Base sequences for mycoplasma genitalium detection and related techniques thereof

Abstract

To provide a base sequence for detection that can comprehensively detect Mycoplasma genitalium regardless of whether it is wild-type or mutant, which does not show any reactivity with other Mycoplasma species.SOLUTION: A base sequence from positions 2096 to 2119 of the 23S rRNA gene sequence of Mycoplasma genitalium, which contains at least 20 consecutive bases, or a base sequence complementary to the base sequence is used as a first primer.SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] The present invention relates to a base sequence for specifically detecting Mycoplasma genitalium and related techniques, and in particular to a method for detecting macrolides in Mycoplasma genitalium and a method and reagent for determining macrolide resistance, as well as a method for detecting macrolides in Mycoplasma genitalium. [Background technology]

[0002] Sexually transmitted diseases are illnesses transmitted through sexual contact. Infections can be asymptomatic, and people can unknowingly infect others. Typical bacteria that cause sexually transmitted diseases include chlamydia (Chlamydia trachomatis), gonococci (Neisseria gonorrhoeae), and Mycoplasma genitalium, with mixed infections frequently observed. Of these, Mycoplasma genitalium is often asymptomatic in women, while a high proportion of men are symptomatic. It has been reported that 14-16% of non-gonococcal urethritis, which accounts for 60-70% of all urethritis cases, is Mycoplasma genitalium urethritis.

[0003] Antibiotics, which are substances that exhibit bacteriostatic or bactericidal effects, are widely used to treat sexually transmitted diseases. Antibiotics include macrolides, tetracyclines, and new quinolones, each with a different mechanism of action and target. However, if a single base mutation or deletion occurs in the target of each antibiotic, these antibiotics may not work and may not be effective.

[0004] Mycoplasma genitalium infections are also difficult to treat with antibiotics, and like chlamydia infections, cases are increasing in difficulty. In Japan, the rate of resistance to macrolide antibiotics is particularly high, estimated at over 40%. It has been reported that mutant strains of Mycoplasma genitalium that are resistant to macrolide antibiotics exhibit resistance due to single-nucleotide mutations at positions 2058 and 2059 (positions determined by the E. coli numbering system) in the gene sequence encoding the 23S rRNA (see Non-Patent Document 1).

[0005] The following two methods have been reported to identify resistance to macrolide antibiotics in Mycoplasma genitalium. (1) A method using sequence analysis using a specific base sequence of the 23S rRNA gene sequence of Mycoplasma genitalium as a sequencing primer (Non-Patent Document 2) (2) A method using a melting curve analysis using a specific base sequence of the 23S rRNA gene sequence of Mycoplasma genitalium as a labeled probe (see Patent Document 1 (JP 2023-130663 A) and Patent Document 2 (JP 2023-534457 A)).

[0006] In the method described in Non-Patent Document 2, sequence analysis is performed after nucleic acid purification, which results in problems such as complicated purification procedures, a lack of speed, and the need for dedicated large-scale equipment for analysis.

[0007] Patent documents 1 and 2 describe the feasibility of identifying resistance to macrolide antibiotics. However, the 23S rRNA sequences of Mycoplasma species are highly conserved, resulting in problems with specificity. In particular, the 23S rRNA gene sequence of Mycoplasma pneumoniae, the causative bacterium of Mycoplasma pneumoniae, is highly homologous to the 23S rRNA gene sequence of Mycoplasma genitalium. Mycoplasma genitalium is a common bacterium responsible for sexually transmitted diseases such as urethritis and cervicitis, but it is also present in the pharynx. When using pharyngeal swabs as samples, a definitive diagnosis of Mycoplasma genitalium requires the design of primer sequences that do not cross-react with other Mycoplasma species.

[0008] In addition, a method for detecting Mycoplasma genitalium has been reported in which the partial repeat of the mgpB gene (MgPar) in the MgPa adhesion operon is used as the target gene (see Patent Document 3 (Japanese Patent No. 7141488)).

[0009] The method described in Patent Document 3 achieves high sensitivity by including multiple target genes, but has the problem that it cannot identify resistance to macrolide antibacterial drugs. [Patent Document 1] Japanese Patent Publication No. 2023-130663 [Patent Document 2] Special Publication No. 2023-534457 [Patent Document 3] Patent No. 7141488 [Non-Patent Document 1] Kaitlin A. Tagg et al., Fluoroquinolone and Macrolide Resistance-Associated Mutations in Mycoplasma genitalium., Journal of Clinical Microbiology July 2013 Volume 51 Number 7 [Non-patent document 2] Ryoichi Hamasuna et al., Mutations in ParC and GyrA of moxifloxacin-resistant and susceptible Mycoplasma genitalium strains., PLOS ONE 13(6):e0198355 DISCLOSURE OF THE INVENTION [Problem to be solved by the invention]

[0010] There is a need for a highly specific method that can comprehensively detect both wild-type and mutant forms of Mycoplasma genitalium, which does not show reactivity with other mycoplasmas. Furthermore, from the viewpoint of drug resistance, a simple, rapid, and highly sensitive method is desired that can not only detect both wild-type and mutant forms, but also detect the presence or absence of resistance to macrolide antibiotics in Mycoplasma genitalium. [Means for solving the problem]

[0011] After extensive research, the present inventors have found that Mycoplasma genitalium can be specifically detected by using a first primer having a specific base sequence. Furthermore, they have found that using a second primer having a specific base sequence and a labeled probe not only enables detection of both wild-type and mutant forms of Mycoplasma genitalium, but also enables specific, simple, highly sensitive, and rapid detection of the presence or absence of macrolide antibiotic resistance in Mycoplasma genitalium.

