Oligonucleotide for detecting trichomonas vaginalis and / or mycoplasma genitalium

Abstract

To provide novel detection means and a method for Trichomonas vaginalis and / or Mycoplasma genitalium.SOLUTION: The present invention provides a probe for detecting a 5.8S rRNA gene of Trichomonas vaginalis, having the following characteristic (a): (a) an oligonucleotide comprising a base sequence S1, which consists of at least 8 consecutive bases within a specific base sequence or its complementary base sequence, or a base sequence S2, where 1 to 3 bases in the base sequence S1 are substituted, deleted, inserted, or added, with either only one of 5'-end or 3'-end is labelled. The present invention also provides a probe for detecting a mgPa gene of Mycoplasma genitalium.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to oligonucleotides for detecting Trichomonas vaginalis and / or Mycoplasma genitalium. [Background technology]

[0002] Sexually transmitted diseases are diseases transmitted by sexual contact, and there are many cases where the symptoms are asymptomatic or mild and unnoticed, leading to the problem that the infection spreads without the person even realizing it. Representative examples of microorganisms that cause sexually transmitted diseases include Chlamydia trachomatis, Neisseria gonorrhoeae, Trichomonas vaginalis, and Mycoplasma genitalium. Other known causative microorganisms include Mycoplasma hominis, Ureaplasma parvum, Ureaplasma urealyticum, and Treponema pallidum.

[0003] Of the microorganisms that cause these sexually transmitted diseases, Trichomonas protozoa has been reported to cause vaginitis, urethritis, cystitis, etc., and it is important for clinical diagnosis to detect it simply, quickly, and with high sensitivity. As methods for testing Trichomonas protozoa, microscopic examination, culture examination, and genetic examination have been developed so far. Of these examination methods, microscopic examination is considered the most mainstream, but there is a problem that the accuracy of the examination depends on the skill of the microscopist and the quality of the equipment. In addition, there has been concern recently that the sensitivity of microscopic examination is insufficient. In contrast, genetic examination has the characteristic that it can test for Trichomonas protozoa quickly and with high sensitivity, and is not easily affected by the skill of the operator (Non-Patent Document 1).

[0004] Chlamydia trachomatis and Neisseria gonorrhoeae are widely known as the main causative microorganisms of male urethritis. On the other hand, Mycoplasma genitalium may be detected in 14-16% of non-gonococcal urethritis patients, and the patient is diagnosed with Mycoplasma genitalium urethritis. It has also been reported that Mycoplasma genitalium not only causes urethritis, but also cervicitis, etc. (Non-Patent Document 2). It is important for clinical diagnosis to detect it simply, quickly, and with high sensitivity. Nucleic acid amplification methods have become the mainstream method for testing Mycoplasma genitalium, and are sold as in vitro diagnostics in Japan. A representative example is the Cobas 6800 / 8800 System TV / MG (registered trademark, manufactured by Roche Diagnostics) that uses PCR. A research reagent is the Aptima Mycoplasma Genitalium Assay (registered trademark, manufactured by Hologic Japan) that uses the TMA method.

[0005] 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 test time is several hours or more. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Issa,RMet al.,Iranian Journal of Clinical Infectious Diseases,2006;1(4):171-175 [Non-Patent Document 2] Japanese Society for Sexually Transmitted Diseases Sexually Transmitted Disease Diagnosis and Treatment Guidelines 2020 Summary of the Invention [Problem to be solved by the invention]

[0007] One object of the present invention is to provide a novel means and method for detecting Trichomonas vaginalis and / or Mycoplasma genitalium. [Means for solving the problem]

[0008] As a result of intensive research in light of the above problems, the inventors have discovered novel means and methods for detecting Trichomonas vaginalis and / or Mycoplasma genitalium, and have completed the present invention through further intensive research.

