleic acid molecule. The temperature for annealing is generally 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 that promotes or optimizes polymerase activity, that is, a temperature that is sufficient to extend from the annealed primer to generate a product complementary to the template nucleic acid. The temperature should be sufficient to synthesize an extension product from each primer annealed to the nucleic acid template, but should not be so high that the extension product is denatured from its complementary template (e.g., the temperature for extension generally ranges from 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).

[0070] PCR assays can employ template nucleic acids, such as RNA or DNA (cDNA). The template nucleic acid does not need to be purified; it can be a small portion of a complex mixture, such as Chlamydia trachomatis nucleic acid contained in human cells. Chlamydia trachomatis nucleic acid molecules can be extracted from biological samples by conventional techniques, such as those 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 many sources, such as plasmids, or natural sources, including bacteria, yeast, viruses, organelles, or higher organisms, such as plants or animals.

[0071] 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, the chain extension reaction typically includes 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 15 mM MgCl2, 0.001% (w / v) gelatin, 0.5-1.0 μg denatured template DNA, 50 pmol of each oligonucleotide primer, 2.5 U Taq polymerase, and 10% DMSO). The reaction typically contains 150 to 320 μM of each of dATP, dCTP, dTTP, dGTP, or one or more analogs thereof.

[0072] The newly synthesized chain forms a double-stranded molecule that can be used for subsequent reaction steps. The steps of chain separation, annealing and extension can be repeated as needed to produce a desired amount of amplified products corresponding to the target nucleic acid molecules. The limiting factor in the reaction is the amount of primers, thermostable enzymes and nucleoside triphosphates present in the reaction. Preferably, at least one cycle step (i.e., denaturation, annealing and extension) is repeated. In order to be used for detection, the number of cycles will depend on the properties of, for example, the sample. If the sample is a complex mixture of nucleic acid, more cycles will be needed to increase the target sequence that is enough to detect. Typically, the cycle step repeats at least about 20 times, but can be repeated up to 40,60 or even 100 times.

[0073] The FRET technique (see, e.g., U.S. Pat. Nos. 4,996,143, 5,565,322, 5,849,489, and 6,162,603) is based on the concept that when a donor fluorescent moiety and a corresponding acceptor fluorescent moiety are located within a certain distance of each other, energy transfer occurs between the two fluorescent moieties, which can be visualized or otherwise detected and / or quantified. When the donor is excited by light radiation having a suitable wavelength, the donor typically transfers energy to the acceptor. The acceptor typically re-emits the transferred energy in the form of light radiation having a different wavelength. In some systems, non-fluorescent energy can be transferred between the donor and acceptor moieties by biomolecules that include a substantially non-fluorescent donor moiety (see, e.g., U.S. Pat. No. 7,741,467).

[0074] In one example, the oligonucleotide probe may contain a donor fluorescent moiety and a corresponding quencher, which may or may not be fluorescent and dissipates the transferred energy in a form other than light. When the probe is intact, energy transfer typically occurs between the two fluorescent moieties, so that the fluorescence emission from the donor fluorescent moiety is quenched. During the extension step of the polymerase chain reaction, the probe bound to the amplification product is cleaved by the 5' to 3' nuclease activity of, for example, Taq polymerase, so that the fluorescence emission of the donor fluorescent moiety is no longer quenched. Exemplary probes for this purpose are described in, for example, U.S. Patent Nos. 5,210,015, 5,994,056 and 6,171,785. Commonly used donor-acceptor pairs include FAM-TAMRA pairs. Commonly used quenchers are DABCYL and TAMRA. Commonly used dark quenchers include BlackHole Quenchers TM (BHQ) (Biosearch Technologies, Inc., Novato, Cal.), Iowa Black TM (Integrated DNATech., Inc., Coralville, Iowa), BlackBerry TM Quencher 650 (BBQ-650) (Berry & Assoc., Dexter, Mich.).

[0075] In another example, two oligonucleotide probes (each comprising a fluorescent moiety) can hybridize to the amplified product at a specific position determined by the complementarity of the oligonucleotide probes to the Chlamydia trachomatis target nucleic acid sequence. After the oligonucleotide probes hybridize to the amplified product nucleic acid at the appropriate position, a FRET signal is generated. The hybridization temperature can be in the range of about 35°C to about 65°C for about 10 seconds to about 1 minute.

