Oligonucleotide for detection of mycoplasma hominis and use thereof

Abstract

To provide an oligonucleotide for detection of Mycoplasma hominis.SOLUTION: A probe for detecting Mycoplasma hominis is provided, comprising either of the base sequences (A) or (B) below: (A) a base sequence A1 of at least 15 consecutive bases in a specific base sequence or its complementary base sequence, or a base sequence A2 in which 1 to 3 bases in the base sequence A1 are substituted, deleted, inserted, or added, with a label attached exclusively to either the 5' or 3' end; (B) a base sequence B1 of at least 15 consecutive bases in a specific base sequence different from the above or its complementary base sequence, or a base sequence B2 in which 1 to 3 bases in the base sequence B1 are substituted, deleted, inserted, or added, with a label attached exclusively to either the 5' or 3' end.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an oligonucleotide for detecting Mycoplasma hominis (MH) that may be contained in a sample, etc. Furthermore, the present invention relates to a method for detecting Mycoplasma hominis that may be contained in a sample using the oligonucleotide, and a reagent, kit, etc. for use in the method. [Background technology]

[0002] Sexually transmitted diseases are diseases transmitted by sexual contact, and many cases are asymptomatic or mild, 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 and Neisseria gonorrhoeae. Other known causative microorganisms include Mycoplasma genitalium, Mycoplasma hominis, Ureaplasma parvum, Ureaplasma urealyticum, Trichomonas vaginalis, and Treponema pallidum.

[0003] Among the microorganisms that cause these sexually transmitted diseases, Mycoplasma hominis 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. A method for testing Mycoplasma hominis is genetic testing, but manual genetic testing is the mainstream and is not actively carried out. In addition to the gonorrhea and chlamydia that have been tested so far, testing for other sexually transmitted disease causative bacteria such as Mycoplasma genitalium and Mycoplasma hominis is also considered important. Summary of the Invention [Problem to be solved by the invention]

[0004] Melting curve analysis is known as a method for detecting nucleic acid amplification products. In melting curve analysis, nucleic acid amplification and detection can be performed in separate steps, and measurements can be performed relatively easily in as little as 30 minutes. Furthermore, melting curve analysis using fluorescently labeled nucleic acid probes has the advantage of being easily adapted to genetic testing using automated analyzers. However, there is still no known nucleic acid probe that can rapidly detect Mycoplasma hominis with high sensitivity using melting curve analysis.

[0005] One object of the present invention is to provide a useful method for detecting Mycoplasma hominis that may be contained in a sample. [Means for solving the problem]

[0006] As a result of intensive research conducted by the present inventors to achieve the above object, they discovered a method for detecting Mycoplasma hominis quickly and easily with high sensitivity by using a specific nucleic acid probe. Based on this knowledge, they further investigated the method and completed the present invention.

[0007] The present invention includes the following aspects. [Item 1] A probe for detecting Mycoplasma hominis, having the following base sequence (A) or (B): (A) a base sequence A1 of at least 15 consecutive bases in the base sequence from 35 to 70 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence A2 in which 1 to 3 bases are substituted, deleted, inserted or added in the base sequence A1, and only either the 5' end or the 3' end is labeled; (B) A base sequence B1 of at least 15 consecutive bases in the base sequence of positions 45 to 95 (e.g., positions 55 to 95, preferably positions 65 to 95) of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence B2 in which 1 to 3 bases have been substituted, deleted, inserted or added in base sequence B1, and only either the 5' end or the 3' end is labeled. [Item 2] The probe according to Item 1, wherein the length of the base sequence of (A) or (B) is 15 to 25 bases. [Item 3] The probe according to item 1 or 2, wherein the base sequence of (A) or (B) comprises a base sequence shown in any one of SEQ ID NOs: 3 to 8 or a complementary base sequence thereto. [Item 4] The probe according to any one of Items 1 to 3, wherein the label is a fluorescent dye label. [Item 5] The probe according to any one of Items 1 to 4, wherein the label is a fluorescent quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe. [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. [Item 7] The probe according to any one of Items 1 to 6, wherein the label is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and derivatives thereof, rhodamine and derivatives thereof, and BODIPY and derivatives thereof. [Item 8] The probe according to any one of Items 1 to 7, wherein the label is a label with at least one fluorescence 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, TAMRA, rhodamine 6G, tetrabromosulfone fluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue). [Item 9] The probe according to any one of Items 1 to 8, wherein the labeled terminal base is cytosine. [Item 10] A method for detecting Mycoplasma hominis that may be contained in a sample, using the probe described in any one of Items 1 to 9. [Item 11] The following steps (1), (2), and (3): (1) providing a sample that may contain Mycoplasma hominis; (2) carrying out a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more of the probes; The method according to claim 10, comprising: [Item 12] The method according to Item 11, wherein the step (2) is carried out by a PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B. [Item 13] The method according to Item 12, wherein the DNA polymerase belonging to Family B is a DNA polymerase derived from KOD or a mutant thereof. [Item 14] The primer set for detecting Mycoplasma hominis used in the nucleic acid amplification reaction in the step (2) is a first primer having a base sequence S1 of at least 18 consecutive bases in the base sequence from positions 1 to 40 of SEQ ID NO: 1 or a complementary base sequence thereto, or a base sequence S2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S1; and a second primer having a base sequence S3 of at least 18 consecutive bases in the base sequence from positions 65 to 150 of SEQ ID NO: 1 or a complementary base sequence thereto, or a base sequence S4 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S3; a first primer having a base sequence S5 of at least 18 consecutive bases in the base sequence of positions 1 to 50 of SEQ ID NO: 2 or a complementary base sequence 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 a second primer having a base sequence S7 of at least 18 consecutive bases in the base sequence of positions 90 to 140 of SEQ ID NO: 2 or a complementary base sequence thereto, or a base sequence S8 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S7; Item 14. The method according to any one of Items 10 to 13, wherein the second primer is complementary to a DNA extension product of the first primer. [Item 15] The method according to Item 14, wherein the first primer has a nucleotide sequence shown in any one of SEQ ID NOs: 9 to 12, a complementary nucleotide sequence thereof, or these nucleotide sequences in which 1 to 3 bases have been substituted, deleted, inserted or added, and the second primer has a nucleotide sequence shown in any one of SEQ ID NOs: 13 to 15, a complementary nucleotide sequence thereof, or these nucleotide sequences in which 1 to 3 bases have been substituted, deleted, inserted or added. [Item 16] The method according to any one of Items 12 to 15, wherein the detection step in step (3) is carried out by melting curve analysis. [Item 17] A kit for detecting Mycoplasma hominis, comprising the probe described in any one of Items 1 to 9. [Item 18] A kit for detecting Mycoplasma hominis, comprising the primer set described in item 14 or 15. Effect of the Invention