[0009] A typical embodiment of the present invention is as follows. [Section 1] A probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis, the probe 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:5 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 2] Item 2. The probe according to Item 1, comprising the base sequence shown in SEQ ID NO: 9 or 10. [Section 3] A probe for detecting the mgPa gene of Mycoplasma genitalium, the probe having the following characteristics (c) and (d): (c) an oligonucleotide comprising a base sequence S3 of at least 8 consecutive bases in a base sequence shown in any one of SEQ ID NOs: 6 to 8 and 30 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 base sequence S3. (d) Only one of the 5' end or the 3' end of the oligonucleotide (c) is labeled. [Section 4] Item 4. The probe according to Item 3, comprising a base sequence shown in any one of SEQ ID NOs: 20 to 22. [Section 5] Item 5. The probe according to any one of Items 1 to 4, wherein the label is a fluorescent dye label. [Section 6] Item 6. The probe according to any one of Items 1 to 5, wherein the label is a label with a fluorescence quenching dye that is quenched by interaction with guanine. [Section 7] Item 7. The probe according to any one of Items 1 to 6, wherein the labeled terminal base is cytosine. [Section 8] A composition for detecting Trichomonas vaginalis, comprising: A composition comprising a primer set consisting of a combination of at least one first primer selected from those shown in (A-1) below and at least one second primer selected from those shown in (B-1) below, said composition being used in combination with a probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis. (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: 11 to 14. (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: 15 to 19. [Section 9] Item 9. The composition according to Item 8, wherein the probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis comprises the base sequence shown in SEQ ID NO: 9 or 10. [Section 10] A composition for detecting Mycoplasma genitalium, 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 mgPa gene of Mycoplasma genitalium. (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: 23 to 25. (D-1) a second primer consisting of a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 26 to 29. [Section 11] Item 11. The composition according to item 10, wherein the probe for detecting the mgPa gene of Mycoplasma genitalium comprises a base sequence represented by any one of SEQ ID NOs: 20 to 22. [Section 12] A kit for detecting Trichomonas vaginalis and Mycoplasma genitalium, comprising the composition according to item 8 or 9 and the composition according to item 10 or 11. Effect of the Invention

[0010] According to the present invention, a novel detection means and method for Trichomonas vaginalis and / or Mycoplasma genitalium are provided. The detection means and method can perform, for example, highly sensitive detection. Furthermore, according to the present invention, even from a specimen 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 Trichomonas vaginalis and / or Mycoplasma genitalium contained in the specimen sample with high sensitivity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] 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".

[0012] In the present 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 Trichomonas vaginalis and / or Mycoplasma genitalium, as well as a method for detecting Trichomonas vaginalis and / or Mycoplasma genitalium using them and a kit for carrying out the method.

[0013] 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.

[0014] In one embodiment of the present invention, the detection target is the 5.8SrRNA gene region of Trichomonas vaginalis and / or a partial region of the mgPa gene of Mycoplasma genitalium. The base sequence of the partial region of the 5.8SrRNA gene of Trichomonas vaginalis is shown in SEQ ID NO: 1. The base sequences of the partial regions of the mgPa gene of Mycoplasma genitalium are shown in SEQ ID NOs: 2 to 4.

[0015] [1] Oligonucleotide probe of the present invention One embodiment of the present invention is an oligonucleotide probe described in (1) and (2) below.

[0016] (1) Probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis The probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis is preferably the following (1-1) or (1-2), more preferably the following (1-2). (1-1) A probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis, 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:5 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 5.8S rRNA gene of Trichomonas vaginalis, comprising the base sequence shown in SEQ ID NO: 9 or 10. The base sequence S1 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: 5 or a base sequence complementary thereto. The base sequence S1 may be the entire length of the base sequence shown in SEQ ID NO: 5 or a base sequence complementary thereto, but may also be, for example, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, or 25 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.