[0076] Fluorescence analysis can be performed using, for example, a photon counting epifluorescence microscope system (containing appropriate dichroic mirrors and filters for monitoring a specific range of fluorescence emission), a photon counting photomultiplier tube system, or a fluorimeter. Excitation can be performed using an argon ion laser, a high intensity mercury (Hg) arc lamp, a fiber optic light source, or other high intensity light source that is appropriately filtered to excite in the desired range to initiate energy transfer or allow direct detection of fluorophores.

[0077] As used herein with respect to donors and corresponding acceptor fluorescent moieties, "corresponding" refers to an acceptor fluorescent moiety or dark quencher having an absorption spectrum that overlaps the emission spectrum of the donor fluorescent moiety. The maximum wavelength of the emission spectrum of the acceptor fluorescent moiety should be at least 100 nm greater than the maximum wavelength of the excitation spectrum of the donor fluorescent moiety. Therefore, efficient non-radiative energy transfer can be generated between them.

[0078] The fluorescent donor and the corresponding acceptor moiety are generally selected for: (a) high efficiency Forster energy transfer; (b) large final Stokes shift (>100 nm); (c) shifting the emission to the red part of the visible spectrum (>600 nm) as much as possible; and (d) shifting the emission to a wavelength higher than the Raman water fluorescence emission produced by excitation at the donor excitation wavelength. For example, the following donor fluorescent moieties can be selected: they have their excitation maximum near a laser line (e.g., helium-cadmium 442 nm or argon 488 nm), have a high extinction coefficient, a high quantum yield, and their fluorescence emission overlaps well with the excitation spectrum of the corresponding acceptor fluorescent moiety. The corresponding acceptor fluorescent moiety can be selected to have 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 part of the visible spectrum (>600 nm).

[0079] Representative donor fluorescent moieties that can be used with various acceptor fluorescent moieties in FRET techniques include fluorescein, fluorescein yellow, B-phycoerythrin, 9-acridine isothiocyanate, fluorescein yellow VS, 4-acetamido-4′-isothio-cyanatostilbene-2,2′-disulfonic acid, 7-di-ethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarin, succinimidyl 1-pyrene butyrate, and 4-acetamido-4′-isothiocyanatostilbene-2,2′-disulfonic acid derivatives. Representative acceptor fluorescent moieties, depending on the donor fluorescent moiety used, include LC Red 640, LC Red 705, Cy5, Cy5.5, lissamine rhodamine B sulfonyl chloride, tetramethylrhodamine isothiocyanate, rhodamine x isothiocyanate, erythrosine isothiocyanate, fluorescein, diethylenetriamine pentaacetate or other chelates of lanthanide ions (e.g., europium or terbium). Donor and acceptor fluorescent moieties can be obtained, for example, from Molecular Probes (Junction City, Oreg.) or Sigma Chemical Co. (St. Louis, Mo.).

[0080] The donor and acceptor fluorescent moieties can be attached to the appropriate probe oligonucleotides via linker arms. The length of each linker arm is important because it affects the distance between the donor and acceptor fluorescent moieties. The length of the linker arm is measured in angstroms. The distance from the nucleotide base to the fluorescent moiety is measured in units of . to about The linker arm may be of the type described in WO 84 / 03285. WO 84 / 03285 also discloses methods for attaching a linker arm to a specific nucleotide base, and for attaching a fluorescent moiety to a linker arm.

[0081] An acceptor fluorescent moiety, such as LC Red 640, can be combined with an oligonucleotide containing an amino linker, such as C6-aminophosphoramidites available from ABI (Foster City, Calif.) or Glen Research (Sterling, VA), to produce, for example, an LC Red 640 labeled oligonucleotide. Frequently used linkers for coupling donor fluorescent moieties, such as fluorescein, to oligonucleotides include thiourea linkers (FITC-derived, such as fluorescein-CPG's from Glen Research or ChemGene (Ashland, Mass.)), amide linkers (fluorescein-NHS-ester derived, such as CX-fluorescein-CPG from BioGenex (San Ramon, Calif.), or 3'-amino-CPGs that require coupling of fluorescein-NHS-ester after oligonucleotide synthesis.

