CN113462794A

CN113462794A - Kit and detection method for detecting mycoplasma pneumoniae nucleic acid and existence of drug-resistant gene variation thereof

Info

Publication number: CN113462794A
Application number: CN202110345670.7A
Authority: CN
Inventors: 川岛洋介
Original assignee: Toyobo Co Ltd
Current assignee: Toyobo Co Ltd
Priority date: 2020-03-31
Filing date: 2021-03-31
Publication date: 2021-10-01
Also published as: JP2021159080A

Abstract

Provided are a kit and a method for detecting Mycoplasma pneumoniae nucleic acid and its presence or absence of drug-resistant genetic variation, which are useful for detecting Mycoplasma pneumoniae nucleic acid and its presence or absence of drug-resistant genetic variation. The kit and the detection method for detecting the mycoplasma pneumoniae nucleic acid and the existence of drug-resistant gene variation are characterized by comprising KOD DNA polymerase, a primer MPN-F, a primer MPN-R and a specific targeted fluorescent probe. The kit and the detection method thereof achieve the aim of quickly, simply and accurately detecting the variation of the 2063 th or 2064 th gene loci of the mycoplasma pneumoniae nucleic acid, the mycoplasma pneumoniae nucleic acid 23S rRNA gene and the 23SrRNA by designing the primer and the probe with the specific sequence.

Description

Kit and detection method for detecting mycoplasma pneumoniae nucleic acid and existence of drug-resistant gene variation thereof

Technical Field

The invention relates to the technical field of molecular biology, in particular to a detection kit and a detection method for mycoplasma pneumoniae nucleic acid and drug-resistant gene variation thereof, wherein the kit is used for qualitatively detecting mycoplasma pneumoniae 23S rRNA gene and variation thereof in sputum samples, throat swab samples or nose swab samples in vitro, and is suitable for auxiliary diagnosis of mycoplasma pneumoniae infection.

Background

Mycoplasma pneumoniae (MP or MP) is the pathogen of human mycoplasma Pneumonia, and is mainly infected by droplets and has a latency period of 2-3 weeks. At present, mycoplasma pneumoniae becomes one of main pathogens of community-acquired pneumonia of children, is a common pathogen of respiratory tract infection of teenagers and old people, can cause mycoplasma pneumonia, upper respiratory tract infection, bronchitis, nephritis and the like, and can cause death in severe cases. MP is sensitive to macrolide antibiotics inhibiting microbial protein synthesis, quinolone antibiotics acting on DNA topoisomerase and tetracycline antibiotics. However, in view of the physiological characteristics of children and the adverse reaction of drugs, tetracycline and quinolone antibiotics are limited to be used for treating mycoplasma infection of children, and macrolide antibiotics become first-line drugs for clinical MP infection. With the wide clinical application of macrolide drugs, the drug resistance phenomenon caused by gene mutation is also emerging. In recent years, MP drug-resistant strains resistant to macrolide antibiotics are clinically isolated in successive reports in various domestic and foreign documents. Germany reports that the drug resistance of MP is 3%; japanese research shows that the drug resistance rate of MP is increased from 5% to more than 30% in 2002-2006; the drug resistance rate of the MP for the children in China is more than 80 percent; further increasing the difficulty of treating MP pneumonia and its extrapulmonary complications in children. Therefore, the clinical development of routine detection of drug resistance genes of children MP infected patients has certain clinical guiding significance for further effective treatment of mycoplasma infected children, reduction of extrapulmonary complications, treatment of later-stage drug-resistant MP infection, research on drug resistance mechanism, drug resistance control and the like.

Currently, the clinical detection of mycoplasma pneumoniae mainly comprises a separation culture method, an antigen detection method, a PCR detection method and a serological detection method.

In the isolation culture method, the cost and the workload required for isolating and culturing Mp from a clinical specimen are large, and a series of subcultures using a special medium are required, and several weeks are required for the culture. Even if the separation efficiency of the culture medium is improved, the diagnostic sensitivity of the method is less than 60% of that of the PCR method, but the specificity of the separation culture method can reach 100%.