[0017] (2) Probe for detecting the mgPa gene of Mycoplasma genitalium The probe for detecting the mgPa gene of Mycoplasma genitalium is preferably the following (2-1) or (2-2), more preferably the following (2-2). (2-1) A probe for detecting the mgPa gene of Mycoplasma genitalium, having the following characteristics (c) and (d): (c) an oligonucleotide comprising a base sequence S3 of at least 8 consecutive bases in a base sequence shown in any one of SEQ ID NOs: 6 to 8 and 30 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 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 the mgPa gene of Mycoplasma genitalium, comprising a base sequence shown in any one of SEQ ID NOs: 20 to 22. The base sequence S3 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 any one of SEQ ID NOs: 6 to 8 and 30 or a base sequence complementary thereto. The base sequence S3 may be the entire length of the base sequence shown in any one of SEQ ID NOs: 6 to 8 or a base sequence complementary thereto, but may be, for example, 50 or less, 45 or less, 40 or less, 35 or less, 30 or less, or 25 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.

[0018] The base sequence shown in SEQ ID NO: 5 is a portion of the base sequence shown in SEQ ID NO: 1. The base sequence shown in SEQ ID NO: 5 is specific to Trichomonas vaginalis. Therefore, by using a portion or the entirety of the base sequence shown in SEQ ID NO: 5 as an oligonucleotide probe, it is possible to specifically detect only Trichomonas vaginalis.

[0019] Although the base sequence shown in SEQ ID NO: 6 is not a part of the base sequence shown in any of SEQ ID NOs: 2 to 4, the mgPa gene is a multicopy gene, so there are multiple copy numbers of conserved regions in the genome of Mycoplasma genitalium, and these conserved regions may have the same nucleic acid sequence as each other, or may have a nucleic acid sequence in which one to several bases (for example, 1 to 3 bases) of nucleic acid are substituted, deleted, inserted or added, so that it may be identical to one of the nucleic acid sequences of such conserved regions. The base sequence of SEQ ID NO: 6 is specific to Mycoplasma genitalium. Therefore, by using a part or all of the base sequence shown in SEQ ID NO: 6 as an oligonucleotide probe, it is possible to specifically detect only Mycoplasma genitalium.

[0020] The base sequence shown in SEQ ID NO: 7 is a part of the base sequence shown in SEQ ID NO: 3. The base sequence shown in SEQ ID NO: 7 is specific to Mycoplasma genitalium. Therefore, by using a part or the whole of the base sequence shown in SEQ ID NO: 7 as an oligonucleotide probe, it is possible to specifically detect only Mycoplasma genitalium.

[0021] The base sequence shown in SEQ ID NO: 8 is a part of the base sequence shown in SEQ ID NO: 4. The base sequence shown in SEQ ID NO: 8 is specific to Mycoplasma genitalium. Therefore, by using a part or the whole of the base sequence shown in SEQ ID NO: 8 as an oligonucleotide probe, it is possible to specifically detect only Mycoplasma genitalium.

[0022] The base sequence shown in SEQ ID NO: 30 is a part of the base sequence shown in SEQ ID NO: 4. The base sequence shown in SEQ ID NO: 30 is specific to Mycoplasma genitalium. Therefore, by using a part or the whole of the base sequence shown in SEQ ID NO: 30 as an oligonucleotide probe, it is possible to specifically detect only Mycoplasma genitalium.

[0023] The above oligonucleotide probes (1) and (2) 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 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) and (2) 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.

[0025] [2] Oligonucleotide primer of the present invention One embodiment of the present invention is an oligonucleotide primer described in (3) and (4) below.

[0026] (3) Primers for detecting Trichomonas vaginalis The primers for detecting Trichomonas vaginalis 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: 11 to 14. (B-1) A second primer consisting of a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 15 to 19. 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: 11 to 14, 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: 11 include SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. 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: 15 to 19, 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: 15 include SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19. The second primer (B-1) may be used alone or in combination of two or more types.