[0012] The first invention provides a first primer comprising a base sequence of at least 20 consecutive bases from positions 2096 to 2119 of the 23S rRNA gene sequence of Mycoplasma genitalium, or a base sequence complementary to this base sequence. This provides a method and reagent for specifically detecting Mycoplasma genitalium, which is unreactive with other Mycoplasma species.

[0013] The second invention, in addition to the first invention, uses as a second primer a base sequence comprising at least 20 consecutive bases from positions 2027 to 2050 of the 23S rRNA gene sequence of Mycoplasma genitalium, or a base sequence complementary to this base sequence, thereby providing a method for detecting Mycoplasma genitalium with high sensitivity and a reagent therefor.

[0014] The third aspect of the present invention uses the nucleotide sequence from positions 2054 to 2078 of the 23S rRNA gene sequence of Mycoplasma genitalium or a nucleotide sequence complementary to this nucleotide sequence as a labeled probe, thereby providing a method and reagents for identifying the presence or absence of resistance to macrolide antibacterial drugs in Mycoplasma genitalium. [Effects of the Invention]

[0015] The present invention enables specific, simple, highly sensitive, and rapid detection of Mycoplasma genitalium. Furthermore, by using a second primer having a specific base sequence and a labeled probe in addition to the first primer of the present invention, it is possible to detect both wild-type and mutant Mycoplasma genitalium, as well as to specifically, simply, highly sensitively, and rapidly detect the presence or absence of macrolide antibiotic resistance in Mycoplasma genitalium. BEST MODE FOR CARRYING OUT THE INVENTION

[0016] 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. In this specification, "to" means "at least or equal to, or equal to, or equal to," and for example, "A to B" in the specification means "at least A and at most B."

[0017] In this specification, the first primer and the second primer may be simply referred to as "primers," and the labeled probe may be simply referred to as "probes," and these may be collectively referred to as "oligonucleotides." Furthermore, a set of a first primer and a second primer may be simply referred to as a "primer set."

[0018] In addition, in this specification, Mycoplasma genitalium that is resistant to macrolide antibiotics may be simply referred to as "resistant type" or "mutant type."

[0019] In one embodiment, the present invention provides a method for specifically detecting Mycoplasma genitalium by using a first primer having a specific base sequence. This method enables specific, simple, highly sensitive, and rapid detection of Mycoplasma genitalium. In addition, by using a second primer having a specific base sequence and a labeled probe and setting the PCR detection temperature to two different temperatures, it is possible to not only detect Mycoplasma genitalium but also determine the presence or absence of a mutation in the 23S rRNA gene sequence associated with macrolide resistance.

[0020] [Methods for detecting Mycoplasma genitalium] In one embodiment, the method for detecting Mycoplasma genitalium that may be contained in a specimen sample is preferably a method using the first primer described below, and may be a method for determining the presence or absence of Mycoplasma genitalium in a specimen sample. The method may also be a method for quantifying Mycoplasma genitalium that may be contained in a specimen sample. In addition, by using the labeled probe described below, it is possible to detect with high sensitivity both wild-type and mutant Mycoplasma genitalium that may be contained in a sample using PCR (RT-PCR). Furthermore, by setting two PCR detection temperatures, it is possible to distinguish and detect either wild-type or mutant Mycoplasma genitalium that may be contained in the sample.

[0021] In a specific embodiment, a method for detecting Mycoplasma genitalium that may be contained in a specimen sample includes at least the following steps (1) and (2): (1) collecting a specimen sample that may contain Mycoplasma genitalium; (2) subjecting the specimen sample of step (1) to a nucleic acid amplification reaction using a primer set having a specific base sequence and a part or all of the 23S rRNA gene sequence of Mycoplasma genitalium as a template; and further detecting the nucleic acid amplification product produced during the amplification reaction in real time using a labeled probe having the specific base sequence. It is preferred that the compound contains:

[0022] [Process (1)] The specimen sample used in step (1) is not particularly limited as long as it may contain Mycoplasma genitalium. Examples of specimen samples include, but are not limited to, blood, blood culture fluid, cervical scraping, vaginal scraping, urethral scraping, urine, throat swab, postnasal swab, sputum, saliva, and oral scraping. Given that Mycoplasma genitalium is a major causative bacterium of sexually transmitted diseases, it is preferable to use cervical scraping, urine, urethral scraping, and throat swab. Depending on the type of biological sample, pretreatments such as dilution, suspension, centrifugation, enzyme treatment, filtration, heat treatment, acid treatment, and alkali treatment, or nucleic acid extraction may be performed.

[0023] The method of collecting and preparing the specimen sample is not particularly limited, and known methods can be used depending on the type and purpose of the specimen sample.

[0024] The method for nucleic acid extraction is not particularly limited, and known methods can be used depending on the type and purpose of the sample. For example, kits sold by various manufacturers may be used for nucleic acid extraction. Nucleic acid extraction may also be performed using an automatic extraction and purification device.

[0025] In a specific preferred embodiment, the test sample may be one that has been prepared by omitting a nucleic acid purification step, which is generally considered essential in conventional nucleic acid amplification reactions. Nucleic acid purification requires specialized reagents and is problematic in that the process is cumbersome, time-consuming, and laborious.