[0082] Detection of L serotype of Chlamydia trachomatis

[0083] The invention provides a method for detecting the presence or absence of the L serotype of Chlamydia trachomatis in a biological sample or a non-biological sample. The method provided avoids the problem of sample contamination, false negative and false positive. These methods include carrying out at least one cycle step and a FRET detection step, and the cycle step includes using at least one pair of primers from a part of the pmpH gene of the L serotype target nucleic acid molecule from sample amplification. Carry out multiple cycle steps, preferably in a thermal cycler. These methods can use gene primers and probes to perform to detect the presence of the pmpH gene from the L serotype, and this indicates that one or more L serotypes exist in the sample.

[0084] As described herein, amplification products can be detected using labeled hybridization probes utilizing FRET technology. One form of FRET utilizes The technique is used to detect the presence or absence of an amplification product, and thus the presence or absence of one or more L serotypes. The technology utilizes a single-stranded hybridization probe that is labeled with, for example, a fluorescent dye and a quencher, which may or may not be fluorescent. When the first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to the second fluorescent moiety according to the FRET principle. The second fluorescent moiety is typically a quencher molecule. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target DNA (i.e., the amplified product) and is degraded by, for example, the 5' to 3' nuclease activity of Taq polymerase during the subsequent extension phase. As a result, the fluorescent moiety and the quencher moiety become spatially separated from each other. Therefore, after exciting the first fluorescent moiety in the absence of a quencher, fluorescent emission from the first fluorescent moiety can be detected. For example, ABI 7700Sequence Detection System (Applied Biosystems) use The invention relates to a method for detecting the presence or absence of an L serotype in a sample and is suitable for performing the method described herein.

[0085] Molecular beacons combined with FRET can also be used to detect the presence of amplification products using real-time PCR methods. Molecular beacon technology uses hybridization probes labeled by a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is usually a quencher, and the fluorescent label is generally located at each end of the probe. Molecular beacon technology uses probe oligonucleotides with sequences that allow the formation of secondary structures (such as hairpins). As a result of the formation of the secondary structure in the probe, when the probe is in solution, the two fluorescent moieties are close in space. After hybridization with the target nucleic acid (i.e., the amplification product), the secondary structure of the probe is destroyed, and the fluorescent moieties become separated from each other, so that after being excited with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.

[0086] Another common form of FRET technology is to use two hybridization probes. Each probe can be labeled with a different fluorescent moiety and is usually designed to hybridize in close proximity to each other in a target DNA molecule (e.g., an amplification product). The donor fluorescent moiety, such as fluorescein, is detected at 470 nm. The instrument's light source excites. During FRET, the fluorescein transfers its energy to an acceptor fluorescent moiety, such as 640 (LC Red 640) or 705 (LC Red 705). The acceptor fluorescent part then emits light of a longer wavelength, The optical detection system of the instrument detects. Effective FRET occurs only when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps the absorption spectrum of the acceptor fluorescent moiety. The intensity of the emitted signal can be related to the number of original target DNA molecules. If amplification of the target nucleic acid occurs and an amplification product is produced, the hybridization step produces a detectable signal based on FRET between the probe pair members.

[0087] Typically, 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 sample collection, shipping delays, improper shipping conditions, or the use of certain collection swabs (calcium alginate or aluminum shafts) are all conditions that may affect the success and / or accuracy of the test results. Using the methods disclosed herein, FRET indicates infection with L serotypes of Chlamydia trachomatis within, for example, 45 cycle steps.

[0088] Representative biological samples that can be used to practice these methods include, but are not limited to, blood, plasma, serum, liver samples, skin swabs, nasal swabs, wound swabs, blood cultures, skin and soft tissue infections. Collection and storage methods of biological samples are known to those skilled in the art. Biological samples can be processed (e.g., by nucleic acid extraction methods and / or test kits as known in the art) to release nucleic acids, or in some cases, biological samples can be directly contacted with PCR reaction components and suitable oligonucleotides.

[0089] Melting curve analysis is an additional step that can be included in the cycle curve. Melting curve analysis is based on the fact that DNA melts at a characteristic temperature called melting temperature (Tm), which is defined as the temperature at which half of the DNA duplex separates into single strands. The melting temperature of DNA depends mainly on its nucleotide composition. Therefore, DNA molecules rich in G and C nucleotides have 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, by detecting the temperature at which the signal is generated, the annealing temperature of the probe can be determined. The melting temperature of the probe from the corresponding amplification product can confirm the presence or absence of the target nucleic acid in the sample.