[0027] (4) Primers for detecting Mycoplasma genitalium The primers for detecting Mycoplasma genitalium are not particularly limited as long as they enable detection, 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: 23 to 25. (D-1) a second primer consisting of a nucleotide sequence showing at least 90% identity to the nucleotide sequence shown in any one of SEQ ID NOs: 26 to 29. The first primer (C-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: 23 to 25, 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: 23 include SEQ ID NO: 24 and SEQ ID NO: 25. The first primer (C-1) may be used alone or in combination of two or more kinds. The second primer (D-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: 26 to 29, 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: 26 include SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29. The second primer (D-1) may be used alone or in combination of two or more types.

[0028] [3-(1)] The method for detecting Trichomonas vaginalis and / or Mycoplasma genitalium according to the present invention The method for detecting Trichomonas vaginalis and / or Mycoplasma genitalium of the present invention preferably comprises the following steps (1) to (3). (1) A step of amplifying nucleic acid derived from Trichomonas vaginalis and / or Mycoplasma genitalium 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).

[0029] 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 the 5.8S rRNA gene of Trichomonas vaginalis and the mgPa gene of Mycoplasma genitalium. (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).

[0030] In one embodiment of the present invention, a primer set and a probe for detecting Trichomonas vaginalis may be used in combination. Also, a primer set and a probe for detecting Mycoplasma genitalium may be used in combination. In principle, the combination may be any combination, but is preferably either of the following (I) and (II). (I) A combination of a pair of primer sets consisting of the first primer (A-1) above and the second primer (B-1) above, and the oligonucleotide probe (1) 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:5 or a base sequence complementary thereto). (II) 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 (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 any of the base sequences shown in SEQ ID NOs: 6 to 8 and 30, or a base sequence complementary thereto).

[0031] In one embodiment of the present invention, a primer set and a probe for detecting Trichomonas vaginalis may be combined with a primer set and a probe for detecting Mycoplasma genitalium to be used to detect both Trichomonas vaginalis and Mycoplasma genitalium. In principle, any combination may be used, but the combination of (I) and (II) above is preferred.

[0032] [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.

[0033] The type of the test nucleic acid is not particularly limited, and examples thereof include DNA, total RNA, mRNA, and other RNA, etc. In addition, examples of the test nucleic acid include nucleic acids contained in samples such as cell culture samples and biological samples parasitized with Trichomonas vaginalis and / or Mycoplasma genitalium.

[0034] 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.

[0035] 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.

[0036] The specific nucleic acid amplification method used in the nucleic acid amplification step in the method for detecting Trichomonas vaginalis and / or Mycoplasma genitalium of the present invention is not particularly limited, and any known method can be used as appropriate. For example, PCR (Polymerase Chain Reaction) method, NASBA (Nucleic acid sequence based amplification) method, TMA (Transcription-mediated amplification) method, SDA (Strand Displacement Amplification) method, etc. can be mentioned, but it is preferable to use PCR method. In each of these methods, the conditions of the amplification reaction are not particularly limited, and the amplification reaction can be performed by a conventionally known method.

[0037] 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.

[0038] When DNA contained in Trichomonas vaginalis and / or Mycoplasma genitalium is amplified in a reaction system containing the probe of the present invention, the oligonucleotide probe may bind to DNA genes contained in the sample Trichomonas vaginalis and / or Mycoplasma genitalium or their amplification products during the nucleic acid amplification process.

[0039] 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.

[0040] 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.

[0041] 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 has achieved such 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.

[0042] [3-(3)] Complex formation between the nucleic acid amplification product derived from Trichomonas vaginalis and / or Mycoplasma genitalium and the probe In the detection method of Trichomonas vaginalis and / or Mycoplasma genitalium 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.

[0043] 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.

[0044] [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.

[0045] Melting curve analysis is carried out, 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 of 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 higher than the absorbance at the start of heating (the absorbance of only double-stranded DNA), and this indicates that melting has been completed.