The method for directly and rapidly detecting the antigen of the Mp infected by the respiratory tract by utilizing the antigen detection technology comprises a direct immunofluorescence method, a reverse phase immunoelectrophoresis technology, an immunoblotting technology and an enzyme-linked antigen immunocapture technology. Considering that the concentration of Mp in the sputum of the patient is about 10²～10⁶The concentration range detectable by the antigen detection technology is 10-100 cfu/ml, and the sensitivity of antigen detection without Mp amplification culture is very limited, so that when the nucleic acid amplification technology can be used for diagnosis, the antigen detection is not recommended to be used for diagnosis.

The application of PCR diagnosis technology has replaced the traditional separation culture method, and the diagnosis capability of PCR technology on Mp infection is proved by simulation of clinical subjects, animal models and clinical experiments in the later 80 s. The conventional Mp PCR detection method now recommended by the U.S. disease control center is based on the PCR detection method originally designed by Bernet et al to amplify a fragment of the MpATP enzyme gene sequence region.

Serological detection methods have long been the primary method of Mp diagnosis and epidemiological studies. The diagnosis method with the highest detection precision for Mp infection of adults is to detect IgM and IgG twice at 2-3 weeks after infection. Mp positivity was observed when the antibody titers differed by more than 4-fold. The continuous rise of IgG antibodies in patients with a history of Mp infection, and the possibility of no IgM response in adults over a prolonged period of time, are all limitations of the use of serological methods to detect Mp infection alone.

With the increase of drug-resistant strains, it is necessary to detect whether the strains are drug-resistant strains before medication, and among various detection methods, only the PCR method can analyze genes of the drug-resistant strains. Therefore, a method for rapidly detecting mycoplasma pneumoniae and/or detecting drug resistance has been studied (patent documents 1 and 2 and non-patent document 1). However, development of more useful detection kits and detection methods is demanded.

Documents of the prior art

Patent document

Patent document 1: japanese laid-open patent publication No. 2015-128400

Patent document 2: japanese patent laid-open publication No. 2014-042459

Non-patent document

Non-patent document 1: antimicrobial AGENTS AND chemocopy oct.2008, p.3542-3549

Disclosure of Invention

Problems to be solved by the invention

The present invention aims to provide a detection kit and a detection method for a mycoplasma pneumoniae nucleic acid and a drug-resistant gene mutation thereof, by which it is possible to quickly and easily detect whether a mutation has occurred in a mycoplasma pneumoniae nucleic acid, a mycoplasma pneumoniae 23S rRNA gene, and a2063 th or 2064 th locus on 23S rRNA.

Means for solving the problems

To achieve the above object, the present invention has the following technical means. The detection kit for the mycoplasma pneumoniae nucleic acid and the drug-resistant gene variation thereof comprises KOD DNA polymerase, a primer MPN-F, a primer MPN-R and a specific targeted fluorescent probe;

the primer MPN-F is a forward primer composed of a base sequence which is continuous and 25 to 36 bases in the base sequence from 1910 to 2039 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or in the base sequence complementary thereto;

the primer MPN-R is a reverse primer composed of a base sequence which is continuous and 25 to 36 bases in the base sequence shown in No. 2091 to No. 2257 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or in the base sequence complementary to the base sequence;

the specific targeted fluorescent probe is a probe comprising a base sequence having a length of 16 to 23 bases which is continuous with or complementary to the base sequence of the 2040 th to 2081 th parts of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33), and a fluorescent dye is labeled on any of the terminal bases.

As described below, the related contents in the above-described technical solutions will be explained.

1. In the above technical scheme, KOD DNA polymerase is extracted and purified from Thermococcus kodakaraensis KOD1 isolated from sulfur-containing stomata of Xiaobaoshima of the island county of deer, and has strong 3 '→ 5' exonuclease activity (correction activity) with an accuracy of about 50 times that of Taq DNA polymerase. Has a DNA synthesis rate of 1Kb/30s or more, which is twice as high as that of the most common Taq polymerase. In addition, the excellent ability to continue synthesis and the like are one of the main reasons for the high elongation rate. With KOD DNA polymerase, the time for one cycle can be shortened from several minutes to several tens of seconds. For example, KOD DNA polymerase available from Toyo Boseki K.K. can be suitably used.