[0008] The present invention can provide a useful means for detecting Mycoplasma hominis that may be contained in a sample. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows a representative example of the results of Test Example 1 (a graph showing the detection results when melting curve analysis was carried out using the probe shown in SEQ ID NO: 4). [Diagram 2] FIG. 1 shows a representative example of the results of Test Example 2 (a graph showing the detection results when melting curve analysis was carried out using the probe shown in SEQ ID NO: 4). [Diagram 3] FIG. 1 shows a representative example of the results of Test Example 3 (a graph showing the detection results when the combination of primers of SEQ ID NOs: 11 and 15 was used). [Figure 4] FIG. 1 shows a representative example of the results of Test Example 4 (a graph showing the detection results when melting curve analysis was carried out using the probe shown in SEQ ID NO: 4). [Diagram 5] 1 is a schematic diagram of primer and probe designs. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 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 in their entirety. In addition, "~" in this specification means "more than or equal to, less than or equal to", for example, if "X~Y" is described in the specification, it means "more than or equal to X, less than or equal to Y". In this specification, "and / or" means any one or any possible combination of two or more of the listed elements. In this specification, "comprising" includes the concepts of "consisting essentially of" and "consisting only of".

[0011] In addition, in this specification, a nucleic acid primer may be simply referred to as a primer, and a nucleic acid probe and a labeled probe may be simply referred to as a probe, and these are also collectively referred to as oligonucleotides.

[0012] In one embodiment, the present invention provides a method for detecting Mycoplasma hominis using a labeled probe containing a specific base sequence. The method can detect Mycoplasma hominis with high sensitivity in a short time, for example. In one embodiment, the present invention can detect Mycoplasma hominis with high sensitivity, for example, in melting curve analysis, by designing a labeled probe targeting a specific base sequence region derived from Mycoplasma hominis. In this specification, SEQ ID NO: 1 and SEQ ID NO: 2 are base sequences derived from Mycoplasma hominis. Specifically, SEQ ID NO: 1 is a part of the base sequence of the 23S rRNA gene of Mycoplasma hominis, and SEQ ID NO: 2 is a part of the base sequence of the yidC gene of Mycoplasma hominis. The present invention encompasses a labeled probe containing a specific base sequence that targets the region of the base sequence shown in SEQ ID NO: 1 or 2 (also referred to as a "nucleic acid probe" in this specification).

[0013] One embodiment of the present invention is a method for detecting Mycoplasma hominis that may be contained in a sample. The method uses a labeled probe having a specific base sequence described below. In a specific embodiment, Mycoplasma hominis that may be contained in a sample can be detected with high sensitivity, for example, in a PCR-melting curve analysis method (including an RT-PCR-melting curve analysis method) by using one specific labeled probe in a reaction solution.

[0014] In a particular embodiment, the method of the present invention comprises at least the following steps (A) and (B): (A) performing a nucleic acid amplification reaction using a nucleic acid primer set consisting of a plurality of nucleic acid primers containing a specific base sequence, using a specific region present in a base sequence derived from Mycoplasma hominis as a template, to generate one or more nucleic acid amplification products; and (B) detecting one or more nucleic acid amplification products obtained in step (A) using one or more labeled probes of the present invention consisting of specific base sequences; Preferably, the method is a PCR-melting curve analysis method in which a PCR reaction is carried out in step (A) and a melting curve analysis is carried out in step (B). Steps (A) and (B) may be carried out in the same reaction solution. Steps (A) and (B) may also be carried out consecutively or simultaneously.

[0015] In a particular embodiment, a method for detecting Mycoplasma hominis that may be contained in a sample includes at least the following steps (1), (2), and (3): (1) providing a sample that may contain Mycoplasma hominis; (2) carrying out a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more labeled probes of the present invention; It is preferable that the method comprises carrying out step (2) by a PCR reaction and carrying out step (3) by melting curve analysis (PCR-melting curve analysis method). Steps (2) and (3) may be carried out in the same reaction solution. Moreover, steps (2) and (3) may be carried out consecutively or simultaneously.

[0016] [Process (1)] In one embodiment, in step (1), it is preferable to prepare a sample that may contain Mycoplasma hominis (for example, biological samples such as animal and plant tissues, body fluids, excrement, and cells; environmental samples such as wipes of facility walls, floors, equipment and fixtures, toilets, and cleaning fluids used to clean them, etc.). Samples that can be used in the present invention are not particularly limited as long as they may contain Mycoplasma hominis. For example, blood, blood culture fluid, cervical scrapings, urethral scrapings, male urethral scrapings, urine, pus, cerebrospinal fluid, pleural fluid, pharyngeal swabs, nasal swabs, sputum, saliva, oral scrapings, tissue sections, skin, vomit, feces, isolated culture colonies, catheter washings, and the like collected from subjects suspected of infection with Mycoplasma hominis are included, but are not limited to these. Considering that Mycoplasma hominis is the main causative bacterium of sexually transmitted diseases, in a particular embodiment, the sample is preferably, for example, tissue section, skin, vomit, urine, isolated culture colony, cervical scraping, urethral scraping, male urethral scraping, urine, or throat swab, and more preferably urine. When a biological sample is used as the measurement target, although there are no particular limitations, pretreatment such as dilution or suspension, centrifugation, enzyme treatment, filtration, heat treatment, acid treatment, alkali treatment, organic solvent treatment, crushing treatment, grinding treatment, or nucleic acid extraction may or may not be performed depending on each biological sample.

[0017] The method of collecting and preparing the sample is not particularly limited, and known methods can be used depending on the type of sample, purpose, etc. In a specific preferred embodiment, the sample does not need to be a sample from which DNA has been isolated or purified, and for example, a specimen collected from a living body may be subjected to proteolytic denaturation treatment using a protease (e.g., proteinase K) and / or heat treatment (e.g., at 60 to 100°C for 1 second to 10 minutes) to previously decompose and remove DNase (DNA decomposition enzyme) present in the specimen, and the resulting sample may be used as is.

[0018] The method for extracting nucleic acid is not particularly limited, and a known method can be used depending on the type of sample, purpose, etc. For nucleic acid extraction, for example, a kit sold by each manufacturer may be used, or an automatic nucleic acid extraction and purification device may be used.