[0046] 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 Trichomonas vaginalis and / or Mycoplasma genitalium of the present invention.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The probe used in the method for detecting Trichomonas vaginalis and / or Mycoplasma genitalium 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] The presence or absence of the test nucleic acid can be determined, for example, by measuring a signal change during hybridization. Specifically, it is preferable to measure a signal change accompanying the temperature drop when the temperature of the reaction solution containing the probe is lowered to form a hybrid.

[0058] 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.

[0059] 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.

[0060] [4] Composition and kit for detecting Trichomonas vaginalis and / or Mycoplasma genitalium In another embodiment, there is provided a composition for detecting Trichomonas vaginalis comprising the probe for detecting Trichomonas vaginalis described above, the composition being used in combination with the probe for detecting Trichomonas vaginalis described above. In another embodiment, there is provided a composition and kit for detecting the 5.8S rRNA gene of Trichomonas vaginalis, comprising a primer set consisting of a combination of the first primer (A-1) above and the second primer (B-1) above, which is used in combination with the probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis described above. In another embodiment, a composition and kit for detecting the mgPa gene of Mycoplasma genitalium are provided, which include a primer set consisting of a combination of the first primer (C-1) above and the second primer (D-1) above, and are used in combination with a probe for detecting the mgPa gene of Mycoplasma genitalium. In another embodiment, compositions and kits for detecting Trichomonas vaginalis and / or Mycoplasma genitalium are provided, comprising a composition for detecting the 5.8S rRNA gene of Trichomonas vaginalis and / or a composition for detecting the mgPa gene of Mycoplasma genitalium. 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

[0061] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0062] [Example 1: Detection of Trichomonas vaginalis] (1-1) Method A combination of primers and probes capable of detecting Trichomonas vaginalis with high sensitivity was explored. 100 (measurement sample 1), 25 (measurement sample 2), or 5 (measurement sample 3) Trichomonas vaginalis protozoa were used per reaction, and detection of Trichomonas vaginalis was confirmed using a set of oligonucleotides and oligonucleotide probes designed based on SEQ ID NOs: 9 to 11, 13 to 15, and 19. Regarding the presence or absence of non-specific amplification, purified water was used as a negative sample for the measurement.

[0063] (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: 11, 13, and 14) 0.4 μL 100 μM Oligonucleotide primer (any one of SEQ ID NO: 15 and 19) 0.2 μL 10 μM oligonucleotide probe (either one of SEQ ID NO: 9 and 10) 0.3 μL KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PP Mix (Genecube® Test Basic, Toyobo) 3 μL

[0064] (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)

[0065] (1-4) Results The oligonucleotide primer and oligonucleotide probe sets designed based on SEQ ID NOs: 9 to 11, 13 to 15, and 19 were able to detect low-concentration samples with high sensitivity. Among them, the combination of SEQ ID NOs: 11 / 15 / 10 in particular showed a high detection rate for low-concentration samples, and was found to be an excellent detection system.

[0066] [Table 1]

[0067] [Example 2: Detection of Mycoplasma genitalium] (2-1) Method In order to explore the range in which Mycoplasma genitalium can be detected with high sensitivity, Mycoplasma genitalium inactivated bacteria liquid was used at 10 copies (measurement sample 1) and 5 copies (measurement sample 2) per reaction, and the detection of Mycoplasma genitalium was confirmed by combining the oligonucleotide probes and oligonucleotide primers shown in SEQ ID NOs: 20 to 29. Regarding the presence or absence of non-specific amplification, purified water was used as a negative sample for the measurement.

[0068] (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: 23 to 25) 0.4 μL 100 μM Oligonucleotide primer (any one of SEQ ID NOs: 26 to 29) 0.2 μL 10 μM Oligonucleotide probe (any one of SEQ ID NOs: 20 to 22) 0.3 μL KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PP Mix (Genecube® Test Basic, Toyobo) 3 μL

[0069] (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)

[0070] (2-4) Results The oligonucleotide and oligonucleotide probe sets designed based on sequence numbers 20 to 25 were able to detect low concentration samples with high sensitivity. Among them, the combinations of sequence numbers 23 / 27 / 20 and 25 / 28 / 22 had better detection sensitivity than other combinations. 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. A detection system capable of detecting Mycoplasma genitalium with high sensitivity was discovered.