2. In the above-mentioned aspect, the fluorescent probe is preferably a Q probe, which is a probe labeled with a fluorescent dye having a characteristic that fluorescence quenching occurs when a specific sequence is mixed. The structure of the Q probe is described in Japanese patent No. 3437816, and by utilizing this feature, it is possible to specifically detect a target nucleic acid sequence without inhibiting high-speed amplification by using a probe labeled with a fluorescent substance without using any other dye inserted into the double-stranded nucleic acid structure such as a DNA intercalator or and without using two kinds of probes causing a FRET phenomenon.

3. In the technical scheme, the kit further comprises an internal control primer IC-F, an internal control primer IC-R, an internal control probe and an internal control template. In a preferred embodiment, the internal control primer IC-F is, for example, a forward primer designed for the matK gene of Grateloupia macrantha (Lagarophon madagascari) and having a sequence of 5'-CCCGGTTATTGTAGAAATTCCTTTCTCCCGTC-3' (SEQ ID NO: 34),

the internal control primer IC-R is, for example, a reverse primer designed for the matK gene of Grateloupia macrantha and having a sequence of 5'-CCCCATCCAGGATTGTAGAATTTGAATCAAG-3' (SEQ ID NO: 35),

the internal control probe is designed using, for example, the matK gene of Grateloupia macrantha as a template, and has a sequence of 5'-GATCTATTCATTCGATATTCC-3' (SEQ ID NO: 36),

the internal control template is, for example, 1 fragment of matK gene of pteris madagassita, and the sequence is 5'-GCGGTTATTGTAGAAATTCCTTTCTCCCGTCCATTTTTTCTTGAAGAAAAAAAAGAAATACCAAAATATCAAAATTTACGATCTATTCATTCGATATTCCCTTTTTTAGAGGACAAATTTTTACATTTAAATTATGTGTCTGATATAGTAATACCTTATCCTATTCATCTCGAAATCTTGATTCAAATTCTACAATCCTGGAT-3' (SEQ ID NO: 37).

The above-mentioned Magassigan Graciliensis (Lagarding literature name is Lagarispon madagascariensis) is aquatic weed and stemmed grass, and is mainly distributed in Magassika.

4. In the above-mentioned technical solution, the positive control reagent 1 and the positive control reagent 2 are both samples designed in advance and having known concentrations and base sequences, and are detected together with other samples to be detected, thereby verifying the accuracy of the detection result.

In order to achieve the above object, the method for detecting mycoplasma pneumoniae nucleic acid and the presence or absence of drug-resistant gene mutation according to the present invention comprises the steps of:

step 1: preparing a test sample collected from sputum, a pharyngeal swab, or a nasal swab and subjected to pretreatment as a test object;

step 2: performing a PCR amplification reaction using the kit, using a nucleic acid contained in the test sample as a template gene strand; and

and step 3: the amplification product obtained in step 2 was detected by a melting curve analysis method, and the presence or absence of the drug-resistant gene mutation in the amplification product was confirmed by using the difference between the melting temperatures of the amplification product having no drug-resistant gene mutation and the probe and the melting temperatures of the amplification product having a drug-resistant gene mutation and the probe.

1. In the above embodiment, the PCR amplification reaction optionally comprises dNTPs, which are a mixture of deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), and deoxythymidine triphosphate (dTTP).

2. In the above technical solution, the reaction process of the PCR amplification reaction of step 2 is, for example, as follows:

(1) pre-denatured at 94.0 deg.c for 30.0 sec to 2.0 min,

(2) denaturating for 1.0-10.0 seconds at 97.0-98.0 ℃,

(3) annealing at 58.0-60.0 ℃ for 3.0-30.0 seconds,

(4) the extension is carried out at 63.0-68.0 ℃ for 5.0-30.0 seconds, and the steps (2) - (4) are circulated 50-70 times (preferably 60 times).

3. In the above technical solution, the melting curve analysis may be an analysis based on a high-resolution melt curve (HRM) analysis technique. The high-resolution melting curve analysis is a latest genetic analysis method for variation scanning and genotyping which is established abroad in recent years, is a new gene analysis technology for forming melting curves with different forms based on different melting temperatures of mononucleotides, has high sensitivity, can detect the difference of single basic groups, and has the advantages of low cost, high flux, high speed, accurate result and no limitation of detected loci.