[0019] [Process (2)] In one embodiment, step (2) is preferably a step of generating one or more nucleic acid amplification products by a nucleic acid amplification method (carrying out a nucleic acid amplification reaction using one or more nucleic acid primer sets). The nucleic acid amplification method is a technique for amplifying several copies of a target nucleic acid to a level at which it can be visualized, i.e., to hundreds of millions of copies or more, and is widely used not only in the field of life science research, but also in fields such as clinical diagnosis, food hygiene inspection, and environmental inspection. Examples of such nucleic acid amplification methods include PCR, LAMP, LCR, TMA, SDA, RT-PCR, RT-LAMP, NASBA, TRC, and TMA. These techniques have already been established in the technical field, and a method can be selected according to the purpose. The nucleic acid amplification method is preferably, but is not limited to, the PCR method (including the RT-PCR method).

[0020] (PCR reaction) PCR reaction is a reaction catalyzed mainly by DNA polymerase. PCR reaction usually includes three steps, namely (i) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (ii) annealing of a primer to a template single-stranded DNA, and (iii) extension of the primer using DNA polymerase, as one cycle, and the cycle is repeated. Examples of DNA polymerase include Taq, Tth, Bst, KOD, Pfu, Pwo, Tbr, Tfi, Tfl, Tma, Tne, Vent, DEEPVENT, and mutants thereof. In the present invention, it is preferable to use a DNA polymerase belonging to family B from the viewpoints of simplicity, speed, high sensitivity, and resistance to amplification inhibition by a sample. In addition, when step (3) is performed by melting curve analysis, it is preferable to use a DNA polymerase belonging to family B that does not have 5'→3' exonuclease activity from the viewpoint of using a fluorescence quenching probe.

[0021] The conditions of the PCR reaction are not particularly limited as long as the reaction proceeds. For example, the first step (i) may be performed at 80 to 100°C for about 0 to 300 seconds (for example, about 0.5 to 300 seconds), the second or subsequent (repeated) step (i) may be performed at 80 to 100°C for about 0.5 to 300 seconds, step (ii) may be performed at 35 to 80°C for about 1 to 300 seconds, and step (iii) may be performed at 35 to 85°C for about 1 to 300 seconds. It is preferable that the cycle from step (i) to (iii) is repeated 30 to 70 times. The temperature and time of the repeated cycle may be changed every 1 to 3 cycles.

[0022] (DNA polymerase) The DNA polymerase that can be used in step (2) is preferably, but is not limited to, a DNA polymerase belonging to family B. The DNA polymerase belonging to family B is not particularly limited, but is preferably a DNA polymerase derived from Archea, and more preferably, includes DNA polymerases derived from bacteria of the genus Pyrococcus and Thermococcus. In addition, suitable DNA polymerases also include mutants thereof that do not lose DNA polymerase activity derived from Archea belonging to family B. Examples of the mutants include mutants in which 1 to 3 amino acids (for example, 1 or 2, or 1) are deleted, substituted, inserted, and / or added in the wild-type amino acid sequence, or mutants that show 80% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more amino acid sequence identity with the wild-type amino acid sequence. Specifically, examples of DNA polymerase mutants include, but are not limited to, mutants aimed at enhancing polymerase activity, deleting exonuclease activity, adjusting substrate specificity, and the like.

[0023] Examples of DNA polymerases derived from the genus Pyrococcus include, but are not limited to, DNA polymerases isolated from Pyrococcus furiosus, Pyrococcus sp. GB-D, Pyrococcus woesei, Pyrococcus abyssi, and Pyrococcus horikoshii, as well as mutants derived therefrom that have not lost their DNA polymerase activity.

[0024] Examples of DNA polymerases derived from the genus Thermococcus include, but are not limited to, DNA polymerases isolated from Thermococcus kodakaraensis, Thermococcus gorgonarius, Thermococcus litoralis, Thermococcus sp. JDF-3, Thermococcus sp. 9 degrees North-7 (Thermococcus sp. 9°N-7), and Thermococcus siculi, and mutants thereof that have not lost their DNA polymerase activity. Preferably, DNA polymerases derived from Thermococcus kodakaraensis and mutants thereof (e.g., KOD-derived DNA polymerase lacking 3'→5' exonuclease activity) can be particularly preferably used in the present invention from the viewpoint of excellent extensibility and thermostability.

[0025] PCR enzymes using these DNA polymerases are commercially available, and examples of such enzymes include Pfu (Staragene), KOD (Toyobo), Pfx (Life Technologies), Vent (New England Biolabs), Deep Vent (New England Biolabs), Tgo (Roche), and Pwo (Roche), any of which can be used in the present invention.

[0026] (DNA polymerase derived from KOD) In the present specification, KOD-derived DNA polymerase (also referred to as KOD DNA polymerase) refers to Thermococcus kodakaraensis-derived DNA polymerase and its variants (e.g., KOD-derived DNA polymerase in which 3'→5' exonuclease activity has been deleted by substituting, deleting, inserting and / or adding 1 to 3 (e.g., 1 or 2, or 1) amino acids in the naturally-occurring amino acid sequence). In one preferred embodiment, step (2) involves performing a nucleic acid amplification reaction using such a KOD-derived DNA polymerase. Compared to Taq DNA polymerase, which is a DNA polymerase belonging to family A, KOD DNA polymerase is superior in accuracy, amplification efficiency, extensibility, and resistance to amplification inhibition by sample-derived inhibitors. In the present invention, the use of such a KOD DNA polymerase is more preferable in terms of simple, rapid and highly sensitive detection of Mycoplasma hominis, as shown in the Examples described later.

[0027] (Nucleic acid primer set) The nucleic acid primer set that can be used in step (2) is not particularly limited as long as it can amplify a nucleic acid fragment derived from Mycoplasma hominis with which the probe described below can form a complex. From the viewpoint of easily obtaining a more sensitive determination result, the nucleic acid primer set is preferably a nucleic acid primer set that can amplify at least the base sequence shown in SEQ ID NO: 1 or 2 by amplifying Mycoplasma hominis.

[0028] In one embodiment, the nucleic acid primer set preferably comprises a first primer having a base sequence S1 of at least 18 consecutive bases (e.g., 18 to 31 bases) in the base sequence of positions 1 to 40 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S2 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added in the base sequence S1, and a second primer having a base sequence S3 of at least 18 consecutive bases (e.g., 18 to 31 bases) in the base sequence of positions 65 to 150 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence S4 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added in the base sequence S3.