[0026] When a specimen sample that does not undergo the nucleic acid purification step is used, the time from specimen collection to obtaining genetic test results can be shortened, for example, a procedure that previously required one day can be reduced to within two hours. Thus, when the method of the present invention is performed using a specimen sample that has not undergone the nucleic acid purification step, Mycoplasma genitalium can be detected simply and quickly because the labor required for nucleic acid purification is eliminated.

[0027] [Process (2)] In one embodiment, the nucleic acid amplification reaction in step (2) is preferably carried out by a nucleic acid amplification method (a nucleic acid amplification reaction using one or more primer sets). The nucleic acid amplification method is a technique for amplifying a few copies of a target nucleic acid to a level at which it can be visualized, i.e., several hundred million 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.

[0028] Examples of such nucleic acid amplification methods include PCR, LAMP, TMA, and SDA. These techniques have already been established in the art, and a method can be selected according to the purpose. The nucleic acid amplification method is preferably PCR (including RT-PCR), but is not limited to this.

[0029] Hereinafter, the PCR reaction performed in step (2) and the oligonucleotides (first primer, second primer, labeled probe) and detection reagents used in the PCR reaction will be described in detail, but the present invention is not limited to these.

[0030] [PCR reaction] PCR reactions (including RT-PCR reactions) are reactions catalyzed primarily by DNA polymerase. PCR reactions typically involve three cycles: (i) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (ii) annealing of primers to template single-stranded DNA, and (iii) extension of the primers using DNA polymerase.

[0031] From the viewpoint of increasing the reaction speed, it is preferable to use reaction conditions that combine the above (ii) and (iii). Furthermore, when an RT reaction is included, a reverse transcription reaction is carried out before (i). PCR enzymes using these DNA polymerases are commercially available, and any of them can be used in the present invention.

[0032] The conditions for the PCR reaction are not particularly limited as long as the reaction proceeds. For example, the first step (i) may be carried out at 90-100°C for about 1-180 seconds, the second and subsequent steps (i) may be carried out at 90-100°C for 1-20 seconds, and steps (ii) and (iii) may be carried out at 50-75°C for about 1-20 seconds. The cycle of steps (i) to (iii) is preferably repeated 30-70 times. The temperature and time of the repeated cycles may be changed for each cycle.

[0033] In a preferred embodiment, two temperatures are set in steps (ii) and (iii): a first temperature (50 to 58°C) and a second temperature (66 to 70°C). By setting two temperatures, it becomes possible to detect and distinguish between both wild-type and mutant forms of Mycoplasma genitalium, which will be described later.

[0034] [Primer] In general, it is known that the Tm value of a primer is preferably 55 to 65° C., more preferably 60 to 65° C. It is known that the length of a primer is preferably 18 to 30 bases, more preferably 18 to 25 bases.

[0035] [First primer] 11 is a list of first primer sequences of the present invention. The first primer of the present invention is a nucleotide sequence containing at least 20 consecutive nucleotides from positions 2096 to 2119 of the 23S rRNA gene sequence of Mycoplasma genitalium, or a nucleotide sequence complementary to this nucleotide sequence.

[0036] In a preferred embodiment, specific examples of the first primer of the present invention include the base sequences shown in any of SEQ ID NOs: 1 to 3 (see FIG. 11) or base sequences complementary thereto.

[0037] [Second primer] 12 is a list of second primer sequences of the present invention. The second primer of the present invention is a nucleotide sequence containing at least 20 consecutive nucleotides from positions 2027 to 2050 of the 23S rRNA gene sequence of Mycoplasma genitalium, or a nucleotide sequence complementary to this nucleotide sequence.

[0038] In a preferred embodiment, specific examples of the second primer of the present invention include the nucleotide sequence shown in any one of SEQ ID NOs: 11 to 15 (see FIG. 12) or a nucleotide sequence complementary thereto. More preferred examples include the nucleotide sequence shown in any one of SEQ ID NOs: 11 to 13 or a nucleotide sequence complementary thereto.

[0039] [Labeled probe] 13 is a list of labeled probe sequences of the present invention. The labeled probe of the present invention is preferably a nucleotide sequence consisting of positions 2054 to 2078 of the 23S rRNA gene sequence of Mycoplasma genitalium, or a nucleotide sequence complementary to this nucleotide sequence.

[0040] Furthermore, when the probe is labeled with a fluorescence quenching dye that is quenched by interaction with guanine, as described below, it is preferable that at least one terminal base labeled with the dye is cytosine. The length of the labeled probe is not particularly limited as long as it is 15 to 30 bases.

[0041] In a preferred embodiment, a specific example of the labeled probe of the present invention is the base sequence shown in SEQ ID NO: 16 (see FIG. 13) or a base sequence complementary thereto.

[0042] The labeled probe is labeled at only either the 5'-end or the 3'-end, and is preferably labeled so as to generate quenching or fluorescence when bound to a nucleic acid containing a base sequence that is 85% or more, preferably 90% or more, more preferably 95% or more identical to a base sequence complementary to the base sequence of the labeled probe, and more preferably is labeled so as to generate quenching.