[0090] In each thermal cycler operation, control samples can also be circulated. Positive control samples can use, for example, control primers and control probes to amplify target nucleic acid control templates (different from the amplified product of the target gene). Positive control samples can also be amplified, for example, plasmid constructs containing target nucleic acid molecules. Such plasmid controls can be used in internal amplification (for example, in a sample) or in a separate sample operation in parallel with a patient sample to amplify the same primers and probes as used to detect the expected target. Such controls are indicators of amplification, hybridization and / or FRET reaction success or failure. Each thermal cycler operation can also include a negative control, for example, lacking target template DNA. Negative controls can measure contamination. This ensures that the system and reagents do not produce false positive signals. Therefore, control reactions can be easily determined, for example, the ability of primers to anneal and initiate extension with sequence specificity, and the ability of probes to hybridize with sequence specificity and FRET to occur.

[0091] In one embodiment, the method includes steps to avoid contamination. For example, enzymatic methods utilizing uracil-DNA glycosylase to reduce or eliminate contamination between one thermal cycler run and the next are described in U.S. Pat. Nos. 5,035,996, 5,683,896, and 5,945,313.

[0092] These methods can be practiced using conventional PCR methods combined with FRET technology. In one embodiment, The following patent application describes Real-time PCR used in the techniques: WO 97 / 46707, WO 97 / 46714 and WO 97 / 46712.

[0093] It can be operated using a PC workstation and can use the Windows NT operating system. When the machine places the capillaries on the optical unit in sequence, the signal from the sample can be obtained. The software can display the fluorescence signal in real time immediately after each measurement. The fluorescence acquisition time is 10-100 milliseconds (msec). After each cycle step, the quantitative display of fluorescence and cycle number can be continuously updated for all samples. The generated data can be stored for further analysis.

[0094] As an alternative to FRET, double-stranded DNA binding dyes such as fluorescent DNA binding dyes (e.g., Green or Gold (Molecular Probes)), can detect amplification products. When interacting with double-stranded nucleic acids, such fluorescent DNA binding dyes emit fluorescent signals after being excited with light of a suitable wavelength. Double-stranded DNA binding dyes (such as nucleic acid intercalating dyes) can also be used. When double-stranded DNA binding dyes are used, melting curve analysis is usually performed to confirm the presence of amplification products.

[0095] It should be understood that embodiments of the present disclosure are not limited to the configuration of one or more commercially available instruments.

[0096] Products / Kits

[0097] Embodiments of the present disclosure further provide products or test kits for detecting L serotypes. Products can include primers and probes for detecting L serotypes, and suitable packaging materials. Representative primers and probes for detecting the pmpH gene of L serotypes can hybridize with target nucleic acid molecules. In addition, the test kit can also include reagents and materials required for DNA fixation, hybridization and detection of suitable packaging, such as solid supports, buffers, enzymes and DNA standards. This paper discloses methods for designing primers and probes, and provides representative examples of primers and probes for amplifying target nucleic acid molecules and hybridizing therewith.

[0098] The article can also include one or more fluorescent moieties for labeling probes, or alternatively, the probe provided with the kit can be labeled. For example, the article can include donor and / or acceptor fluorescent moieties for labeling target gene probes. Examples of suitable FRET donor fluorescent moieties and corresponding acceptor fluorescent moieties are provided above.

[0099] The article of manufacture may also include a package insert or package label having instructions for detecting the L serotype in a sample using primers and probes for the pmpH gene from the L serotype. The article of manufacture may additionally include reagents (e.g., buffers, polymerases, cofactors, or agents to prevent contamination) for implementing the methods disclosed herein. Such reagents may be specific to one of the commercially available instruments described herein.

[0100] Embodiments of the present disclosure will be further described in the following examples, which do not limit the scope of the invention described in the claims.

[0101] It is noteworthy that embodiments according to the method of the present disclosure may include one or more additional steps or omit one or more described steps of the method. In general, the method may be modified in any suitable manner that still achieves the result of selectively and specifically detecting one or more L serotypes of Chlamydia trachomatis in a sample. Other variations of the method falling within the scope of the present disclosure will become apparent from the additional examples and descriptions included herein.

[0102] Examples

[0103] The following examples are intended to be illustrative and are not intended to be limiting in any way.

