Oligonucleotide for detecting chlamydia trachomatis and / or neisseria gonorrhoeae

Abstract

To provide a novel detection approach and method for Chlamydia trachomatis and / or Neisseria gonorrhoeae.SOLUTION: The present invention provides: a probe for detecting the porB gene of Chlamydia trachomatis; a composition for detecting Chlamydia trachomatis, comprising the probe, wherein it is used in combination with a probe for detecting the endogenous plasmid of Chlamydia trachomatis; and a probe for detecting the opa gene of Neisseria gonorrhoeae.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to oligonucleotides for detecting Chlamydia trachomatis (hereinafter sometimes abbreviated as CT) and / or Neisseria gonorrhoeae. [Background technology]

[0002] Chlamydia trachomatis is the bacterium that primarily causes genital chlamydia infections, causing urethritis and epididymitis in men and cervicitis and pelvic inflammatory disease in women. These diseases are transmitted through sexual intercourse. Chlamydia primarily infects the urogenital tract, and the number of patients is the largest of all sexually transmitted diseases (Non-Patent Document 1). Neisseria gonorrhoeae is the bacterium that primarily causes urethritis in men and cervicitis in women. The infection transmission rate from a single sexual act is high, thought to be around 30% (Non-Patent Document 2).

[0003] Chlamydia trachomatis is an intracellular parasitic pathogen, and cultured cells are required for isolation and culture. The sensitivity of the culture method is significantly reduced depending on the storage conditions of the specimen, because gonococci are sensitive to environmental changes such as temperature (Non-Patent Document 2). Culture tests are difficult and are not performed in general laboratories.

[0004] Other testing methods include methods that use antigen-antibody reactions and nucleic acid amplification methods, but it has been pointed out that the antigen-antibody method has poor sensitivity.

[0005] Currently, nucleic acid amplification is the mainstream method for testing Chlamydia trachomatis, and is sold in Japan as an in vitro diagnostic. Representative methods include the Cobas 6800 / 8800 System CT / NG (registered trademark, manufactured by Roche Diagnostics), which uses PCR, and the Aptima Combo2 Chlamydia / Gonorrhea (registered trademark, manufactured by Hologic Japan), which uses the TMA method.

[0006] All of the above nucleic acid amplification methods are said to be excellent in terms of sensitivity and specificity, but all of them have the problem that the tests take several hours or more.

[0007] Patent Document 1 discloses a nucleic acid amplification method capable of rapidly detecting Chlamydia trachomatis, an oligonucleotide primer and an oligonucleotide probe for use in the nucleic acid amplification method, and a kit for carrying out the nucleic acid amplification method.

[0008] Genes that can detect Chlamydia trachomatis with high sensitivity are generally endogenous plasmids that exist in multiple locations in Chlamydia cells. On the other hand, although there are a very small number of cases overseas, as shown in Non-Patent Document 3, Chlamydia trachomatis that do not have endogenous plasmids have been reported. [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Japanese Journal of Sexually Transmitted Diseases Vol. 27 No. 1 Supplement [Non-Patent Document 2] Japanese Society for Sexually Transmitted Diseases Sexually Transmitted Disease Diagnosis and Treatment Guidelines 2020 [Non-Patent Document 3] Yeow, TC, Wong, WF, Sabet, NS et al. Prevalence of plasmid-bearing and plasmid-free Chlamydia trachomatis infection women among who visited obstetrics and gynecology clinics in Malaysia. BMC Microbiol 16, 45 (2016). [Patent documents]

[0010] [Patent Document 1] Patent No. 6182844 Summary of the Invention [Problem to be solved by the invention]

[0011] One object of the present invention is to provide a novel means and method for detecting Chlamydia trachomatis and / or Neisseria gonorrhoeae. [Means for solving the problem]

[0012] Means for Solving the Problems The present inventors conducted intensive research in view of the above problems and discovered novel means and methods for detecting Chlamydia trachomatis and / or Neisseria gonorrhoeae. Further intensive research led to the completion of the present invention.

[0013] A typical embodiment of the present invention is as follows. [Section 1] Probe for detecting the pоrB gene of Chlamydia trachomatis. [Section 2] A probe according to item 1, having the following characteristics (a) and (b): (a) An oligonucleotide comprising a base sequence S1 of at least 8 consecutive bases in the base sequence shown in SEQ ID NO: 9 or a base sequence complementary thereto, or a base sequence S2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S1. (b) Only one of the 5' end or the 3' end of the oligonucleotide (a) is labeled. [Section 3] 3. The probe according to item 1 or 2, comprising a base sequence shown in any one of SEQ ID NOs: 27 to 32. [Section 4] A composition for detecting Chlamydia trachomatis, comprising the probe according to any one of Items 1 to 3, which is used in combination with a probe for detecting an endogenous plasmid of Chlamydia trachomatis. [Section 5] Item 5. The composition according to item 4, wherein the probe for detecting an endogenous plasmid of Chlamydia trachomatis has the following characteristics (c) and (d): (c) an oligonucleotide comprising a base sequence S3 of at least 8 consecutive bases in the base sequence shown in SEQ ID NO: 8 or a base sequence complementary thereto, or a base sequence S4 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S3. (d) Only one of the 5' end or the 3' end of the oligonucleotide (c) is labeled. [Section 6] Item 6. The composition according to item 4 or 5, wherein the probe for detecting an endogenous plasmid of Chlamydia trachomatis comprises a base sequence represented by any one of SEQ ID NOs: 12 to 15, 56, and 57. [Section 7] A probe for detecting the opa gene of Neisseria gonorrhoeae, the probe having the following characteristics (e) and (f): (e) an oligonucleotide comprising a base sequence S5 of at least 8 consecutive bases in the base sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 or a base sequence complementary thereto, or a base sequence S6 in which 1 to 3 bases have been substituted, deleted, inserted or added in base sequence S5, and comprising the base sequence of the 37th to 43rd bases in the base sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 or a base sequence complementary thereto. (f) Only one of the 5' end or the 3' end of the oligonucleotide (e) is labeled. [Section 8] Item 8. The probe according to Item 7, comprising a base sequence shown in any one of SEQ ID NOs: 40 to 42. [Section 9] Item 9. The probe according to any one of Items 1 to 8, wherein the label is a fluorescent dye label. [Section 10] 10. The probe according to claim 1, wherein the label is a fluorescent quenching dye that is quenched by interaction with guanine. [Section 11] Item 11. The probe according to any one of Items 1 to 10, wherein the labeled terminal base is cytosine. [Section 12] A composition for detecting Chlamydia trachomatis, comprising: The composition includes a primer set consisting of a combination of at least one first primer selected from those shown in the following (A-1) and at least one second primer selected from those shown in the following (B-1), and is used in combination with a probe for detecting the porB gene of Chlamydia trachomatis; and / or The composition comprises a primer set consisting of a combination of at least one first primer selected from those shown in (A-2) below and at least one second primer selected from those shown in (B-2) below, and is used in combination with a probe for detecting an endogenous plasmid of Chlamydia trachomatis. (A-1) a first primer consisting of a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 33 to 35. (A-2) a first primer consisting of a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 16 to 18. (B-1) A second primer consisting of a nucleotide sequence showing at least 90% identity to any of the nucleotide sequences shown in SEQ ID NOs: 36 to 38. (B-2) a second primer consisting of a nucleotide sequence showing at least 90% identity to any of the nucleotide sequences shown in SEQ ID NOs: 19 to 21. [Section 13] The composition comprises a primer set consisting of a combination of at least one first primer selected from those shown in (A-1) and at least one second primer selected from those shown in (B-1), and the probe for detecting the porB gene of Chlamydia trachomatis comprises a base sequence represented by any one of SEQ ID NOs: 27 to 32. The composition according to item 12. [Section 14] The composition comprises a primer set consisting of a combination of at least one first primer selected from those shown in (A-2) and at least one second primer selected from those shown in (B-2), and the probe for detecting an endogenous plasmid of Chlamydia trachomatis comprises any of the base sequences shown in SEQ ID NOs: 12 to 15, 56, and 57. The composition according to claim 12 or 13. [Section 15] A composition for detecting Neisseria gonorrhoeae, comprising: A composition comprising a primer set consisting of a combination of at least one first primer selected from those shown in (C-1) below and at least one second primer selected from those shown in (D-1) below, which is used in combination with a probe for detecting the opa gene of Neisseria gonorrhoeae. (C-1) a first primer consisting of a nucleotide sequence showing at least 90% identity to any of the nucleotide sequences shown in SEQ ID NOs: 43 to 45. (D-1) a second primer consisting of a nucleotide sequence showing at least 90% identity to any of the nucleotide sequences shown in SEQ ID NOs: 46 to 48. [Section 16] Item 16. The composition according to item 15, wherein the probe for detecting the opa gene of Neisseria gonorrhoeae comprises a base sequence represented by any one of SEQ ID NOs: 39 to 42. [Section 17] A kit for detecting Chlamydia trachomatis and Neisseria gonorrhoeae, comprising the composition according to any one of items 12 to 14 and the composition according to item 15 or 16. Effect of the Invention