[0043] The fluorescent dye may be either a fluorescent substance that emits 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 upon hybridization with a target nucleic acid amplification product, and is particularly preferably a fluorescence quenching dye that quenches fluorescence by interacting with guanine upon hybridization with a target nucleic acid amplification product. Specific examples of the fluorescent dye include, but are not limited to, at least one fluorescence 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)), and BODIPY and its derivatives (e.g., BODIPY-FL).

[0044] In a particularly preferred embodiment, a probe labeled with a fluorescence quenching dye and having a terminal base of cytosine is more preferred. When such a probe hybridizes to a target 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 changes in the fluorescence intensity of the reaction solution.

[0045] In this way, the use of the labeled probe of the present invention makes it possible to detect both wild-type and mutant Mycoplasma genitalium. In a more preferred embodiment, the detected Mycoplasma genitalium can be distinguished and detected as either the wild-type or the mutant.

[0046] [Reagents for detecting Mycoplasma genitalium] The present invention provides a reagent for detecting Mycoplasma genitalium and a reagent for identifying macrolide resistance. The reagent preferably contains at least components necessary for nucleic acid amplification reaction and detection, in addition to the first primer, second primer, and labeled probe of the present invention described above.

[0047] Known components can be used for each of the necessary components. For example, the reagent of the present invention preferably contains at least a DNA polymerase, deoxyribonucleoside triphosphates (dNTPs), and inorganic salts such as magnesium salts or manganese salts. The concentrations of each component can be adjusted as appropriate. For example, the concentrations of the first and second primers are preferably 0.01 to 5 μM, more preferably 0.05 to 2 μM. The concentration of the labeled probe is preferably 0.01 to 0.50 μM, more preferably 0.02 to 0.10 μM. The concentration of the DNA polymerase is preferably 0.1 to 5 U / Test. The concentration of the dNTPs is preferably 0.02 to 1 mM, more preferably 0.1 to 0.5 mM. The concentration of the inorganic salts such as magnesium salts or manganese salts is preferably 0.1 to 10 mM, more preferably 1 to 5 mM.

[0048] Furthermore, the reagent of the present invention may contain additives known in the art for the purposes of suppressing nonspecific amplification and promoting the reaction. Examples of additives for suppressing nonspecific amplification include known anti-DNA polymerase antibodies. Examples of additives for promoting the reaction include bovine serum albumin (BSA), dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, propylene glycol, trimethylene glycol, betaine, trehalose, polyvinylpyrrolidone (PVP), tetramethylammonium acetate (TMAA), polyethylene glycol, Triton, Tween, and Nonidet P40.

[0049] In addition, 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.

[0050] The present invention will be described in detail below with reference to examples, but is not limited to these examples. Figure 1 is a graph showing temperature changes in Example 1 of the present invention, and Figure 2 is an enlarged view of a portion of Figure 1. As shown in Figure 2, fluorescence measurement is performed at both the top (thermal denaturation temperature) and bottom (primer annealing and primer extension temperatures) of the temperature range.

[0051] (Examples 1 to 3, Comparative Examples 1 to 7) Confirmation of specificity

[0052] Materials and Methods The oligonucleotides used in the PCR method in Examples 1 to 3 and Comparative Examples 1 to 7 are as shown in Table 1.

[0053] [Table 1]

[0054] In Examples 1 to 3 and Comparative Examples 1 to 7, the melting temperature (Tm value) and chain length of the first primer used in the PCR method are as shown in (Table 2). The Tm value of the primer is calculated using the Nearest Neighbor method.

[0055] [Table 2]

[0056] <PCR reaction solution composition> The PCR reaction solution composition is prepared in the manner shown in (Table 3).

[0057] [Table 3]

[0058] <PCR reaction conditions> For PCR and fluorescence measurement, a fully automated gene analyzer Smart Gene (registered trademark of Mizho Medy Co., Ltd.) sold by Mizho Medy Co., Ltd. is used. The excitation wavelength and fluorescence wavelength during fluorescence measurement are set to a central wavelength of 525 nm. Note that measurement is also possible with a general-purpose PCR device that can set and use the excitation wavelength and fluorescence wavelength during fluorescence measurement under conditions close to a central wavelength of 525 nm. Examples of general-purpose PCR devices include Lightcycler (registered trademark) nano (Roche Diagnostics Co., Ltd.). The PCR reaction conditions are as shown in (Table 4).

[0059] [Table 4]

[0060] <Nucleic acid sample> The nucleic acid samples used in the PCR methods of Examples 1 to 3 and Comparative Examples 1 to 7 are shown below.

[0061] We used purified DNA from Mycoplasma pneumoniae (FH strain, ATCC No. 15531) purchased from ATCC using the QIAamp DNA Mini Kit.

[0062] <Data analysis method> The data analysis method using QProbe is based on the method described in Japanese Patent No. 4724380, and involves corrective calculation processing of the obtained raw data.

[0063] Calculations according to Equation 1 are performed for all cycles of the amplification reaction. f[n]=fhyb.[n] / fden.[n] (Formula 1) where: fn: Fluorescence intensity value at n cycles calculated by (Equation 1) fhyb.[n]: Fluorescence intensity value of the nth cycle extension and detection step fden.[n]: Fluorescence intensity value of the nth cycle of denaturation step Next, for all cycles of the amplification reaction, calculation is performed using the following (Equation 2). F[n]=f[n] / f

[22] (Formula 2) where: F[n]: The relative value at the nth cycle when the fluorescence intensity value obtained by (Equation 1) at the 22nd cycle is set to 1 The value of F[n] in the final cycle of the amplification reaction, F

[46] , is used to determine Mycoplasma genitalium.