[0071] [Table 2]

[0072] [Example 3: Detection of multiple bacterial species in one reaction system] (3-1) Method In order to search for a combination of primers and probes that can detect Trichomonas vaginalis and Mycoplasma genitalium with high sensitivity in a single reaction system, 5 copies of gDNA of Trichomonas vaginalis and 5 copies of gDNA of Mycoplasma genitalium were used per reaction, and detection of Trichomonas vaginalis or Mycoplasma genitalium was confirmed with a set of oligonucleotide primers and oligonucleotide probes designed based on SEQ ID NOs: 9, 10, 13-15, and 19-28. Purified water was used as a negative sample for the measurement to check for the presence or absence of nonspecific amplification.

[0073] (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: 11, 14, and 23 to 25) 0.4 μL 100 μM Oligonucleotide primer (any one of SEQ ID NOs: 15, 19, and 26 to 28) 0.2 μL 10 μM oligonucleotide probe (any one of SEQ ID NOs: 9, 10, and 20 to 22) 0.3 μL KOD Mix (GeneCube(R) Test Basic, Toyobo) 3μL PP Mix (Genecube® Test Basic, Toyobo) 3 μL

[0074] (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)

[0075] (3-4) Results The combinations of SEQ ID NO:25 / 28 / 22 and 11 / 15 / 10 had better detection sensitivity than other combinations. 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.

[0076] [Table 3] [Industrial Applicability]

[0077] By utilizing the present invention in a test for Trichomonas vaginalis and / or Mycoplasma genitalium, it is possible to detect Trichomonas vaginalis and / or Mycoplasma genitalium with high sensitivity and with excellent speed and accuracy. In addition, the present invention allows multiple bacterial species to be detected simultaneously in one reaction system by arbitrarily combining primer sets and probes, so that it becomes possible to easily test for Trichomonas vaginalis and / or Mycoplasma genitalium.

Claims

1. A probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis, the probe 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:5 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.

2. The probe according to claim 1 , comprising the base sequence shown in SEQ ID NO: 9 or 10.

3. A probe for detecting the mgPa gene of Mycoplasma genitalium, the probe having the following characteristics (c) and (d): (c) an oligonucleotide comprising a base sequence S3 of at least 8 consecutive bases in a base sequence shown in any one of SEQ ID NOs: 6 to 8 and 30 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.

4. The probe according to claim 3, comprising a base sequence shown in any one of SEQ ID NOs: 20 to 22.

5. The probe according to any one of claims 1 to 4, wherein the label is a fluorescent dye label.

6. The probe according to any one of claims 1 to 4, wherein the label is a label with a fluorescence quenching dye that is quenched by interaction with guanine.

7. The probe according to any one of claims 1 to 4, wherein the labeled terminal base is cytosine.

8. A composition for detecting Trichomonas vaginalis, comprising: A composition comprising a primer set consisting of a combination of at least one first primer selected from those shown in (A-1) below and at least one second primer selected from those shown in (B-1) below, said composition being used in combination with a probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis. (A-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: 11 to 14. (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: 15 to 19.

9. The composition according to claim 8, wherein the probe for detecting the 5.8S rRNA gene of Trichomonas vaginalis comprises the base sequence shown in SEQ ID NO: 9 or 10.

10. A composition for detecting Mycoplasma genitalium, 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 mgPa gene of Mycoplasma genitalium. (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: 23 to 25. (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: 26 to 29.

11. The composition according to claim 10, wherein the probe for detecting the mgPa gene of Mycoplasma genitalium comprises a base sequence represented by any one of SEQ ID NOs: 20 to 22.

12. A kit for detecting Trichomonas vaginalis and Mycoplasma genitalium, comprising a composition according to claim 8 or 9 and a composition according to claim 10 or 11.

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