[0029] In another embodiment, the nucleic acid primer set preferably comprises a first primer having a base sequence S5 of at least 18 consecutive bases (e.g., 18 to 31 bases) in the base sequence of positions 1 to 50 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added in the base sequence S5, and a second primer having a base sequence S7 of at least 18 consecutive bases (e.g., 18 to 31 bases) in the base sequence of positions 90 to 140 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added in the base sequence S7.

[0030] The second primer is preferably complementary to the DNA extension product of the first primer.

[0031] In a further preferred embodiment, the nucleic acid primer set includes a first primer having a base sequence shown in any of SEQ ID NOs: 9 to 12, a complementary base sequence thereof, or a base sequence in which one to three (e.g., one or two, or one) bases have been substituted, deleted, inserted or added, and a second primer having a base sequence shown in any of SEQ ID NOs: 13 to 15, a complementary base sequence thereof, or a base sequence in which one to three (e.g., one or two, or one) bases have been substituted, deleted, inserted or added, wherein one primer is complementary to a DNA extension product of the other primer. From the viewpoint of facilitating high-sensitivity detection with a high rate of fluorescence change even when detecting small amounts (e.g., about 1 to 20 copies) of Mycoplasma hominis, it is particularly preferable that the nucleic acid primer set that can be used in the present invention is a nucleic acid primer set that includes a first primer having the base sequence shown in SEQ ID NO: 10 or 11, its complementary base sequence, or said base sequence in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added, and a second primer having the base sequence shown in SEQ ID NO: 14 or 15, its complementary base sequence, or said base sequence in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted or added, wherein one primer is complementary to the DNA extension product of the other primer.

[0032] [Process (3)] The embodiment of step (3) is not particularly limited, and can be carried out by any method known in the art. The Mycoplasma hominis to be detected can mutate in the same way as other infectious microorganisms. Here, if a mismatch occurs between the base sequence of the primer or probe and the base sequence of the target Mycoplasma hominis mutant, the binding strength of the primer or probe to the target gene or the nucleic acid amplification product derived therefrom (target nucleic acid) can be reduced. In particular, when there are many mismatches between the target nucleic acid and the probe in the real-time PCR method, the probe cannot sufficiently bind to the target nucleic acid, so that the rise of the amplification curve may be delayed or may not rise at all, and such a situation may induce a false negative. In the case of the melting curve analysis method, since step (3) is performed after the completion of PCR, even if there is a mismatch in the primer or probe, detection is possible as long as the nucleic acid amplification product is finally obtained, so that the effect of mismatch can be suppressed more than in the real-time PCR method. Therefore, in step (3), it is particularly preferable to detect the nucleic acid amplification product by melting curve analysis. Furthermore, by detecting the nucleic acid amplification products using melting curve analysis, it is possible to detect them in a shorter time (for example, 45 minutes or less, preferably 40 minutes or less, and more preferably 35 minutes or less from the start of the nucleic acid amplification reaction to the completion of melting curve analysis).

[0033] In one embodiment, step (3) comprises the following steps (3-1) and (3-2): (3-1) a step of hybridizing one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of the step (2) with one or more nucleic acid probes of the present invention to form a complex; and (3-2) detecting the complex of step (3-1) In order to obtain a highly sensitive determination result in the detection of Mycoplasma hominis, it is preferable to use a nucleic acid probe described below that can specifically react with a nucleic acid amplification product that can be generated in the nucleic acid amplification reaction in step (2) to form a complex. The hybridization in step (3-1) is preferably carried out under temperature conditions that allow sufficient hybridization of the nucleic acid amplification product with the nucleic acid probe, such as, but not limited to, a temperature at least 5°C lower than the Tm value of the nucleic acid probe, more preferably at least 10°C lower.

[0034] (Nucleic acid probe) The nucleic acid probe of the present invention is not particularly limited as long as it has the base sequence of (A) or (B). When the nucleic acid probe of the present invention is a nucleic acid probe labeled with a fluorescent quenching dye that is quenched by interaction with guanine, which will be described later, it is preferable that at least one terminal base labeled with the dye is cytosine.

[0035] In one embodiment, the nucleic acid probe of the present invention can detect Mycoplasma hominis (including Mycoplasma hominis mutants) having a base sequence that shows 85% or more identity to the base sequence of bases 35 to 70 of SEQ ID NO: 1 or bases 45 to 95 of SEQ ID NO: 2, and can be a nucleic acid probe that can detect Mycoplasma hominis (including Mycoplasma hominis mutants) having a base sequence that shows 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity to the base sequence of bases 35 to 70 of SEQ ID NO: 1 or bases 45 to 95 of SEQ ID NO: 2.

[0036] In a specific embodiment, the nucleic acid probe of the present invention may be a probe containing a base sequence in which 1 to 3 bases are substituted, deleted, inserted or added in the base sequence A1 or B1. The number of bases that may be substituted, deleted, inserted or added may preferably be 1 or 2. When a base is substituted, deleted, inserted or added in this way, the probe is said to contain a mismatched base, and it has been confirmed in the examples described below that a probe containing such a mismatched base can also be suitably used in the present invention.

[0037] In this specification, containing a mismatched base (or simply referred to as "mismatch") means containing a base that is not complementary to the base sequence of the target nucleic acid (when the target nucleic acid becomes double-stranded after the nucleic acid amplification reaction, the base sequence of any single-stranded nucleic acid resulting from dissociation of the double strand). For example, when a cytosine base is present in the base sequence of the target nucleic acid, the base at the position corresponding to the cytosine base in the probe is a base other than guanine (e.g., an adenine base, a cytosine base, a thymine base, and a universal base). For example, when detecting a region of the target nucleic acid that is prone to mutation, a mismatched base (e.g., a universal base) can be selected at the position of the probe that corresponds to the base that is prone to mutation.

[0038] When the nucleic acid probe of the present invention contains a mismatched base, the position of the mismatched base is not particularly limited as long as it does not inhibit the effect of the present invention. From the viewpoint of more reliable detection of the nucleic acid amplification product, it is preferable that the mismatched base is not the terminal base of each probe. For example, the position of the mismatched base is preferably within 5 mers around the center of the total length n of the base sequence constituting the probe ((n+1) / 2 when n is an odd number, n / 2 when n is an even number), more preferably within 4 mers around, even more preferably within 3 mers around, even more preferably within 2 mers around, and particularly preferably within 1 mer around.