[0064] <Mycoplasma genitalium detection> To determine whether Mycoplasma genitalium is present, the F

[46] value of the test sample is compared with the threshold value. If the F

[46] of the test sample is less than the threshold value, the result is considered positive; otherwise, the result is considered negative. The threshold value used is the mean value of the F

[46] of negative samples that do not contain Mycoplasma genitalium minus five times the standard deviation (hereinafter referred to as the mean - 5SD), i.e., 0.9910.

[0065] <Flowchart of the determination method> FIG. 3 is a flowchart showing a determination method according to the first embodiment of the present invention.

[0066] Figure 4 is a graph showing the change in relative fluorescence value in Example 1 of the present invention. As shown in Figure 4, in Example 1, the relative fluorescence value (F

[46] ) in the final cycle does not fall below the judgment threshold, and the test is determined to be negative for Mycoplasma genitalium. Table 5 shows the Mycoplasma genitalium judgment results and the relative fluorescence value (F

[46] ) in the final cycle. In Examples 1 to 3, the relative fluorescence value (F

[46] ) does not fall below the judgment threshold, and the test is determined to be negative for Mycoplasma genitalium.

[0067] Figure 5 is a graph showing the change in relative value in Comparative Example 1. Table 5 shows the Mycoplasma genitalium determination results and the relative fluorescence value (F

[46] ) in the final cycle. As shown in Figure 5, in Comparative Examples 1 to 7, the relative fluorescence value (F

[46] ) was below the determination threshold, and the result was determined to be positive for Mycoplasma genitalium.

[0068] [Table 5]

[0069] (result) As is clear from Table 5, when a purified DNA product of Mycoplasma pneumoniae was used as a nucleic acid sample, no quenching of the labeled probe was observed under the conditions using the first primers shown in Examples 1 to 3 (first primers using oligonucleotides containing at least 20 consecutive bases, the base sequence from positions 2096 to 2119 of the 23S RNA gene sequence of Mycoplasma genitalium or a base sequence complementary to this base sequence). This demonstrates that the first primers used in Examples 1 to 3 have high specificity for Mycoplasma genitalium. On the other hand, under the conditions using the first primers in Comparative Examples 1 to 7, obvious quenching of the labeled probe was confirmed. This indicates that the first primers used in Comparative Examples 1 to 7 exhibit cross-reactivity with Mycoplasma pneumoniae, that is, they have low specificity with Mycoplasma genitalium. For SEQ ID NO: 2, which has the lowest Tm value among the first primers used in Examples 1 to 3, the Tm value is 57.0 °C and the chain length is 20 bases. If the 3'-terminal of SEQ ID NO: 2 is truncated by 1 base, the Tm value of the base sequence is 55.2 °C, and if it is truncated by 2 bases, the Tm value is 52.8 °C, which is below the lower limit or the lower limit of the preferred range. Therefore, it is considered preferable that the length of the first primer includes 20 bases. (Examples 4 to 6, Comparative Examples 8 to 9) Confirmation of the detection sensitivity of wild-type Mycoplasma genitalium <Materials and Methods> The oligonucleotides used in the PCR method in Examples 4 to 6 and Comparative Examples 8 to 9 are as shown in (Table 6).

[0070] [Table 6]

[0071] The melting temperature (Tm value) and chain length of the second primers used in the PCR method in Examples 4 to 6 and Comparative Examples 8 to 9 are as shown in (Table 7). The calculation of the Tm value of the primers is performed using the Nearest Neighbor method.

[0072] [Table 7]

[0073] <Composition of PCR reaction solution> The composition of the PCR reaction solution is prepared in the same manner as in Example 1.

[0074] <PCR reaction conditions> The PCR reaction conditions are the same as in Example 1.

[0075] <Nucleic acid sample> The nucleic acid samples used in the PCR methods of Examples 4 to 6 and Comparative Examples 8 and 9 are shown below.

[0076] (Wild-type Mycoplasma genitalium artificially synthesized RNA) This is an artificially synthesized RNA produced by artificially synthesizing a gene fragment of 23S rRNA, a ribosomal RNA derived from Mycoplasma genitalium, followed by a transcription reaction.

[0077] The artificially synthesized RNA has a sequence including positions 2058 and 2059 of 23S rRNA, and is derived from the G37 strain. The G37 strain is a macrolide-susceptible strain.

[0078] (Preparation of nucleic acid samples) The length of the above artificial synthetic RNA is 380 nt.

[0079] Calculate the number of copies per μL from the length of the artificially synthesized RNA and the concentration of the solution (μg / μL), and then dilute it with TE buffer solution as necessary.

[0080] <Data analysis method> The data analysis method is carried out in the same manner as in Example 1.

[0081] <Mycoplasma genitalium detection> The method for determining Mycoplasma genitalium is carried out in the same manner as in Example 1.

[0082] The Mycoplasma genitalium determination results and the relative fluorescence values ​​(F

[46] ) in the final cycle for Examples 4 to 6 and Comparative Examples 8 and 9 are shown in Table 8.

[0083] When the RNA amount was 800 copies / test, the relative fluorescence value (F

[46] ) was below the judgment threshold in all of Examples 4 to 6 and Comparative Examples 8 and 9, and the test was judged to be positive for Mycoplasma genitalium.