[0104] Example 1:

[0105] This example describes the PCR experimental conditions used for RT-PCR detection of the pmpH gene target. The 6800 / 8800 system (Roche Molecular Systems, Inc., Pleasanton, CA) detected the pmpH gene target by RT-PCR. The final concentrations and thermal profiles of the amplification reagents used for the PCR amplification reaction are shown in Table 2:

[0106] Table 2: PCR reagents and thermal profiles Mg reagent (R1)

[0107]

[0108] Master Mix (R2) - General

[0109]

[0110] PCR thermal cycle parameters

[0111]

[0112] As shown in Table 2, Test Conc = test concentration; rxn = reaction; Temp = temperature; mm = minutes; ss = seconds; sec = seconds.

[0113] The preparative PCR program includes initial denaturation and incubation at 55°C, 60°C and 65°C for reverse transcription of the RNA template. Incubation at three temperatures has the following advantageous effects at the same time: at lower temperatures, slightly mismatched target sequences (such as genetic variants of an organism) are also transcribed, while at higher temperatures, the formation of RNA secondary structures is inhibited, making transcription more efficient. The PCR cycles are divided into two measurements, both of which apply a one-step setup (combining annealing and extension). The first 5 cycles at 55°C allow increased inclusiveness by preamplifying slightly mismatched target sequences, while the 45 cycles of the second measurement provide increased specificity by using an annealing / extension temperature of 58°C.

[0114] Example 2:

[0115] The results of PCR assays for detecting L serotypes of Chlamydia trachomatis using a set of primers and probes are shown in Table 3. Master mix (MMx) 1 used a forward primer of SEQ ID NO: 1, a reverse primer of SEQ ID NO: 3, and a probe of SEQ ID NO: 5. MMx 2 used a forward primer of SEQ ID NO: 2, a reverse primer of SEQ ID NO: 4, and a probe of SEQ ID NO: 5. MMx 3 used a forward primer of SEQ ID NO: 7, a reverse primer of SEQ ID NO: 9, and a probe of SEQ ID NO: 11. Template plasmids carrying consensus nucleic acid sequences 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 ranging from 1.00E+01 copies / reaction to 1.00E+07 copies / reaction. Preliminary testing indicated that these primer / probe pairs produced good results in terms of sensitivity and specificity for L serotypes (ie, serotypes L1, L2, and L3).

[0116] Table 3: PCR assay results

[0117]

[0118] Referring to Table 3, NaN = not a number; Conc = concentration; Avg = average; cp / rxn = copy number / reaction; Ct = cycle threshold; RFI = relative fluorescence intensity.

[0119] The present invention is presented in several different embodiments in the following description with reference to the accompanying drawings, wherein like reference numerals represent the same or similar elements. References throughout this specification to "one embodiment," "an embodiment," and similar language mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in at least one embodiment of the present invention. Thus, phrases "in one embodiment," "in an embodiment," and similar language appearing throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0120] In one or more embodiments, the described features, structures or characteristics of the present invention may be combined in any suitable manner. In the following description, many specific details are listed to provide a thorough understanding of the embodiments of the system. However, those skilled in the relevant art will recognize that the system and method can be practiced without one or more specific details or using other methods, parts, materials, etc. In other cases, well-known structures, materials or operations are not shown or described in detail to avoid blurring aspects of the present invention. Therefore, the foregoing description is intended to be exemplary and does not limit the scope of the inventive concept.

[0121] Each reference identified in this application is incorporated herein by reference in its entirety.

Claims

1. A method for detecting Chlamydia trachomatis causing lymphogranuloma venereum in a sample trachomatis) serovar (L serovar), the method comprising: (a) performing an amplification step, the amplification step comprising contacting the sample with at least one set of primers designed to target the polymorphic membrane protein H (pmpH) gene of the L serovar to produce an amplification product if the L serovar is present in the sample; (b) performing a hybridization step comprising contacting said amplification product in the presence of said L serovar in said sample with at least one detectable probe targeting said pmpH gene of said L serovar; and (c) performing a detection step, said detection step comprising detecting the presence or absence of said amplification product, wherein the presence of said amplification product indicates the presence of said L serovar in said sample, and wherein the absence of said amplification product indicates the absence of said L serovar in said sample, wherein the at least one set of primers and the at least one detectable probe comprise: a forward primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 6 and 7, or a combination thereof; a reverse primer comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 4, 8, and 9, or a combination thereof; and A probe comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 5, 10 and 11, or their complementary sequences.