[0014] According to the present invention, a novel detection means and method for Chlamydia trachomatis and / or Neisseria gonorrhoeae are provided. The detection means and method can perform, for example, highly sensitive detection. Furthermore, according to the present invention, even from a sample that has not been subjected to a nucleic acid purification step and may contain biological contaminants that inhibit nucleic acid amplification reaction, it is possible to detect Chlamydia trachomatis and / or Neisseria gonorrhoeae contained in the sample with high sensitivity. Furthermore, according to the present invention, it is possible to detect Chlamydia trachomatis that does not have an endogenous plasmid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention will be described in further detail below with reference to embodiments of the present invention, but the present invention is not limited thereto. All non-patent and patent documents described in this specification are incorporated herein by reference and are incorporated in their entirety into the specification. In addition, in this specification, "~" means "more than or equal to, less than or equal to", for example, if the specification states "X~Y", it means "more than or equal to X, less than or equal to Y". In this specification, "and / or" means part or all. In this specification, "comprise" includes the concepts of "consisting essentially of" and "consisting only of".

[0016] In addition, in this specification, the nucleic acid primer may be simply referred to as a primer, and the nucleic acid probe and the labeled probe may be simply referred to as a probe, and these are also collectively referred to as oligonucleotides. The present invention includes an oligonucleotide primer (primer) and an oligonucleotide probe (probe) for detecting Chlamydia trachomatis and / or Neisseria gonorrhoeae, as well as a method for detecting Chlamydia trachomatis using them and a kit for carrying out the method.

[0017] As used herein, sequence identity can be calculated using an analytical tool that is commercially available or available via an electric communication line (Internet), for example, using the commercially available software GENETYX (GENETYX Corporation) or the National Center for Biotechnology Information (NCBI) homology algorithm BLAST (Basic local alignment search tool) (http: / / www.ncbi.nlm.nih.gov / BLAST / ) using default (initial setting) parameters.

[0018] In one embodiment of the present invention, the detection targets are an endogenous plasmid of Chlamydia trachomatis, a partial region of the porB gene of Chlamydia trachomatis, and / or a partial region of the opa gene of Neisseria gonorrhoeae. The base sequence of the partial region of the endogenous plasmid of Chlamydia trachomatis is shown in SEQ ID NO: 1. The base sequence of the partial region of the porB gene of Chlamydia trachomatis is shown in SEQ ID NO: 2. The base sequences of the partial regions of the opa gene of Neisseria gonorrhoeae are shown in SEQ ID NOs: 3 to 7.

[0019] [1] Oligonucleotide probe of the present invention One embodiment of the present invention is an oligonucleotide probe described in any one of (1) to (3) below.

[0020] (1) A probe for detecting the pоrB gene of Chlamydia trachomatis. The probe for detecting the porB gene of Chlamydia trachomatis is preferably the following (1-1) or (1-2), more preferably the following (1-2). (1-1) A probe for detecting the pоrB gene of Chlamydia trachomatis, having the following characteristics (a) and (b). (a) An oligonucleotide comprising a base sequence S1 of at least 8 consecutive bases in the base sequence shown in SEQ ID NO: 9 or a base sequence complementary thereto, or a base sequence S2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S1. (b) Only one of the 5' end or the 3' end of the oligonucleotide (a) is labeled. (1-2) A probe for detecting the pоrB gene of Chlamydia trachomatis, comprising a base sequence shown in any one of SEQ ID NOs: 27 to 32. The base sequence S1 is preferably a base sequence of 10 or more, 12 or more, 14 or more, 16 or more, 18 or more, or 20 or more consecutive bases in the base sequence shown in SEQ ID NO: 9 or a base sequence complementary thereto. The base sequence S1 may be the entire length of the base sequence shown in SEQ ID NO: 9 or a base sequence complementary thereto, but may also be, for example, 50 or less, 45 or less, 40 or less, 35 or less, or 30 or less bases. The base sequence S2 is preferably a base sequence in which one or two (for example, one) bases have been substituted, deleted, inserted or added in the base sequence S1.