[0039] From the viewpoint of enabling more sensitive detection, the fluorescently labeled probe having the base sequence of (A) or (B) is preferably a fluorescently labeled probe having the base sequence shown in any one of SEQ ID NOs: 3 to 8 or a complementary base sequence thereto. Furthermore, from the viewpoint of enabling higher detection through a synergistic effect together with the fluorescently labeled probe of (B), the fluorescently labeled probe of (A) is preferably a fluorescently labeled probe having the base sequence shown in any one of SEQ ID NOs: 3 to 5 and 8 or a complementary base sequence thereto, more preferably a fluorescently labeled probe having the base sequence shown in any one of SEQ ID NOs: 3 to 5 or a complementary base sequence thereto, and particularly preferably a fluorescently labeled probe having the base sequence shown in SEQ ID NO: 4 or a complementary base sequence thereto.

[0040] The length of the nucleic acid probe of the present invention is not particularly limited as long as it is 15 bases or more, for example, 16 bases or more, and is usually 25 bases or less, preferably 23 bases or less, more preferably 22 bases or less, even more preferably 21 bases or less, and even more preferably 20 bases or less (for example, 19 bases or less, or 18 bases or less). The length of the probe may be, for example, 15 to 25 bases, more preferably 16 to 22 bases, and even more preferably 16 to 21 bases. In a specific embodiment, the length of the nucleic acid probe of the present invention may be 16 to 20 bases, or may be 16 to 18 bases. By using a probe of such a length, Mycoplasma hominis can be detected with higher sensitivity.

[0041] In a specific preferred embodiment, specific examples of the nucleic acid probe of the present invention include a probe containing a base sequence shown in any one of SEQ ID NOs: 3 to 8, a complementary base sequence thereof, or a base sequence in which 1 to 3 (e.g., 1 or 2, or 1) bases have been substituted, deleted, inserted, or added in the base sequence. By using a probe having such a specific base sequence, Mycoplasma hominis can be detected with even higher sensitivity.

[0042] The above-mentioned probe is preferably labeled at only one of the 5'-end or 3'-end. In one embodiment, the nucleic acid probe of the present invention is preferably labeled to generate quenching or fluorescence when bound to a nucleic acid containing a base sequence that shows 85% or more, preferably 90% or more, more preferably 93% or more, even more preferably 95% or more, and even more preferably 98% or more identity with a base sequence complementary to the base sequence of the nucleic acid probe, and more preferably labeled to generate quenching. The labeling substance is not particularly limited, but is more preferably a fluorescent dye.

[0043] The fluorescent dye may be either a fluorescent substance that generates fluorescence or a fluorescent substance that quenches fluorescence by hybridizing with a target nucleic acid amplification product to form a complex, but is preferably a fluorescent substance that quenches fluorescence when hybridized with a target nucleic acid amplification product, and is particularly preferably a fluorescence quenching dye that is quenched by interaction with guanine when hybridized with a target nucleic acid amplification product (for example, a fluorescence quenching dye that is quenched by interaction with guanine). Specific examples of the fluorescent quenching dye include, but are not limited to, at least one fluorescent quenching dye selected from the group consisting of fluorescein and its derivatives (e.g., fluorescein isothiocyanate (FITC)), rhodamine and its derivatives (e.g., 5-carboxyrhodamine 6G (GR6G), tetramethylrhodamine (TAMRA), carboxyrhodamine, x-rhodamine, sulforhodamine 101 acid chloride), and BODIPY and its derivatives (e.g., BODIPY-FL, BODIPY-FL / C3, BODIPY-FL / C6, BODIPY-5-FAM, BODIPY-TMR, BODIPY-TR, BODIPY-R6G, BODIPY-564, BODIPY-581, BODIPY-591, BODIPY-630, BODIPY-650, BODIPY-665).

[0044] More specifically, examples of the fluorescence quenching dye that is quenched by interaction with guanine include at least one fluorescence 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, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue), and these fluorescence quenching dyes can be suitably used in the present invention.

[0045] In a particular preferred embodiment, a probe in which the terminal base labeled with a fluorescence quenching dye is cytosine is more preferred. When such a probe hybridizes to a nucleic acid amplification product, it can form a base pair with a guanine base in the nucleic acid amplification product and interact with it to quench the fluorescence, making it very easy to measure the change in the fluorescence intensity of the reaction solution.

[0046] In addition, when the probe is hybridized, even if the cytosine base of the probe and the guanine base in the nucleic acid amplification product do not form a base pair, the fluorescence can be quenched as long as the distance between these bases is close. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. That is, when the probe is hybridized, the fluorescence can be quenched if the guanine base in the nucleic acid amplification product is present within a range of, for example, 1 to 3 bases relative to the cytosine base of the probe (a base that forms a base pair with the cytosine base is considered to be 1).

[0047] Therefore, even if at least one terminal base labeled with a fluorescence quenching dye is not cytosine, the change in the fluorescence intensity of the reaction solution can be measured. For example, details are described in Japanese Patent No. 5354216, and the present invention can also refer to this technology. For example, when the probe is hybridized, quenching can be achieved if a guanine base in the nucleic acid amplification product is present within a range of, for example, 1 to 3 bases relative to the terminal base labeled with the fluorescence quenching dye (the base that forms a base pair with the terminal base is counted as 1).

[0048] In a particular preferred embodiment, the nucleic acid probe of the present invention is used in step (3). In this manner, Mycoplasma hominis can be detected by using the nucleic acid probe of the present invention. In the method of the present invention, one type of the probe of the present invention may be used, or two or more types of the probe may be used in combination.

[0049] In one embodiment, the step (3) may include at least one of the following steps (3-a), (3-b), and (3-c): (3-a) Simultaneously with step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution, thereby monitoring the progress of the reaction (nucleic acid amplification reaction) of step (2) in real time. (3-b) After completion of step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the fluorescence intensity of the reaction solution, thereby monitoring the progress of the reaction (nucleic acid amplification reaction) of step (2) at an endpoint. (3-c) after completion of the step (2), a step of hybridizing the nucleic acid probe of the present invention to the nucleic acid amplification product in the reaction solution and measuring the temperature dependence of the fluorescence intensity of the reaction solution. By the step (3-a), (3-b), or (3-c), the formation of a complex formed between a nucleic acid amplification product and a nucleic acid probe can be detected simply, quickly, and with high sensitivity. The steps (3-a), (3-b), and (3-c) may be performed in combination, for example, both steps (3-a) and (3-b), or both steps (3-a) and (3-c) can be performed. In one embodiment, from the viewpoint of more rapid detection of a nucleic acid amplification product, the step (3-b) or (3-c) is preferred. In particular, the step (3-c), i.e., the melting curve analysis method, is preferred.

[0050] (Step (3-a)) Step (3-a) is a method for monitoring the progress of a nucleic acid amplification reaction in real time (so-called real-time PCR method), and quantitative analysis is possible by comparing with a control substance of known concentration.