[0084] When the RNA amount was 200 copies / test, the relative fluorescence values ​​(F

[46] ) of Examples 4 to 6 and Comparative Example 8 were below the judgment threshold and were determined to be Mycoplasma genitalium positive, but the relative fluorescence value (F

[46] ) of Comparative Example 9 was not below the judgment threshold and was determined to be Mycoplasma genitalium negative. Furthermore, Comparative Example 8 was determined to be Mycoplasma genitalium positive, but its relative fluorescence value (F

[46] ) was confirmed to be larger than that of Examples 4 to 6.

[0085] [Table 8]

[0086] (result) As is clear from Table 8, good results were obtained when artificially synthesized RNA from wild-type Mycoplasma genitalium was used as a nucleic acid sample. Among these, the conditions using the second primers shown in Examples 4 to 6 (second primers using oligonucleotides containing at least 20 consecutive bases, either the base sequence from positions 2027 to 2050 of the Mycoplasma genitalium 23S rRNA gene sequence or a base sequence complementary to this base sequence) were confirmed to be highly sensitive. This is thought to be due to improved PCR efficiency due to shorter amplification products or optimized primer Tm values. Among the second primers used in Examples 4 to 6, SEQ ID NO: 13 had the lowest Tm value, a Tm value of 59.5°C and a chain length of 20 bases. On the other hand, SEQ ID NO: 15 used in Comparative Example 9 had a Tm value of 57.2°C and a chain length of 18 bases, which is at the lower end of the preferred range of Tm values ​​and chain lengths. Therefore, it is considered preferable that the length of the second primer contains 20 bases. (Examples 7 to 9, Comparative Examples 10 to 11) Confirmation of the detection sensitivity of mutant Mycoplasma genitalium Materials and Methods The oligonucleotides used in the PCR method in Examples 7 to 9 and Comparative Examples 10 and 11 are as shown in Table 9.

[0087]

Table 9

[0088] <PCR reaction solution composition> The PCR reaction solution composition is prepared in the same manner as in Example 1.

[0089] <PCR reaction conditions> The PCR reaction conditions are the same as those in Example 1.

[0090] <Nucleic acid sample> The nucleic acid samples used in the PCR methods of Examples 7 to 9 and Comparative Examples 10 to 11 are shown below.

[0091] (Artificial synthetic RNA of mutant Mycoplasma genitalium) It is artificial synthetic RNA prepared by transcription reaction after artificially synthesizing a gene fragment of 23S rRNA, which is ribosomal RNA derived from Mycoplasma genitalium.

[0092] The artificial synthetic RNA is a sequence containing positions 2058 and 2059 of 23S rRNA, and the sequence derived from strain M6320 is used. Strain M6320 is a macrolide-resistant strain.

[0093] (Preparation of nucleic acid sample) The length of the above artificial synthetic RNA is 380 nt.

[0094] After calculating the copy number per 1 μL from the length of the artificial synthetic RNA and the concentration of the solution (μg / μL), it is diluted with TE buffer solution as necessary.

[0095] <Data analysis method> The data analysis method is the same as that in Example 1.

[0096] <Determination of Mycoplasma genitalium> The method for determining Mycoplasma genitalium is the same as that in Example 1.

[0097] The Mycoplasma genitalium determination results and the relative fluorescence values ​​(F

[46] ) in the final cycle for Examples 7 to 9 and Comparative Examples 10 to 11 are shown in Table 10.

[0098] When the RNA amount was 800 copies / test, the relative fluorescence value (F

[46] ) was below the judgment threshold in all of Examples 7 to 9 and Comparative Examples 10 to 11, and the test was judged to be positive for Mycoplasma genitalium.

[0099] When the RNA amount was 200 copies / test, the relative fluorescence values ​​(F

[46] ) of Examples 7 to 9 and Comparative Example 11 were below the judgment threshold and were determined to be Mycoplasma genitalium positive, but the relative fluorescence value (F

[46] ) of Comparative Example 10 was not below the judgment threshold and was determined to be Mycoplasma genitalium negative. Furthermore, Comparative Example 10 was determined to be Mycoplasma genitalium positive, but its relative fluorescence value (F

[46] ) was confirmed to be higher than that of Examples 7 to 9.

[0100] [Table 10]

[0101] (result) As is clear from (Table 10), when synthetic RNA of mutant Mycoplasma genitalium was used as a nucleic acid sample, good results were shown in all cases. Among them, the conditions using the second primer shown in Examples 7 to 9 (an oligonucleotide having a nucleotide sequence at positions 2027 to 2050 of the 23S rRNA gene sequence of Mycoplasma genitalium or a nucleotide sequence complementary to this nucleotide sequence and containing at least 20 consecutive bases) were confirmed to be the most sensitive. This is presumably because the PCR efficiency was improved due to the shortening of the amplification product or the optimization of the Tm value of the primer. The primer of SEQ ID NO: 13 having the lowest Tm value among the second primers used in Examples 7 to 9 has a Tm value of 59.5 °C and a chain length of 20 bases. On the other hand, the Tm value of SEQ ID NO: 15 used in Comparative Example 11 is 57.2 °C and the chain length is 18 bases, which is the lower limit of the preferable Tm value and chain length range. Therefore, it is considered preferable that the length of the second primer includes 20 bases. (Examples 10 to 16, Comparative Example 12) Confirmation of macrolide resistance mutation discrimination Here, Fig. 6 is a graph showing the temperature change in Example 10 of the present invention, and Fig. 7 is a partially enlarged view of Fig. 6. <Materials and methods> The oligonucleotides used in the PCR method in Examples 10 to 16 and Comparative Example 12 are as shown in (Table 11).