[0021] (2) Probes for detecting endogenous plasmids of Chlamydia trachomatis The probe for detecting an endogenous plasmid of Chlamydia trachomatis is preferably the following (2-1) or (2-2), more preferably the following (2-2). (2-1) A probe for detecting an endogenous plasmid of Chlamydia trachomatis having the following characteristics (c) and (d): (c) an oligonucleotide comprising a base sequence S3 of at least 8 consecutive bases in the base sequence shown in SEQ ID NO: 8 or a base sequence complementary thereto, or a base sequence S4 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S3. (d) Only one of the 5' end or the 3' end of the oligonucleotide (c) is labeled. (2-2) A probe for detecting an endogenous plasmid of Chlamydia trachomatis, comprising a base sequence shown in any one of SEQ ID NOs: 12 to 15, 56, and 57. The base sequence S3 is preferably a base sequence of 10 or more, 15 or more, 20 or more, or 25 or more consecutive bases in the base sequence shown in SEQ ID NO: 8 or a base sequence complementary thereto. The base sequence S3 may be the entire length of the base sequence shown in SEQ ID NO: 8 or a base sequence complementary thereto, but may be, for example, 50 or less, 45 or less, 40 or less, or 35 or less bases. The base sequence S4 is preferably a base sequence in which one or two (for example, one) bases have been substituted, deleted, inserted or added in the base sequence S3.

[0022] (3) Probe for detecting the opa gene of Neisseria gonorrhoeae The probe for detecting the opa gene of N. gonorrhoeae is preferably the following (3-1) or (3-2), more preferably the following (3-2). (3-1) A probe for detecting the opa gene of Neisseria gonorrhoeae having the following characteristics (e) and (f). (e) an oligonucleotide comprising a base sequence S5 of at least 8 consecutive bases in the base sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 or a base sequence complementary thereto, or a base sequence S6 in which 1 to 3 bases have been substituted, deleted, inserted or added in base sequence S5, and comprising the base sequence of the 37th to 43rd bases in the base sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 or a base sequence complementary thereto. (f) Only one of the 5' end or the 3' end of the oligonucleotide (e) is labeled. (3-2) A probe for detecting the opa gene of Neisseria gonorrhoeae, comprising a base sequence shown in any one of SEQ ID NOs: 40 to 42. The base sequence S5 is preferably a base sequence of 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or 15 or more consecutive bases in the base sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 or a base sequence complementary thereto. The base sequence S5 may be the entire length of the base sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 or a base sequence complementary thereto, but may be, for example, 50 bases or less, 45 bases or less, 40 bases or less, 35 bases or less, 30 bases or less, or 25 bases or less. The base sequence S6 is preferably a base sequence in which one or two (for example, one) bases have been substituted, deleted, inserted or added in the base sequence S5.

[0023] The above oligonucleotide probes (1) to (3) may or may not have labeled nucleic acids. When labeled, there is no particular limit to the number of nucleic acids to be labeled. Preferably, the nucleic acid at the end of the oligonucleotide probe, preferably the cytosine base at the end, is labeled. More preferably, only one of the nucleic acids at the end of the oligonucleotide probe is labeled. There is no particular limit to the labeling substance, but it is more preferably a fluorescent dye. As the fluorescent dye, in particular, a fluorescent dye that is quenched upon hybridization with the target nucleic acid is preferred, and for example, a fluorescent quenching dye that is quenched by interaction with guanine can be mentioned. Specific examples of such fluorescent dyes include fluorescein or its derivatives (e.g., fluorescein isothiocyanate (FITC)), BODIPY (trademark) series, and rhodamine or its derivatives (e.g., 5-carboxyrhodamine 6G (GR6G) and tetramethylrhodamine (TAMRA)).

[0024] The oligonucleotide probes (1) to (3) above may be used alone or in combination of two or more. For example, the oligonucleotide probe (1) above may be used in combination with the oligonucleotide probe (2) above to improve sensitivity. To detect both Chlamydia trachomatis and Neisseria gonorrhoeae, the oligonucleotide probe (1) and / or the oligonucleotide probe (2) above may be used in combination with the oligonucleotide probe (3) above.

[0025] [2] Oligonucleotide primer of the present invention One embodiment of the present invention is an oligonucleotide primer described in (4) to (6) below.

[0026] (4) Primers for detecting the porB gene of Chlamydia trachomatis The primers for detecting the porB gene of Chlamydia trachomatis are not particularly limited as long as they enable detection, but a primer set combining the following (A-1) and (B-1) is preferable. (A-1) a first primer consisting of a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 33 to 35. (B-1) A second primer consisting of a nucleotide sequence showing at least 90% identity to any of the nucleotide sequences shown in SEQ ID NOs: 36 to 38. The first primer (A-1) is not particularly limited as long as it shows at least 90% identity to the base sequence shown in any one of SEQ ID NOs: 33 to 35, and may be composed of a base sequence showing 95% or more, 97% or more, or 99% or more identity. Examples of the first primer showing at least 90% identity to SEQ ID NO: 33 include SEQ ID NO: 34 and SEQ ID NO: 35. The first primer (A-1) may be used alone or in combination of two or more kinds. The second primer (B-1) is not particularly limited as long as it shows at least 90% identity to the base sequence shown in any one of SEQ ID NOs: 36 to 38, and may be composed of a base sequence showing 95% or more, 97% or more, or 99% or more identity. Examples of the base sequence showing at least 90% identity to SEQ ID NO: 36 include SEQ ID NO: 37 and SEQ ID NO: 38. The second primer (B-1) may be used alone or in combination of two or more types.

[0027] (5) Primers for detecting endogenous plasmids of Chlamydia trachomatis The primers for detecting an endogenous plasmid of Chlamydia trachomatis are not particularly limited as long as they enable detection, but a primer set combining the following (A-2) and (B-2) is preferable. (A-2) a first primer consisting of a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 16 to 18. (B-2) a second primer consisting of a nucleotide sequence showing at least 90% identity to any of the nucleotide sequences shown in SEQ ID NOs: 19 to 21. The first primer (A-2) is not particularly limited as long as it shows at least 90% identity to the base sequence shown in any one of SEQ ID NOs: 16 to 18, and may be composed of a base sequence showing 95% or more, 97% or more, or 99% or more identity. Examples of a primer showing at least 90% identity to SEQ ID NO: 16 include SEQ ID NO: 17 and SEQ ID NO: 18. The first primer (A-2) may be used alone or in combination of two or more kinds. The second primer (B-2) is not particularly limited as long as it shows at least 90% identity to the base sequence shown in any one of SEQ ID NOs: 19 to 21, and may be composed of a base sequence showing 95% or more, 97% or more, or 99% or more identity. Examples of the base sequence showing at least 90% identity to SEQ ID NO: 19 include SEQ ID NO: 20 and SEQ ID NO: 21. The second primer (B-2) may be used alone or in combination of two or more types.