[0051] (Step (3-b)) In step (3-b), the progress of the nucleic acid amplification reaction is monitored at the end point, thereby enabling rapid detection of the target nucleic acid contained in the sample. Furthermore, the approximate amount of the target nucleic acid can be estimated by comparing the fluorescence intensity at the end point. For example, the progress of the nucleic acid amplification reaction is monitored at an end point by measuring the fluorescence intensity of a reaction solution containing a nucleic acid probe labeled with a fluorescent quenching dye. After the nucleic acid amplification reaction is completed, the fluorescence intensity of the reaction solution is measured, and the fluorescence intensity of the reaction solution after the reaction is compared with the fluorescence intensity of the reaction solution before the reaction, thereby confirming the presence or absence of amplification of the target nucleic acid. Alternatively, the presence or absence of the target nucleic acid contained in the sample can also be confirmed by comparing the fluorescence intensity of the reaction solution after the reaction with the fluorescence intensity of a control reaction solution. The control reaction solution is a reaction solution to which a sample known to be negative or positive is added instead of the sample to be measured. The progress of the nucleic acid amplification reaction generally needs to be monitored in real time, but for the purpose of more rapid and convenient detection, it is preferable to measure it at an end point.

[0052] (Step (3-c)) In step (3-c), measuring the temperature dependency of the fluorescence intensity may specifically mean measuring the fluorescence intensity at each temperature while changing the temperature of the reaction solution from low to high. The melting temperature (Tm value) specific to the nucleic acid probe used can be obtained by first differentiating the obtained fluorescence intensity with respect to temperature. The fluorescence intensity may also be converted into a fluorescence quenching rate or the like according to the purpose. The detection and analysis of target nucleic acids using the Tm value is called melting curve analysis. In general, the Tm value refers to the temperature at which the proportion of an oligonucleotide that forms a double strand with its complementary strand is equal to the proportion that does not form a double strand and is single-stranded. Since the Tm value is a value specific to a base sequence, melting curve analysis can be used as a method for analyzing base sequence polymorphisms of target nucleic acids. Base sequence polymorphisms referred to here include single nucleotide polymorphisms, base substitutions, base deletions, base insertions, etc.

[0053] As an example, melting curve analysis is also applied to SNP analysis. When there is a mutation in the base sequence of the target nucleic acid relative to the probe, the bases mismatch when the probe hybridizes, and the Tm value is generally low. Therefore, single nucleotide polymorphism analysis (SNP analysis) can also be performed by comparing the magnitude of the Tm value.

[0054] [Reagents for detecting Mycoplasma hominis] In another embodiment, the present invention provides a reagent for detecting Mycoplasma hominis. The reagent preferably contains at least components necessary for nucleic acid amplification reaction and detection in addition to the nucleic acid probe of the present invention described above. The necessary components can be those known in the art. For example, the reagent of the present invention preferably contains at least a nucleic acid primer set for PCR, a DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and inorganic salts such as magnesium salts. The nucleic acid primer set for PCR and the nucleic acid probe for detection can contain a plurality of sets in order to amplify a plurality of regions of Mycoplasma hominis. The concentration of each component can be appropriately adjusted, but for example, the nucleic acid probe is preferably 0.01 to 1 μM, more preferably 0.02 to 0.5 μM. When used as a nucleic acid probe set, each nucleic acid probe contained in the probe set is preferably within the above concentration range. The nucleic acid primer is preferably 0.01 to 10 μM. The DNA polymerase is preferably 0.01 to 1 U / μL, more preferably 0.02 to 0.5 U / μL. The concentration of deoxyribonucleoside triphosphates (dNTPs) is preferably 0.02 to 1 mM, and more preferably 0.1 to 0.5 mM. The concentration of inorganic salts such as magnesium salts is preferably 0.1 to 10 mM, and more preferably 1 to 5 mM.

[0055] Furthermore, the reagent of the present invention may contain additives known in the art for the purpose of suppressing non-specific amplification and promoting the reaction. Examples of additives for suppressing non-specific amplification include known anti-DNA polymerase antibodies and phosphoric acid. Examples of additives for promoting the reaction include bovine serum albumin (BSA), protease inhibitors, single-strand binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), gelatin, tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, carnitine, Triton, Tween 20, Nonidet (registered trademark) P40, and the like. In order to facilitate the determination of false negatives, the reagent of the present invention preferably contains an internal control known in the art. In the present invention, these additives may be used alone or in combination of two or more.

[0056] In a specific embodiment, the reagent of the present invention preferably includes an internal control. By using the internal control in combination, it is possible to easily confirm that the nucleic acid amplification reaction is performed normally, reducing the risk of false negatives, and enabling a more accurate Mycoplasma hominis test. When using the internal control in combination in the melting curve analysis method, it is preferable that the detection temperature of the internal control and the detection temperature of the Mycoplasma hominis detection probe of the present invention are separated by a certain degree (for example, 5°C or more). For example, when using the internal control and / or the Mycoplasma hominis detection probe of the present invention in the melting curve analysis method, the detection temperature can be determined by a method known in the art, and those having detection temperatures different from each other, for example, 5°C or more, can be appropriately selected and used by those skilled in the art.

[0057] [Kit for detecting Mycoplasma hominis] In another embodiment, the present invention provides a kit for detecting Mycoplasma hominis. The kit of the present invention is not particularly limited as long as it contains the nucleic acid probe of the present invention or the reagent of the present invention described above and is configured to detect (including differentiation) Mycoplasma hominis. For example, the kit of the present invention can optionally contain a reagent capable of detecting (including quantification) the presence of the target substance and / or an instruction manual for use that explains the method of use, etc. For example, the kit of the present invention can be provided in a form in which the nucleic acid probe, components necessary for the nucleic acid amplification reaction, and components necessary for detecting the amplification product are sealed in the same container or in separate containers, and packaged in, for example, a single package, and includes information on the method of use of the kit. The kit of the present invention can also contain a positive control solution and / or a negative control solution. EXAMPLES

[0058] The present invention will be specifically described below by showing examples, but the present invention is not limited to these examples.