[0102] [Table 11]

[0103] <PCR reaction solution composition> The PCR reaction solution composition is prepared in the same manner as in Example 1.

[0104] <PCR reaction conditions> PCR and fluorescence measurements were performed using the Smart Gene (registered trademark, Japan) fully automated genetic analyzer sold by Mizuho Medi Co., Ltd. The excitation and emission wavelengths for fluorescence measurements were set to a central wavelength of 525 nm. Measurements can also be performed using a general-purpose PCR instrument that can be set and used under conditions similar to the central wavelength of 525 nm. Examples of general-purpose PCR instruments include the Lightcycler (registered trademark) nano (Roche Diagnostics). PCR reaction conditions are shown in Table 12. Steps 1 and 2 were performed alternately, one cycle at a time, and the temperatures corresponding to A and B are shown in Tables 13 and 14.

[0105] [Table 12]

[0106] [Table 13]

[0107] [Table 14]

[0108] <Nucleic acid sample> As in Examples 4 and 7, the nucleic acid samples used are artificially synthesized RNA of wild-type Mycoplasma genitalium and artificially synthesized RNA of mutant Mycoplasma genitalium. <Data analysis method> The data analysis method using QProbe is based on the method described in Japanese Patent No. 7221491, and involves corrective calculation processing of the obtained raw data.

[0109] For all cycles of the amplification reaction, calculations are performed using the following (Equation 3) and (Equation 3'). f1[n]=fhyb.1[n] / fden.1[n] (Formula 3) f2[n]=fhyb.2[n] / fden.2[n]...(Formula 3') where: f1[n]: Fluorescence intensity value at step 1 of the nth cycle calculated by (Equation 3). fhyb.1[n]: Fluorescence intensity value in the extension and detection step of step 1 of the nth cycle. fden.1[n]: Fluorescence intensity value at the denaturation step of step 1 in the nth cycle. f2[n]: Fluorescence intensity value at step 2 of the nth cycle calculated by (Equation 3'). fhyb.2[n]: Fluorescence intensity value in the extension and detection step of step 2 of the nth cycle. fden.2[n]: Fluorescence intensity value at the denaturation step of step 2 in the nth cycle Furthermore, the calculation is performed using the following formula. F1[n]=f1[n] / f1

[12] (Formula 4) F2[n]=f2[n] / f2

[12] ...(Formula 4') Fr'[n]=(a-F2[n]) / (a-F1[n]) (Equation 5) where: f1

[12] : The fluorescence intensity value at the 12th cycle when the fluorescence intensity before the target nucleic acid is amplified is stable, and is used as a reference for the change in the fluorescence intensity value in step 1. f2

[12] : The fluorescence intensity value at the 12th cycle when the fluorescence intensity before the target nucleic acid is amplified is stable, and is used as a reference for the change in the fluorescence intensity value in step 2. F1[n]: The relative value at the nth cycle when the fluorescence intensity value obtained by (Equation 4) at the 12th cycle is set to 1. F2[n]: The relative value at the nth cycle when the fluorescence intensity value obtained by (Equation 4') at the 12th cycle is set to 1. a: A constant that ensures that Fr' does not become less than 0 Fr'[n]: calculation value for mutation detection

[0110] <Mycoplasma genitalium detection> To determine Mycoplasma genitalium, the F

[23] value of the test sample is compared with the threshold value. If the F

[23] of the test sample is less than the threshold value, the result is positive; otherwise, the result is negative. The threshold value is the mean value of the F

[23] of negative samples that do not contain Mycoplasma genitalium minus five times the standard deviation (hereinafter referred to as the mean - 5SD), i.e., 0.9910.

[0111] <Mutation identification judgment> Mutation identification is determined by comparing the Fr'

[23] value of the measurement sample with the judgment threshold. If the Fr'

[23] value of the measurement sample is less than the judgment threshold, a mutation is detected. If the Fr'

[23] value of the measurement sample is greater than the judgment threshold, a mutation is detected. The judgment threshold is set to 0.2000. Note that the calculation is performed with the constant a in Equation 5 set to 1.00.

[0112] <Flowchart of the determination method> FIG. 8 is a flowchart showing a determination method according to a tenth embodiment of the present invention.

[0113] Table 15 shows the Mycoplasma genitalium determination results, relative fluorescence values ​​(F

[23] ) and calculated values ​​for mutation determination (Fr'

[23] ) for Examples 10 to 12 and Comparative Example 12, measured at the detection temperatures shown in Table 13.

[0114] In all of Examples 10 to 12, when the artificially synthesized RNA of wild-type Mycoplasma genitalium was measured, Fr'

[23] was not below the judgment threshold, indicating that no mutation was present; when the artificially synthesized RNA of mutant Mycoplasma genitalium was measured, Fr'

[23] was below the judgment threshold, indicating that a mutation was present.

[0115] In Comparative Example 12, when the artificially synthesized RNA of wild-type Mycoplasma genitalium was measured, Fr'

[23] did not fall below the judgment threshold, and it was correctly determined that there was no mutation; however, when the artificially synthesized RNA of mutant Mycoplasma genitalium was measured, Fr'

[23] did not fall below the judgment threshold, and it was erroneously determined that there was no mutation.