[0028] (6) Primers for detecting Neisseria gonorrhoeae The primers for detecting gonococci are not particularly limited as long as they enable detection of gonococci, but a primer set combining the following (C-1) and (D-1) is preferable. (C-1) a first primer consisting of a nucleotide sequence showing at least 90% identity to any of the nucleotide sequences shown in SEQ ID NOs: 43 to 45. (D-1) a second primer consisting of a nucleotide sequence showing at least 90% identity to any of the nucleotide sequences shown in SEQ ID NOs: 46 to 48. The first primer (C-1) may be one that consists of a base sequence that shows at least 90% identity to the base sequence shown in any one of SEQ ID NOs: 43 to 45. Examples of a primer that shows at least 90% identity to SEQ ID NO: 43 include SEQ ID NO: 44 and SEQ ID NO: 45. The first primer (C-1) may be used alone or in combination of two or more types. The second primer (D-1) may be one that consists of a base sequence that shows at least 90% identity to the base sequence shown in any one of SEQ ID NOs: 46 to 48. Examples of a primer that shows at least 90% identity to SEQ ID NO: 46 include SEQ ID NO: 47 and SEQ ID NO: 48. The second primer (D-1) may be used alone or in combination of two or more types.

[0029] [3-(1)] Method for detecting Chlamydia trachomatis and / or Neisseria gonorrhoeae according to the present invention The method for detecting Chlamydia trachomatis or Neisseria gonorrhoeae of the present invention preferably comprises the following steps (1) to (3). (1) A step of amplifying nucleic acid derived from Chlamydia trachomatis and / or Neisseria gonorrhoeae that may be present in a sample using at least one of the oligonucleotide primer sets described above. (2) A step of hybridizing the nucleic acid amplification product obtained in the step (1) with at least one of the oligonucleotide probes described above to form a complex. (3) detecting the complex obtained in step (2).

[0030] More preferably, the above method includes the steps shown in (1A), (1B), (2A), and (3A) below. (1A) A step of preparing an oligonucleotide primer set for amplifying at least one of an endogenous plasmid of Chlamydia trachomatis, a porB gene of Chlamydia trachomatis, and an opa gene of Neisseria gonorrhoeae. (1B) A step of amplifying the test nucleic acid using a reaction solution containing the test nucleic acid and the oligonucleotide primer set of step (1A). (2A) A step of hybridizing the nucleic acid amplification product obtained in step (1B) with at least one of the oligonucleotide probes described above to form a complex. (3A) detecting the complex obtained in step (2A).

[0031] As one embodiment of the present invention, a primer set and a probe for detecting Chlamydia trachomatis may be used in combination. Also, a primer set and a probe for detecting Neisseria gonorrhoeae may be used in combination. In principle, the combination is arbitrary, but preferably, it is any of the following (I) to (III). (I) A combination of a pair of primer sets consisting of the first primer (A-2) and the second primer (B-2) above, and the oligonucleotide probe (2) above (preferably an oligonucleotide probe containing a base sequence of at least 8 consecutive bases in a base sequence showing 90% or more identity to the base sequence shown in SEQ ID NO:8 or a base sequence complementary thereto). (II) A combination of a pair of primer sets consisting of the first primer (A-1) described above in (A-1) and the second primer (B-1) described above, and the oligonucleotide probe described above in (1) (preferably an oligonucleotide probe containing a base sequence of at least 8 consecutive bases in a base sequence showing 90% or more identity to the base sequence shown in SEQ ID NO: 9 or a base sequence complementary thereto). (III) A combination of a pair of primer sets consisting of the first primer (C-1) above and the second primer (D-1) above, and the oligonucleotide probe (3) above (preferably an oligonucleotide probe containing a base sequence of at least 8 consecutive bases in a base sequence showing 90% or more identity to the base sequence shown in SEQ ID NO: 10 or 11, or a base sequence complementary thereto).

[0032] In addition, as one embodiment of the present invention, a primer set and a probe for detecting Chlamydia trachomatis may be combined with a primer set and a probe for detecting Neisseria gonorrhoeae to be used for detecting both Chlamydia trachomatis and Neisseria gonorrhoeae. In principle, any combination may be used, but the combination of (I) and (III), the combination of (II) and (III), or the combination of (I) to (III) is preferable.

[0033] [3-(2)] Amplification of test nucleic acid The test nucleic acid in the present invention may be, for example, single-stranded or double-stranded. In the case of a double-stranded nucleic acid, it is preferable to include a step of dissociating the double stranded nucleic acid into single stranded nucleic acids by heating, for example, in order to hybridize the test nucleic acid with a probe to form a hybrid.

[0034] The type of the test nucleic acid is not particularly limited, and examples thereof include DNA, total RNA, mRNA, and other RNA, etc. Examples of the test nucleic acid include nucleic acids contained in samples such as cell culture samples and biological samples parasitized with Chlamydia trachomatis and / or Neisseria gonorrhoeae.

[0035] The biological sample is not particularly limited, but examples thereof include a swab used to wipe the genitals or pharynx, or a swab liquid attached to the swab, urine, and gargle liquid. There are no limitations on the method of collecting samples and the method of preparing nucleic acids such as DNA and RNA, and conventionally known methods can be used.

[0036] It is preferable to amplify a sequence containing a base site to be detected by a nucleic acid amplification method such as PCR using the above-mentioned oligonucleotide primer set with the test nucleic acid as a template. The conditions for PCR are not particularly limited, and the PCR can be performed by a conventional method.

[0037] The specific nucleic acid amplification method used in the nucleic acid amplification step in the detection method for Chlamydia trachomatis and / or Neisseria gonorrhoeae of the present invention is not particularly limited, and any known method can be used as appropriate. For example, the PCR (Polymerase Chain Reaction) method, the NASBA (Nucleic acid sequence based amplification) method, the TMA (Transcription-mediated amplification) method, the SDA (Strand Displacement Amplification) method, etc. are included, but it is preferable to use the PCR method. In each of these methods, the conditions for the amplification reaction are not particularly limited, and the amplification reaction can be performed by a conventionally known method.

[0038] When PCR is used for nucleic acid amplification, the DNA polymerase to be used is not particularly limited, but is preferably an α-type DNA polymerase, for the reasons explained below.

[0039] When DNA contained in Chlamydia trachomatis and / or Neisseria gonorrhoeae is amplified in a reaction system containing the probe of the present invention, the oligonucleotide probe may bind to DNA genes or their amplification products contained in the sample of Chlamydia trachomatis and / or Neisseria gonorrhoeae during the nucleic acid amplification process.

[0040] Alpha-type DNA polymerases such as KOD DNA Polymerase (derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1) do not have 5'-3' exonuclease activity, but have 3'-5' exonuclease activity. Therefore, the use of alpha-type DNA polymerase can prevent probe degradation, and the 3'-5' exonuclease activity provides high accuracy in the nucleic acid amplification process.

[0041] Although KOD DNA Polymerase is an α-type DNA polymerase, it has high DNA synthesis activity, a DNA synthesis rate of 100 bases / second or more, and excellent elongation efficiency. Therefore, among α-type DNA polymerases, it is preferable to use KOD DNA Polymerase (manufactured by Toyobo, trademark) in carrying out the present invention.