2. The method of claim 1 , wherein the hybridization step comprises contacting the amplification product with the detectable probe, the detectable probe being labeled with a donor fluorescent moiety and a corresponding acceptor moiety; and the detection step comprises detecting the presence or absence of fluorescence resonance energy transfer (FRET) between the donor fluorescent moiety and the acceptor moiety of the probe, wherein the presence or absence of fluorescence indicates the presence or absence of the L serovar in the sample.

3. The method according to any one of claims 1 to 2, wherein the amplification step employs a polymerase having 5' to 3' nuclease activity.

4. The method according to any one of claims 1 to 3, wherein the sample is a biological sample.

5. The method of claim 4, wherein the biological sample is a vaginal swab sample, a clinician-collected vaginal swab sample, an endocervical swab sample, an oropharyngeal (throat) swab sample, or an anorectal swab sample.

6. The method according to any one of claims 1 to 5, wherein the forward primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 and 2, or a combination thereof; the reverse primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3 and 4, or a combination thereof; and the probe comprises a nucleic acid sequence of SEQ ID NO: 5 or its complementary sequence.

7. according to the method described in any one of claims 1 to 5, wherein the forward primer comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:6 and 7, or a combination thereof; the reverse primer comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:8 and 9, or a combination thereof; and the probe comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:10 and 11, or its complementary sequence.

8. 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 its complementary sequence.

9. A method for detecting a serovar of Chlamydia trachomatis that causes lymphogranuloma venereum (L serovar) in a sample, the method comprising: (a) performing an amplification step, the 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 serovar to produce an amplification product if the L serovar is present in the sample; (b) performing a hybridization step comprising contacting said amplification product in the presence of said L serovar in said sample with at least one detectable probe targeting said pmpH gene of said L serovar; and (c) performing a detection step, said detection step comprising detecting the presence or absence of said amplification product, wherein the presence of said amplification product indicates the presence of said L serovar in said sample, and wherein the absence of said amplification product indicates the absence of said L serovar in said sample, wherein said at least one set of primers is designed to target a region comprising at least a portion of the last 1000 nucleotides at the 3' end of said pmpH gene of said L serovar.

10. The method of claim 9, wherein the at least one set of primers is designed to target a region comprising at least a portion of the last 500 nucleotides of the 3' end of the pmpH gene of the L serovar.

11. The method of claim 9, wherein the at least one set of primers is designed to target a region comprising at least a portion of nucleotides 2600 to 2900 of the pmpH gene of an L1 serovar or a corresponding region of a different L serovar; or 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 L1 serovar or a corresponding region of a different L serovar.

12. A kit for detecting the pmpH gene of a serovar of Chlamydia trachomatis that causes lymphogranuloma venereum (L serovar) in a sample, the kit comprising an amplification reagent, the amplification reagent comprising: (a) a DNA polymerase having 5' to 3' nuclease activity; (b) Nucleotide monomers; (c) at least one pair of primers and at least one detectable probe, wherein the at least one pair of primers and the at least one detectable probe comprise: i. a forward primer comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1, 2, 6 and 7, or any combination thereof; ii. a reverse primer comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 3, 4, 8 and 9, or a combination thereof; and iii. a detectable probe comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 5, 10 and 11, or a complementary sequence thereof.

13. The kit of claim 12, wherein the detectable probe is labeled with a donor fluorescent moiety and a corresponding acceptor moiety.

14. A kit according to any one of claims 12 to 13, wherein the forward primer comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 1 and 2, or a combination thereof; the reverse primer comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NOs: 3 and 4, or a combination thereof; and the probe comprises a nucleic acid sequence of SEQ ID NO: 5 or its complementary sequence.

15. A kit according to any one of claims 12 to 13, wherein the forward primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7, or a combination thereof; the reverse primer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8 and 9, or a combination thereof; and the probe comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11, or a complementary sequence thereof.

16. The kit according to claim 15, 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 complementary sequence.

17. An oligonucleotide comprising or consisting of a nucleotide sequence selected from SEQ ID NO: 1 to 11 or a complementary sequence thereof.

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