The design principle of the present invention is as follows. The kit of the invention uses a PCR melting curve method to detect whether the 23S rRNA gene of the Mycoplasma pneumoniae nucleic acid and the 2063 or 2064 locus thereof have variation. The detection principle is divided into two parts, one part is that a real-time fluorescence quantitative PCR technology is utilized, the 23S rRNA gene field of the mycoplasma pneumoniae is taken as a target template gene chain, mycoplasma pneumoniae specific amplification primers are added for denaturation and annealing, and KOD DNA polymerase capable of amplifying at high speed is utilized to catalyze each primer so as to extend the gene chain. In this case, the target gene strand is amplified by repeating the extension of the primer annealed by the action of the enzyme activity of the dNTP substrate and the magnesium ion. Then, the amplified target gene chain is hybridized with a specific matched mycoplasma pneumoniae fluorescent probe (the probe can be quickly and conveniently detected), so that fluorescence change is caused, and the existence of mycoplasma pneumoniae is detected. The other part is a method of determining whether or not the 2063 or 2064 locus of Mycoplasma pneumoniae 23S rRNA gene has a mutation by drawing a melting curve by detecting a change in fluorescence using the difference in melting temperature between a wild-type Mycoplasma pneumoniae nucleic acid and the gene at 2063 or 2064 locus. In addition, an internal control sample is added to the reaction system, so that the influence of an interfering substance of a clinical sample on the PCR reaction can be known, and the false negative of the detection of the mycoplasma pneumoniae can be further prevented.

In a specific embodiment, a fully automated nucleic acid purification/fluorescence PCR analyzer is used to perform PCR amplification, and after hybridization with a fluorescent probe specific to Mycoplasma pneumoniae, the change in fluorescence is detected. The software system of the device automatically monitors the fluorescence intensity in real time, and calculates the real-time fluorescence variation, so that a melting curve is drawn, the fluorescence differential value and the melting temperature of an unknown sample are obtained, and the detection of the unknown sample is realized.

The detection kit (PCR melting curve method) for the mycoplasma pneumoniae nucleic acid and the drug-resistant gene variation thereof is used for qualitatively detecting the mycoplasma pneumoniae 23S rRNA gene and the drug-resistant variation thereof in sputum, a throat swab sample or a nose swab sample by using a PCR amplification method in vitro, and is suitable for auxiliary diagnosis of mycoplasma pneumoniae infection. The detection of the 23S rRNA gene of Mycoplasma pneumoniae and the variation of the 2063 or 2064 locus thereof by using a full-automatic nucleic acid purification/fluorescence PCR analyzer (preferably GENECUBE (registered trademark)) can assist a clinician in determining the optimal treatment scheme for treating Mycoplasma pneumoniae, so that the clinician can determine the medicine, dosage and economic treatment scheme suitable for patients according to the optimal treatment scheme, and powerful means are provided for clinical rational medication, personalized medication and treatment.

ADVANTAGEOUS EFFECTS OF INVENTION

The advantageous effects of the present invention are as follows.

(1) According to the present invention, by using a combination of KOD DNA polymerase, primer MPN-F, primer MPN-R and a specifically labeled fluorescent probe, it is possible to amplify Mycoplasma pneumoniae nucleic acid 23S rRNA efficiently and detect it with high sensitivity while distinguishing between wild type and variant types.

(2) The conventional PCR reaction system usually requires 40. mu.L, and the amount of the fluorescent quantitative PCR amplification reaction system of the present invention is only 13.2. mu.L at the minimum.

(3) The fluorescent quantitative PCR amplification reaction time is greatly shortened, and 60 cycles of denaturation, annealing and extension can be completed within 40 minutes at least.

(4) The conventional detection kit for the drug-resistant gene variation of the mycoplasma pneumoniae nucleic acid needs to be stored at the temperature of-20 ℃, and the kit can be stored at the temperature of 2-8 ℃.

(5) The invention adopts the complete sealed PCR amplification, and can prevent false positive results caused by residual pollution (carry-over) and the like.