[0042] Furthermore, a mutant type of α-type DNA polymerase that has been mutated to achieve a deoxyribonucleic acid synthesis rate of 100 bases / second or more, or a DNA polymerase composition that achieves said performance by combining a wild type and / or a mutant type, can also be used as a DNA polymerase suitable for carrying out the present invention. For example, in addition to the above-mentioned KOD DNA Polymerase, other DNA polymerases having a deoxyribonucleic acid synthesis rate of 100 bases / second or more can be used, such as "KOD FX (manufactured by Toyobo, registered trademark)", "KOD -Plus- (manufactured by Toyobo, trademark)", "KOD Dash (manufactured by Toyobo, registered trademark)", "KOD -Multi&Epi- (manufactured by Toyobo, trademark)", "KOD One (manufactured by Toyobo, registered trademark)", and PrimeSTAR HS DNA polymerase (manufactured by Takara Bio, registered trademark). Among these, KOD-Plus-, which combines high accuracy and DNA synthesis activity, is preferable.

[0043] [3-(3)] Complex formation between nucleic acid amplification products derived from Chlamydia trachomatis and / or Neisseria gonorrhoeae and a probe In the detection method for Chlamydia trachomatis and / or Neisseria gonorrhoeae of the present invention, it is preferable to hybridize the amplification product obtained by amplifying the test nucleic acid described above with at least one of the oligonucleotide probes described above to form a complex.

[0044] The timing of adding the oligonucleotide probe to the sample containing the nucleic acid amplification product is not particularly limited, and may be added to any reaction system before, during, or after the nucleic acid amplification reaction. Among them, it is preferable to add the probe before the amplification reaction, since the amplification reaction and the detection reaction described below can be performed continuously. When the probe is added before the nucleic acid amplification reaction, it is preferable to add a fluorescent dye or a phosphate group to the 3' end of the probe, for example, as described below.

[0045] [3-(4)] Detection of complexes between nucleic acid amplification products and probes The method for detecting the complex obtained above is not particularly limited, and may be, for example, a method based on melting curve analysis.

[0046] Melting curve analysis is performed, for example, as follows. When a solution containing double-stranded DNA is heated, the absorbance at 260 nm increases. This is because the hydrogen bonds between the two strands in the double-stranded DNA are broken by heating, causing the DNA to dissociate into single-stranded DNA (melting of the DNA). When all of the double-stranded DNA has dissociated into single-stranded DNA, the absorbance is about 1.5 times the absorbance at the start of heating (the absorbance of only double-stranded DNA), and this indicates that melting has been completed.

[0047] In the present invention, the measurement of the signal change accompanying the temperature change for the melting curve analysis can be performed by measuring the absorbance at 260 nm based on the above-mentioned principle, but when a label is added to the oligonucleotide probe of the present invention, it is preferable to measure the signal of the label. Therefore, it is preferable to use a labeled probe as the oligonucleotide probe used in the method for detecting Chlamydia trachomatis and / or Neisseria gonorrhoeae of the present invention.

[0048] Examples of the labeled probe include a labeled probe that shows a signal by itself and does not show a signal by hybridization, and a labeled probe that does not show a signal by itself and shows a signal by hybridization. The former probe does not show a signal when it forms a hybrid (double strand) with the detection target sequence, but shows a signal when the probe is released by heating. The latter probe shows a signal by forming a hybrid (double strand) with the detection target sequence, and the signal decreases (disappears) when the probe is released by heating. Therefore, by detecting the signal by this label under signal-specific conditions (such as absorbance), the progress of melting can be grasped in the same way as the above-mentioned 260 nm absorbance measurement.

[0049] A specific example of a labeled probe is preferably a probe that is labeled with a fluorescent dye, that is fluorescent by itself, and that reduces (e.g., quenches) its fluorescence upon hybridization. Such a phenomenon is generally called the fluorescence quenching phenomenon. As a probe utilizing this fluorescence quenching phenomenon, a probe generally called a guanine quenching probe is preferable. Such a probe is known as a so-called QProbe (registered trademark). A guanine quenching probe is, for example, a probe designed such that the base at the 3' or 5' end of an oligonucleotide is cytosine, and the end is labeled with a fluorescent dye that emits weaker light when the terminal base cytosine approaches the complementary base guanine. In the probe of the present invention, for example, a fluorescent dye that exhibits the fluorescence quenching phenomenon may be bound to the cytosine at the 3' end of the oligonucleotide, or the 5' end of the oligonucleotide may be designed to be cytosine and bound to it.

[0050] The fluorescent dye is not particularly limited, but examples of the fluorescent quenching dye that is quenched by interaction with guanine include at least one fluorescent quenching dye selected from the group consisting of 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL), carboxyrhodamine 6G (CR6G), TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue), and these fluorescent quenching dyes can be suitably used in the present invention. The detection conditions of the probe are not particularly limited and can be appropriately determined depending on the fluorescent dye used, but for example, Pacific Blue can be detected at a detection wavelength of 450 to 480 nm, TAMRA at a detection wavelength of 585 to 700 nm, and BODIPYFL at a detection wavelength of 515 to 555 nm. By using such a probe, hybridization and dissociation can be easily confirmed by detecting changes in the signal.

[0051] The probe used in the method for detecting Chlamydia trachomatis and / or Neisseria gonorrhoeae of the present invention may have, for example, a phosphate group added to the 3' end. As described below, a test nucleic acid (target nucleic acid) for detecting the presence or absence of a gene can be prepared by a nucleic acid amplification method such as PCR, and in this case, the probe of the present invention can be made to coexist in the reaction system of the nucleic acid amplification reaction. In such a case, if a phosphate group is added to the 3' end of the probe, the probe itself can be sufficiently prevented from being elongated by the nucleic acid amplification reaction. In addition, the same effect can be obtained by adding a labeling substance as described above to the 3' end.

[0052] Dissociation of the resulting amplification product and hybridization of the resulting single-stranded nucleic acid with the labeled probe can be carried out, for example, by changing the temperature of the reaction solution.

[0053] The heating temperature in the dissociation step is not particularly limited as long as the amplification product can be dissociated at the temperature, and is, for example, 85 to 98° C. The heating time is also not particularly limited, and is usually 1 second to 10 minutes, and preferably 1 second to 5 minutes.

[0054] Furthermore, the hybridization of the dissociated single-stranded nucleic acid with the labeled probe can be carried out, for example, by lowering the heating temperature in the dissociation step after the dissociation step. The temperature condition is, for example, 35 to 50°C.