[0059] Test Example 1: Confirmatory test for detection of Mycoplasma hominis (1-1) Method In order to search for a nucleic acid probe that can specifically detect Mycoplasma hominis, 50 copies of Mycoplasma hominis DNA were used per reaction, and the presence or absence of non-specific amplification and the detection of Mycoplasma hominis were confirmed using a total of six nucleic acid probes designed based on the base sequences shown in SEQ ID NOs: 3 to 8. Purified water was used as a negative sample for the measurement of the presence or absence of non-specific amplification. The nucleic acid probes used were oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 3 to 8, which were synthesized according to a conventional method (only either the 5' or 3' end was labeled with CR6G. See FIG. 5 for the labeling position in each probe). In addition, a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 10 or 11 and a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 14 or 15 were used as the nucleic acid primer set.

[0060] (1-2) Reaction solution A reaction solution containing the components shown below was prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.5 μM of a primer consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 10 or 11 3.0 μM primer shown in SEQ ID NO: 14 or 15 0.4 μM of a probe represented by any one of SEQ ID NOs: 3 to 8

[0061] (1-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. 94℃ 30 seconds 97℃ 1 sec -58℃ 5 sec -68℃ 5 sec (50 cycles)

[0062] (1-4) Results Figure 1 shows a detection graph obtained when 50 copies of Mycoplasma hominis were detected by nucleic acid amplification and melting curve analysis using the probe shown in SEQ ID NO: 4 as a representative example. Similarly, the measurement results using the probe used in this example are summarized in Table 1. The fluorescence change rate (represented as the quenching rate in this example) was calculated by the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0063] As shown in the results of Table 1, it was found that all of the probes were capable of detecting Mycoplasma hominis. Furthermore, the probes shown in SEQ ID NO: 5 and 7 were designed to contain a mismatch base different from the base sequence shown in SEQ ID NO: 2 near the center of the base sequence constituting the probe. From these results, it was confirmed that the Mycoplasma hominis detection probe of the present invention may contain a mismatch base. Furthermore, when non-specific amplification was confirmed using purified water as a negative sample, no non-specific amplification was observed when any of the probes was used.

[0064] [Table 1]

[0065] Test Example 2: Confirmatory test for detection of Mycoplasma hominis at low copy numbers (2-1) Method In order to search for a nucleic acid probe that can specifically detect Mycoplasma hominis, 10 copies of Mycoplasma hominis DNA were used per reaction, and the presence or absence of non-specific amplification and the detection of Mycoplasma hominis were confirmed using a total of six nucleic acid probes designed based on the base sequences shown in SEQ ID NOs: 3 to 8. Purified water was used as a negative sample for the measurement of the presence or absence of non-specific amplification. The nucleic acid probes used were oligonucleotides consisting of the base sequences shown in SEQ ID NOs: 3 to 8, which were synthesized according to a conventional method (only either the 5' or 3' end was labeled with CR6G. See FIG. 5 for the labeling position in each probe). In addition, a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 10 or 11 and a set of nucleic acid primers consisting of the base sequence shown in SEQ ID NO: 14 or 15 were used as the nucleic acid primer set.

[0066] (2-2) Reaction solution A reaction solution containing the components shown below was prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.5 μM of a primer consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 10 or 11 3.0 μM primer shown in SEQ ID NO: 14 or 15 0.4 μM of a probe represented by any one of SEQ ID NOs: 3 to 8

[0067] (2-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. 94℃ 30 seconds 97℃ 1 sec -58℃ 5 sec -68℃ 5 sec (50 cycles)

[0068] (2-4) Results Figure 2 shows a detection graph obtained when 10 copies of Mycoplasma hominis were detected by nucleic acid amplification and melting curve analysis using the probe shown in SEQ ID NO: 4 as a representative example. Similarly, the measurement results using the probe used in this example are summarized in Table 2. The fluorescence change rate (represented as the quenching rate in this example) was calculated by the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0069] As shown in the results in Table 2, it was found that Mycoplasma hominis can be detected with high sensitivity depending on the probe designed. In addition, the probe shown in SEQ ID NO: 5 was designed to contain a mismatch base different from the base sequence shown in SEQ ID NO: 2 near the center of the base sequence constituting the probe, and it was confirmed that Mycoplasma hominis can be detected down to low copies even if it contains a mismatch base. 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 probes was used.

[0070] [Table 2]

[0071] Example 3: Primer combination test for detection of Mycoplasma hominis (3-1) Method In order to verify the combination of nucleic acid primers (primer set) used together with the probe of the present invention confirmed in Examples 1 and 2, 10 copies of Mycoplasma hominis-derived DNA were used per reaction and verification was performed. In this test example, a nucleic acid consisting of the base sequence shown in SEQ ID NO: 3, 4, or 8, synthesized according to a conventional method, was used as the nucleic acid probe (labeled with CR6G at the 3' end; see FIG. 5). The nucleic acid primers used were a set of a nucleic acid primer consisting of a base sequence complementary to the base sequence shown in any of SEQ ID NOs: 9 to 12 and a nucleic acid primer consisting of a base sequence shown in any of SEQ ID NOs: 13 to 15.

[0072] (3-2) Reaction solution A reaction solution containing the components shown below was prepared using Genecube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.) An internal control (IC) with a known sequence was also added to the reagent to confirm whether nucleic acid amplification was performed normally. 0.5 μM of a primer consisting of a base sequence complementary to any of the base sequences shown in SEQ ID NOs: 9 to 12 3.0 μM of a primer represented by any one of SEQ ID NOs: 13 to 15 0.4 μM probe shown in SEQ ID NO: 3, 4, or 8

[0073] (3-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. 94℃ 30 seconds 97℃ 1 sec -58℃ 5 sec -68℃ 5 sec (50 cycles)

[0074] (3-4) Results 3 is a detection graph obtained when Mycoplasma hominis was detected by nucleic acid amplification and melting curve analysis using the probe shown in SEQ ID NO: 8 and the primers shown in the combination of SEQ ID NO: 11 and 15. Similarly, the measurement results using the nucleic acid probe used in this example are summarized in Table 3. The fluorescence change rate (represented as the quenching rate in this example) was calculated by the formula (fluorescence intensity before amplification - fluorescence intensity after amplification) / (fluorescence intensity before amplification).

[0075] As shown in the results, when the probe of the present invention is used, Mycoplasma hominis in a sample can be detected regardless of the combination of primers, regardless of the primer set used. However, when detecting very low copy Mycoplasma hominis DNA as in this test example, the use of the primer of SEQ ID NO: 12 showed a tendency for the fluorescence value to be lower than that of the primer of SEQ ID NO: 11. From these results, it was confirmed that the combination of nucleic acid primers selected also has an effect on the detection of Mycoplasma hominis with higher sensitivity, and in particular, when the probe shown in SEQ ID NO: 8 is used, the combination of SEQ ID NO: 11 and 15 is preferable because the fluorescence change rate is high even when detecting low copy Mycoplasma hominis DNA. On the other hand, it was suggested that the effect of the combination of nucleic acid primers selected is small when the probe of SEQ ID NO: 3 or 4 is used.