[0116] [Table 15]

[0117] (result) As is clear from Table 15, when the detection temperature B in step 2 is 66°C, it was confirmed that mutations can be correctly identified and determined when the detection temperature A in step 1 is 50 to 58°C.

[0118] Table 16 shows the Mycoplasma genitalium determination results, relative fluorescence values ​​(F

[23] ) and calculated values ​​for mutation determination (Fr'

[23] ) for Examples 13 to 16 measured at the detection temperatures shown in Table 14.

[0119] In all of Examples 13 to 16, when the artificially synthesized RNA of wild-type Mycoplasma genitalium was measured, Fr'

[23] was not below the judgment threshold, indicating that no mutation was present; when the artificially synthesized RNA of mutant Mycoplasma genitalium was measured, Fr'

[23] was below the judgment threshold, indicating that a mutation was present.

[0120] [Table 16]

[0121] (result) Figure 9 is a graph showing the relative fluorescence values ​​and calculated values ​​for each cycle of the wild-type artificial synthetic RNA (Table 16), and Figure 10 is a graph showing the relative fluorescence values ​​and calculated values ​​for each cycle of the mutant artificial synthetic RNA (Table 16). For the wild-type Mycoplasma genitalium artificial synthetic RNA, the relative fluorescence value (F

[23] ) in the final cycle was below the decision threshold, and the calculated value for determining mutation (Fr'

[23] ) was higher than the decision threshold, so it was determined to be Mycoplasma genitalium positive and no mutation was present. On the other hand, for the mutant Mycoplasma genitalium artificial synthetic RNA, the relative fluorescence value (F

[23] ) in the final cycle was below the decision threshold, and the calculated value for determining mutation (Fr'

[23] ) was lower than the decision threshold, so it was determined to be Mycoplasma genitalium positive and mutation was present.

[0122] As is clear from Table 16, when detection temperature B in step 2 is 70°C, it was confirmed that mutations can be correctly identified and determined when detection temperature A in step 1 is 50 to 60°C. As is clear from Tables 15 and 16, when detection temperature A in step 1 is 50 to 58°C and detection temperature B in step 2 is 66 to 70°C, it can be seen that not only can Mycoplasma genitalium be detected, but mutations can also be identified and determined. [Brief explanation of the drawings]

[0123] [Figure 1] FIG. 1 is a graph showing temperature changes in Example 1 of the present invention. [Figure 2] Figure 2 is an enlarged view of a portion of Figure 1. [Figure 3] FIG. 3 is a flowchart showing a determination method according to the first embodiment of the present invention. [Figure 4] FIG. 4 is a graph showing the amplification curve (F[n]) of Example 1 as a representative example of a negative determination of Mycoplasma genitalium. [Figure 5] FIG. 5 is a graph showing the amplification curve (F[n]) of Comparative Example 1 as a representative example of a positive determination of Mycoplasma genitalium. [Figure 6] FIG. 6 is a graph showing temperature changes in Example 10 of the present invention. [Figure 7] Figure 7 is an enlarged view of a portion of Figure 6. [Figure 8] FIG. 8 is a flowchart showing a determination method according to a tenth embodiment of the present invention. [Figure 9] FIG. 9 is a graph showing the values ​​of wild-type artificial synthetic RNAs in Table 16. [Figure 10] FIG. 10 is a graph showing the values ​​of the mutant artificial synthetic RNAs in Table 16. [Figure 11] FIG. 11 is a list of first primer sequences of the present invention. [Figure 12] FIG. 12 is a list of second primer sequences of the present invention. [Figure 13]FIG. 13 is a list of labeled probe sequences of the present invention.

Claims

1. A base sequence from positions 2096 to 2119 of the 23S rRNA gene sequence of Mycoplasma genitalium, which comprises at least 20 consecutive bases, or a base sequence complementary to said base sequence.

2. A method for detecting Mycoplasma genitalium, which comprises using the base sequence according to claim 1 as a first primer.

3. Furthermore, the method for detecting Mycoplasma genitalium according to claim 2, wherein a base sequence from positions 2027 to 2050 of the 23S rRNA gene sequence of Mycoplasma genitalium, which base sequence contains at least 20 consecutive bases, or a base sequence complementary to said base sequence, is used as a second primer.

4. The method for detecting Mycoplasma genitalium according to claim 3, further comprising using a base sequence from positions 2054 to 2078 of the 23S rRNA gene sequence of Mycoplasma genitalium or a base sequence complementary to said base sequence as a labeled probe.

5. 5. The method for detecting Mycoplasma genitalium according to claim 4, wherein the labeled probe is labeled with a fluorescent quenching dye that is quenched when hybridized with a target.

6. 6. The method for detecting Mycoplasma genitalium according to claim 5, wherein the PCR detection temperature conditions are set using a first temperature and a second temperature different from the first temperature, and Mycoplasma genitalium is detected and the presence or absence of a macrolide resistance mutation in the 23S rRNA gene is detected.

7. The method for detecting Mycoplasma genitalium according to claim 6, wherein the first temperature is set in the range of 50 to 58°C, and the second temperature is set in the range of 66 to 70°C.

8. A reagent for detecting Mycoplasma genitalium and identifying macrolide resistance, using a first primer consisting of the base sequence described in claim 1, a second primer consisting of the base sequence described in claim 3, and a labeled probe consisting of the base sequence described in claim 4.

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