[0055] The volume and concentration of each component in the reaction system (reaction system) in the hybridization step are not particularly limited. Specifically, in the reaction system, the concentration of the nucleic acid is, for example, 0.01 to 100 μmol / L, preferably 0.1 to 10 μmol / L, and the concentration of the labeled probe is, for example, preferably within a range that satisfies the addition ratio to the nucleic acid, for example, 0.01 to 100 μmol / L, preferably 0.01 to 10 μmol / L.

[0056] It is preferable to change the temperature of the reaction solution and measure a signal value indicating the melting state of the hybridization product between the amplification product and the labeled probe. Specifically, for example, the reaction solution (the hybridization product between the single-stranded nucleic acid and the labeled probe) is heated, and the change in the signal value accompanying the temperature increase is measured. As described above, when a probe (guanine quenching probe) with a terminal C base labeled is used, the fluorescence decreases (or is quenched) when hybridized with the single-stranded nucleic acid, and the fluorescence is emitted when dissociated. Therefore, for example, the hybridization product with decreased (or quenched) fluorescence is gradually heated, and the increase in the fluorescence intensity accompanying the temperature increase is measured.

[0057] The temperature range when measuring the change in fluorescence intensity is not particularly limited, but for example, the starting temperature is room temperature to 85° C., and preferably 25 to 70° C., and the ending temperature is, for example, 40 to 105° C. In addition, the rate of temperature increase is not particularly limited, but for example, 0.05 to 20° C. / sec, and preferably 0.08 to 5° C. / sec.

[0058] The presence or absence of the test nucleic acid can be determined, for example, by measuring a change in a signal during hybridization. Specifically, it is preferable to measure a change in a signal accompanying a decrease in temperature when the temperature of the reaction solution containing the probe is decreased to form a hybrid.

[0059] As a specific example, when a labeled probe that shows a signal by itself but does not show a signal by hybridization (e.g., a guanine quenching probe) is used, the probe emits fluorescence when the single-stranded nucleic acid and the labeled probe are dissociated, but when a hybrid is formed by lowering the temperature, the fluorescence decreases (or is quenched). Therefore, for example, the temperature of the reaction solution is gradually lowered, and the decrease in fluorescence intensity accompanying the temperature decrease is measured. On the other hand, when a labeled probe that does not show a signal by itself but shows a signal by hybridization is used, the probe does not emit fluorescence when the single-stranded nucleic acid and the labeled probe are dissociated, but when a hybrid is formed by lowering the temperature, the probe emits fluorescence. Therefore, for example, the temperature of the reaction solution is gradually lowered, and the increase in fluorescence intensity accompanying the temperature decrease is measured.

[0060] In the present invention, the change in the signal may be analyzed to determine the genotype at a target base site, and the Tm (melting temperature) value may be determined.

[0061] [4] Composition and kit for detecting Chlamydia trachomatis and / or Neisseria gonorrhoeae In another embodiment, a composition for detecting Chlamydia trachomatis is provided, comprising a probe for detecting Chlamydia trachomatis as described above, the composition being used in combination with a probe for detecting an endogenous plasmid of Chlamydia trachomatis as described above. In another embodiment, a composition and kit for detecting the porB gene of Chlamydia trachomatis are provided, which comprise a primer set consisting of a combination of the first primer (A-1) above and the second primer (B-1) above, and are used in combination with the probe for detecting the porB gene of Chlamydia trachomatis described above. In another embodiment, a composition and kit for detecting an endogenous plasmid of Chlamydia trachomatis are provided, which comprises a primer set consisting of a combination of the first primer (A-2) above and the second primer (B-2) above, and is used in combination with the probe for detecting an endogenous plasmid of Chlamydia trachomatis described above. In another embodiment, there is provided a composition and kit for detecting the opa gene of N. gonorrhoeae, comprising a primer set consisting of a combination of the first primer (C-1) above and the second primer (D-1) above, used in combination with a probe for detecting the opa gene of N. gonorrhoeae. In another embodiment, compositions and kits for detecting Chlamydia trachomatis and / or Neisseria gonorrhoeae are provided, comprising a composition for detecting the porB gene of Chlamydia trachomatis, a composition for detecting an endogenous plasmid of Chlamydia trachomatis, and / or a composition for detecting the opa gene of Neisseria gonorrhoeae. The above kit can be provided in a form in which some or all of the components are enclosed in the same container or in separate containers, packaged in a single package, and including information on how to use the kit, etc. The above kit can also include a positive control solution and / or a negative control solution. EXAMPLES

[0062] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0063] [Example 1: Detection of endogenous plasmids in Chlamydia trachomatis] (1-1) Method In order to search for a region capable of detecting Chlamydia trachomatis with high sensitivity, 20 copies of an inactivated Chlamydia trachomatis solution were used per reaction, and detection of Chlamydia trachomatis was confirmed using a total of four oligonucleotide probes shown in SEQ ID NOs: 12 to 15. The region of the endogenous plasmid of Chlamydia trachomatis reported in Patent Document 1 was used as a comparison for detection. Regarding the presence or absence of non-specific amplification, purified water was used as a negative sample for measurement.

[0064] (1-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube Test Basic (manufactured by Toyobo). The combinations of oligonucleotide primers and oligonucleotide probes are shown in Table 1. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 10 μM oligonucleotide primer (any one of SEQ ID NOs: 17, 22, and 23) 0.4 μL 100 μM oligonucleotide primer (any one of SEQ ID NOs: 19, 24, and 25) 0.2 μL 10 μM Oligonucleotide probe (any one of SEQ ID NOs: 12 to 15 and 26) 0.3 μL KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PP Mix (Genecube® Test Basic, Toyobo) 3 μL

[0065] (1-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycle, and the presence or absence of amplification was confirmed by melting curve analysis. 94℃ 30 seconds (One cycle) 97℃ 1 second 58℃ 5 seconds 68℃ 5 seconds (more than 50 cycles) 94℃ 30 seconds 39℃ 30 seconds 39~75℃ (Temperature rises at 0.09℃ / sec)

[0066] (1-4) Results A set of four oligonucleotides and oligonucleotide probes, including probes designed based on SEQ ID NOs: 12 to 15, showed a higher detection rate for low-concentration samples than the control, and was found to be superior as a detection system. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed when any of the primers and probes were used. The possibility of detecting Chlamydia trachomatis with higher sensitivity was found by constructing an endogenous plasmid detection system outside the region different from Patent Document 1.

[0067] [Table 1]

[0068] [Example 2: Detection of endogenous plasmids in Chlamydia trachomatis] (2-1) Method A primer set capable of detecting Chlamydia trachomatis with higher sensitivity in the region of the endogenous plasmid of Chlamydia trachomatis found in Example 1 was searched for. 10 copies of Chlamydia trachomatis inactivated bacteria solution were used per reaction, and detection of Chlamydia trachomatis was confirmed with a set of oligonucleotides and oligonucleotide probes designed based on SEQ ID NOs: 16 to 21. As a comparison, it was investigated whether the region of the endogenous plasmid of Chlamydia trachomatis reported in Patent Document 1 could be detected with high sensitivity by a combination of primer sets. Regarding the presence or absence of non-specific amplification, purified water was used as a negative sample for measurement.