[0076] [Table 3]

[0077] Example 4: Detection of Mycoplasma hominis in urine samples after simple pretreatment (4-1) Method Negative urine samples were centrifuged at 13,000 rpm, and the supernatant was spiked with Mycoplasma hominis DNA at 10 copies / test.

[0078] (4-2) Reaction solution A reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (manufactured by Toyobo Co., Ltd.). 0.5 μM of a primer consisting of a base sequence complementary to the base sequence shown in SEQ ID NO: 10 3.0 μM primer shown in SEQ ID NO:14 0.4 μM probe shown in SEQ ID NO:4

[0079] (4-3) Reaction Using GENECUBE (registered trademark), the reaction solution was reacted in the following temperature cycles, and the fluorescence intensity in each cycle was measured. 94℃ 30 seconds 97℃ 1 sec -58℃ 5 sec -68℃ 5 sec (50 cycles)

[0080] (4-4) Results FIG. 4 is a detection graph obtained when a negative urine sample was centrifuged at 13,000 rpm and Mycoplasma hominis DNA was added to the supernatant to give 10 copies / test. It can be seen that Mycoplasma hominis can be detected with sufficient sensitivity when the primers and probes of the present invention are used. The centrifugal supernatant of the urine sample used in this test example did not undergo a nucleic acid purification process, and therefore contains biological impurities such as various proteins that inhibit nucleic acid amplification reactions. From the results of this test example, it was confirmed that according to the present invention, even samples obtained by simply processing urine samples in this way can be measured with high sensitivity without any problems. [Industrial Applicability]

[0081] By using the method, reagent, or kit using the probe of the present invention, Mycoplasma hominis that may be contained in a sample can be detected easily, quickly, and with high sensitivity. Therefore, the present invention is not only useful for research purposes, but also realizes early detection and early treatment of Mycoplasma hominis, which leads to the prevention of the spread of sexually transmitted diseases and can greatly contribute to clinical diagnosis, environmental testing, etc.

Claims

1. A probe for detecting Mycoplasma hominis having the following base sequence (A) or (B): (A) a base sequence A1 of at least 15 consecutive bases in the base sequence from 35 to 70 of SEQ ID NO: 1 or a complementary base sequence thereof, or a base sequence A2 in which 1 to 3 bases are substituted, deleted, inserted or added in the base sequence A1, and only either the 5' end or the 3' end is labeled; (B) A base sequence B1 of at least 15 consecutive bases in the base sequence from base 45 to base 95 of SEQ ID NO: 2 or its complementary base sequence, or a base sequence B2 in which 1 to 3 bases have been substituted, deleted, inserted or added in base sequence B1, and only either the 5' end or the 3' end is labeled.

2. The probe according to claim 1, wherein the length of the base sequence of (A) or (B) is 15 to 25 bases.

3. The probe according to claim 1, wherein the base sequence of (A) or (B) comprises a base sequence shown in any one of SEQ ID NOs: 3 to 8 or a complementary base sequence thereto.

4. The probe of claim 1 , wherein the label is a fluorescent dye label.

5. The probe according to claim 1 , wherein the label is a fluorescent quenching dye that is quenched when bound to a nucleic acid containing a base sequence that is 90% or more identical to a base sequence complementary to the base sequence of the probe.

6. The probe according to claim 1 , wherein the label is a fluorescent quenching dye that is quenched by interaction with guanine.

7. 2. The probe of claim 1, wherein the label is a label with at least one fluorescence quenching dye selected from the group consisting of fluorescein and its derivatives, rhodamine and its derivatives, and BODIPY and its derivatives.

8. The probe according to claim 1, wherein the label is a label with at least one fluorescence 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, TAMRA, rhodamine 6G, tetrabromosulfonefluorescein (TBSF), and 2-oxo-6,8-difluoro-7-dihydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue).

9. The probe of claim 1 , wherein the labeled terminal base is a cytosine.

10. A method for detecting Mycoplasma hominis which may be contained in a sample, using the probe according to any one of claims 1 to 9.

11. The following steps (1), (2), and (3): (1) providing a sample that may contain Mycoplasma hominis; (2) performing a nucleic acid amplification reaction in a reaction solution containing the sample provided in step (1); and (3) detecting one or more nucleic acid amplification products obtained in the nucleic acid amplification reaction of step (2) using one or more of the probes; The method of claim 10, comprising:

12. The method according to claim 11, wherein the step (2) is carried out by a PCR reaction, and the nucleic acid amplification enzyme used in the PCR reaction is a DNA polymerase belonging to family B.

13. The method according to claim 12, wherein the DNA polymerase belonging to family B is a KOD-derived DNA polymerase or a mutant thereof.

14. The primer set for detecting Mycoplasma hominis used in the nucleic acid amplification reaction in the step (2) is a first primer having a base sequence S1 of at least 18 consecutive bases in the base sequence from 1 to 40 of SEQ ID NO: 1 or a complementary base sequence thereto, or a base sequence S2 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S1; and a second primer having a base sequence S3 of at least 18 consecutive bases in the base sequence from 65 to 150 of SEQ ID NO: 1 or a complementary base sequence thereto, or a base sequence S4 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S3; a first primer having a base sequence S5 of at least 18 consecutive bases in the base sequence from 1 to 50 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S6 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S5; and a second primer having a base sequence S7 of at least 18 consecutive bases in the base sequence from 90 to 140 of SEQ ID NO: 2 or a complementary base sequence thereof, or a base sequence S8 in which 1 to 3 bases have been substituted, deleted, inserted or added in the base sequence S7; The method of claim 10, wherein the second primer is complementary to the DNA extension product of the first primer.

15. The method according to claim 14, wherein the first primer has a nucleotide sequence shown in any one of SEQ ID NOs: 9 to 12, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in said nucleotide sequence, and the second primer has a nucleotide sequence shown in any one of SEQ ID NOs: 13 to 15, a complementary nucleotide sequence thereof, or a nucleotide sequence in which 1 to 3 bases have been substituted, deleted, inserted or added in said nucleotide sequence.

16. 13. The method of claim 12, wherein the detecting step in step (3) is carried out by melting curve analysis.

17. A kit for detecting Mycoplasma hominis, comprising the probe according to any one of claims 1 to 9.

18. A kit for detecting Mycoplasma hominis, comprising the primer according to claim 14 or 15.

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