[0069] (2-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube Test Basic (manufactured by Toyobo). The combinations of oligonucleotide primers and oligonucleotide probes are shown in Table 1. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 10 μM Oligonucleotide primer (any one of SEQ ID NOs: 16 to 18 and 49 to 51) 0.4 μL 100 μM oligonucleotide primer (any one of SEQ ID NOs: 19, 20, 52, and 53) 0.2 μL 10 μM Oligonucleotide probe (any one of SEQ ID NOs: 15 and 54 to 57) 0.3 μL KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PP Mix (Genecube® Test Basic, Toyobo) 3 μL

[0070] (2-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycle, and the presence or absence of amplification was confirmed by melting curve analysis. 94℃ 30 seconds (One cycle) 97℃ 1 second 58℃ 5 seconds 68℃ 5 seconds (more than 50 cycles) 94℃ 30 seconds 39℃ 30 seconds 39~75℃ (Temperature rises at 0.09℃ / sec)

[0071] (2-4) Results It was found that a set of oligonucleotide primers designed based on sequence numbers 16 to 21 and oligonucleotide probes designed based on sequence numbers 15, 56, and 57 had a higher detection rate for low-concentration samples than the comparison control, making it an excellent detection system.

[0072] [Table 2]

[0073] [Example 3: Detection of the porB gene of Chlamydia trachomatis] (3-1) Method In order to search for a region in which Chlamydia trachomatis can be detected with high sensitivity, 20 copies of inactivated Chlamydia trachomatis bacteria solution were used per reaction, and detection of Chlamydia trachomatis was confirmed using a primer set combining oligonucleotide primers designed based on SEQ ID NOs: 33 to 38. Regarding the presence or absence of non-specific amplification, purified water was used as a negative sample for measurement.

[0074] (3-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube Test Basic (manufactured by Toyobo). The combinations of oligonucleotide primers and oligonucleotide probes are shown in Table 1. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 10 μM oligonucleotide primer (any one of SEQ ID NOs: 33 to 35) 0.4 μL 100 μM Oligonucleotide primer (any one of SEQ ID NOs: 36 to 38) 0.2 μL 10 μM oligonucleotide probe (SEQ ID NO: 32) 0.3 μL KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PP Mix (Genecube® Test Basic, Toyobo) 3 μL

[0075] (3-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycle, and the presence or absence of amplification was confirmed by melting curve analysis. 94℃ 30 seconds (One cycle) 97℃ 1 second 58℃ 5 seconds 68℃ 5 seconds (more than 50 cycles) 94℃ 30 seconds 39℃ 30 seconds 39~75℃ (Temperature rises at 0.09℃ / sec)

[0076] (3-4) Results Among the six oligonucleotide primers designed based on SEQ ID NOs: 33 to 38, a combination with excellent detection sensitivity for low-concentration samples was found. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed when any of the primers and probes were used. It was shown that the porB gene detection system can be used to detect Chlamydia trachomatis, which does not have an endogenous plasmid.

[0077] [Table 3]

[0078] [Example 4: Detection of opa gene of Neisseria gonorrhoeae] (4-1) Method In order to search for a region where gonococci can be detected with high sensitivity, 5 copies of gonococcal gDNA were used per reaction, and detection of gonococci was confirmed using a set of oligonucleotide primers designed based on SEQ ID NOs: 43 and 45 to 47 and oligonucleotide probes of SEQ ID NOs: 39 to 42. Regarding the presence or absence of non-specific amplification, purified water was used as a negative sample for the measurement.

[0079] (4-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube Test Basic (manufactured by Toyobo). The combinations of oligonucleotide primers and oligonucleotide probes are shown in Table 1. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 10 μM oligonucleotide primer (either one of SEQ ID NO: 43 and 45) 0.4 μL 100 μM Oligonucleotide primer (any one of SEQ ID NO: 46 and 47) 0.2 μL 10 μM Oligonucleotide probe (any one of SEQ ID NOs: 39 to 42) 0.3 μL KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PP Mix (Genecube® Test Basic, Toyobo) 3 μL

[0080] (4-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycle, and the presence or absence of amplification was confirmed by melting curve analysis. 94℃ 30 seconds (One cycle) 97℃ 1 second 58℃ 5 seconds 68℃ 5 seconds (more than 50 cycles) 94℃ 30 seconds 39℃ 30 seconds 39~75℃ (Temperature rises at 0.09℃ / sec)

[0081] (4-4) Results It was found that the combination of oligonucleotide primers designed based on SEQ ID NOs: 43 and 45 to 47 and oligonucleotide probes of SEQ ID NOs: 39 to 42 had excellent detection sensitivity for low concentration samples. In addition, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed when any of the primers and probes were used.

[0082] [Table 4]

[0083] [Example 5: Detection of multiple bacterial species in one reaction system] (5-1) Method To explore the combination of primers and probes that can detect Neisseria gonorrhoeae and Chlamydia trachomatis with high sensitivity in a single reaction system, 5 copies of gDNA of Neisseria gonorrhoeae or Chlamydia trachomatis were used per reaction, and detection of Neisseria gonorrhoeae or Chlamydia trachomatis was confirmed using sets of oligonucleotide primers and oligonucleotide probes designed based on SEQ ID NOs: 41-48, 14, 15, 17-19, 21, 31-34, 36, and 38. Purified water was used as a negative sample for the measurement to check for the presence or absence of non-specific amplification.

[0084] (5-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube Test Basic (manufactured by Toyobo). The combinations of oligonucleotide primers and oligonucleotide probes are shown in Table 1. An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification had been performed normally. 10 μM Oligonucleotide primer for amplifying the opa gene of Neisseria gonorrhoeae (either one of SEQ ID NO: 41 or 42) 0.4 μL 100 μM Oligonucleotide primer for amplifying the opa gene of Neisseria gonorrhoeae (any one of SEQ ID NOs: 43 to 45) 0.2 μL 10 μM Oligonucleotide probe for detecting the opa gene of Neisseria gonorrhoeae (any one of SEQ ID NOs: 46 to 48) 0.3 μL 10 μM Chlamydia trachomatis endogenous plasmid amplification oligonucleotide primer (either one of SEQ ID NO: 14 and 15) 0.4 μL 100 μM Chlamydia trachomatis endogenous plasmid amplification oligonucleotide primer (any one of SEQ ID NOs: 17 and 18) 0.2 μL 10 μM Chlamydia trachomatis endogenous plasmid detection oligonucleotide probe (any one of SEQ ID NOs: 19 and 21) 0.3 μL 10 μM Chlamydia trachomatis pоrB gene amplification oligonucleotide primer (either one of SEQ ID NO: 31 and 32) 0.4 μL 100 μM Chlamydia trachomatis pоrB gene amplification oligonucleotide primer (any one of SEQ ID NOs: 33 and 34) 0.2 μL 10 μM Chlamydia trachomatis pоrB gene detection oligonucleotide probe (any one of SEQ ID NOs: 36 and 38) 0.3 μL KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PP Mix (Genecube® Test Basic, Toyobo) 3 μL

[0085] (5-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycle, and the presence or absence of amplification was confirmed by melting curve analysis. 94℃ 30 seconds (One cycle) 97℃ 1 second 58℃ 5 seconds 68℃ 5 seconds (more than 50 cycles) 94℃ 30 seconds 39℃ 30 seconds 39~75℃ (Temperature rises at 0.09℃ / sec)

[0086] (5-4) Results Among the oligonucleotide combinations of the N. gonorrhoeae opa gene detection system, the Chlamydia trachomatis endogenous plasmid detection system, and the Chlamydia trachomatis pоrB gene detection system, we found a combination that had excellent detection sensitivity for low-concentration samples. In addition, when we confirmed non-specific amplification using purified water as a negative sample, no non-specific amplification was observed when any of the primers and probes were used.

[0087] [Table 5] [Industrial Applicability]

[0088] By utilizing the present invention in a test for Chlamydia trachomatis and / or Neisseria gonorrhoeae, for example, Chlamydia trachomatis and / or Neisseria gonorrhoeae can be detected with high speed, accuracy, and high sensitivity. In addition, the present invention can simultaneously detect multiple bacterial species in one reaction system using any combination of primer sets and probes, making it possible to simply test for Chlamydia trachomatis and / or Neisseria gonorrhoeae.

Claims

1. A probe for detecting the pоrB gene of Chlamydia trachomatis.

2. The probe according to claim 1 , having the following characteristics (a) and (b): (a) An oligonucleotide comprising a base sequence S1 of at least 8 consecutive bases in the base sequence shown in SEQ ID NO:9 or a base sequence complementary thereto, or a base sequence S2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S1. (b) Only one of the 5' end or the 3' end of the oligonucleotide (a) is labeled.

3. The probe according to claim 1, comprising a base sequence shown in any one of SEQ ID NOs: 27 to 32.

4. A composition for detecting Chlamydia trachomatis, comprising the probe according to any one of claims 1 to 3, said composition being used in combination with a probe for detecting an endogenous plasmid of Chlamydia trachomatis.

5. The composition according to claim 4, wherein the probe for detecting an endogenous plasmid of Chlamydia trachomatis has the following characteristics (c) and (d): (c) an oligonucleotide comprising a base sequence S3 of at least 8 consecutive bases in the base sequence shown in SEQ ID NO: 8 or a base sequence complementary thereto, or a base sequence S4 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S3. (d) Only one of the 5' end or the 3' end of the oligonucleotide (c) is labeled.

6. The composition according to claim 4, wherein the probe for detecting an endogenous plasmid of Chlamydia trachomatis comprises a base sequence represented by any one of SEQ ID NOs: 12 to 15, 56, and 57.

7. A probe for detecting the opa gene of Neisseria gonorrhoeae, the probe having the following characteristics (e) and (f): (e) An oligonucleotide comprising a base sequence S5 of at least 8 consecutive bases in the base sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 or a base sequence complementary thereto, or a base sequence S6 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S5, and comprising the 37th to 43rd bases in the base sequence shown in SEQ ID NO: 10 or SEQ ID NO: 11 or a base sequence complementary thereto. (f) Only one of the 5' end or the 3' end of the oligonucleotide (e) is labeled.

8. The probe according to claim 7, comprising a base sequence shown in any one of SEQ ID NOs: 40 to 42.

9. The probe according to any one of claims 1 to 3, 7 and 8, wherein the label is a fluorescent dye label.

10. The probe according to any one of claims 1 to 3, 7 and 8, wherein the label is a label with a fluorescence quenching dye that is quenched by interaction with guanine.

11. The probe according to any one of claims 1 to 3, 7 and 8, wherein the labeled terminal base is cytosine.

12. A composition for detecting Chlamydia trachomatis, comprising: The composition includes a primer set consisting of a combination of at least one first primer selected from the following (A-1) and at least one second primer selected from the following (B-1), and is used in combination with a probe for detecting the porB gene of Chlamydia trachomatis; and / or The composition comprises a primer set consisting of a combination of at least one first primer selected from those shown in (A-2) below and at least one second primer selected from those shown in (B-2) below, and is used in combination with a probe for detecting an endogenous plasmid of Chlamydia trachomatis. (A-1) A first primer consisting of a base sequence showing at least 90% identity to a base sequence shown in any one of SEQ ID NOs: 33 to 35. (A-2) A first primer consisting of a base sequence showing at least 90% identity to any one of the base sequences shown in SEQ ID NOs: 16 to 18. (B-1) A second primer consisting of a base sequence showing at least 90% identity to any one of the base sequences shown in SEQ ID NOs: 36 to 38. (B-2) A second primer consisting of a base sequence showing at least 90% identity to any one of the base sequences shown in SEQ ID NOs: 19 to 21.

13. The composition comprises a primer set consisting of a combination of at least one first primer selected from those shown in (A-1) and at least one second primer selected from those shown in (B-1), and a probe for detecting the porB gene of Chlamydia trachomatis comprises a base sequence represented by any one of SEQ ID NOs: 27 to 32. The composition according to claim 12.

14. The composition comprises a primer set consisting of a combination of at least one first primer selected from those shown in (A-2) and at least one second primer selected from those shown in (B-2), and the probe for detecting an endogenous plasmid of Chlamydia trachomatis comprises any of the base sequences shown in SEQ ID NOs: 12 to 15, 56, and 57. The composition according to claim 12.

15. A composition for detecting Neisseria gonorrhoeae, comprising: A composition comprising a primer set consisting of a combination of at least one first primer selected from those shown in (C-1) below and at least one second primer selected from those shown in (D-1) below, said composition being used in combination with a probe for detecting the opa gene of gonorrhea. (C-1) A first primer consisting of a base sequence showing at least 90% identity to any one of the base sequences shown in SEQ ID NOs: 43 to 45. (D-1) A second primer consisting of a base sequence showing at least 90% identity to any one of the base sequences shown in SEQ ID NOs: 46 to 48.

16. The composition according to claim 15, wherein the probe for detecting the opa gene of Neisseria gonorrhoeae comprises a base sequence represented by any one of SEQ ID NOs: 39 to 42.

17. A kit for detecting Chlamydia trachomatis and Neisseria gonorrhoeae, comprising a composition according to any one of claims 12 to 14 and a composition according to claim 15 or 16.

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