(6) The positive control sample of the present invention is essentially a sample designed in advance and having a known concentration and base sequence, and is detected together with other test samples (e.g., throat swab samples) using a detection kit for detecting relevant mycoplasma pneumoniae nucleic acids and drug-resistant gene variations thereof, thereby verifying the accuracy of the detection result of the test sample. That is, when a test sample is detected by using a detection kit for mycoplasma pneumoniae nucleic acid and drug-resistant gene variation thereof, the positive control sample of the present invention is equivalent to a positive sample with a known concentration, and is used for verifying whether the detection result of the detection kit for mycoplasma pneumoniae nucleic acid and drug-resistant gene variation thereof is true and effective, thereby solving the quality detection problem of mycoplasma pneumoniae nucleic acid and drug-resistant gene variation thereof.

In conclusion, the kit and the detection method thereof achieve the purpose of rapidly, simply and accurately detecting the mycoplasma pneumoniae nucleic acid, the mycoplasma pneumoniae nucleic acid 23S rRNA gene and the mutation of the 2063 th or 2064 th gene locus on the 23S rRNA by designing the primer MPN-F, the primer MPN-R and the specific targeting fluorescent probe with specific sequences.

Drawings

FIG. 1 is a graph showing fluorescence quantitative PCR amplification melting curves when the 2063 th or 2064 th locus of the Mycoplasma pneumoniae nucleic acid-positive 23S rRNA gene is not mutated, and the abscissa shows temperature and the ordinate shows differential fluorescence values.

FIG. 2 is a graph showing fluorescence quantitative PCR amplification melting curves when the 2063 locus of the 23S rRNA gene is changed in a case where the Mycoplasma pneumoniae nucleic acid is positive, and the abscissa shows temperature and the ordinate shows differential fluorescence values.

FIG. 3 is a graph showing fluorescence quantitative PCR amplification melting curves when the 2064 locus of the 23S rRNA gene is changed in a case where the Mycoplasma pneumoniae nucleic acid is positive, and the abscissa shows temperature and the ordinate shows differential fluorescence values.

FIG. 4 is a graph showing the melting curve of fluorescent quantitative PCR amplification in the case of Mycoplasma pneumoniae nucleic acid negativity, with the horizontal axis showing temperature and the vertical axis showing differential fluorescence.

FIG. 5 is a graph showing the melting curve of the internal control sample in the fluorescence quantitative PCR amplification, with the horizontal axis showing temperature and the vertical axis showing differential fluorescence.

FIG. 6 is a PCR amplification melting graph showing the results of evaluation test 2 of primers and probes. The upper panel shows the result of using the specific-labeled fluorescent probe QP-1, and the lower panel shows the result of using the specific-labeled fluorescent probe QP-2. Each line represents the case of using the mycoplasma pneumoniae genome template at various dilution ratios and the case of using purified water as a comparative example (including no template).

FIG. 7 is a PCR amplification melting graph showing the results of evaluation test 3 of primers and probes.

FIG. 8 is a PCR amplification melting graph showing the results of evaluation test 4 of primers and probes.

FIG. 9 is a PCR amplification melting graph showing the results of evaluation test 5 of primers and probes.

Detailed Description

The present invention will be further described with reference to the drawings and examples.

Example, kit and method for detecting Mycoplasma pneumoniae nucleic acid and presence or absence of drug-resistant gene mutation thereof

The wild-type base sequence of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene that can be a subject of detection of the present invention is the base sequence represented by SEQ ID NO. 33 of the sequence Listing.

The detection kit for the mycoplasma pneumoniae nucleic acid and the drug-resistant gene variation thereof comprises KOD DNA polymerase, a primer MPN-F, a primer MPN-R and a specific targeted fluorescent probe,

the primer MPN-F is a forward primer composed of a base sequence having a length of 25 to 36 bases which is continuous in a base sequence from 1910 to 2039 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or in a base sequence complementary thereto,

the primer MPN-R is a reverse primer composed of a base sequence having a length of 25 to 36 continuous bases in the base sequence represented by SEQ ID NO. 2091 to SEQ ID NO. 2257 or in the base sequence complementary thereto of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO. 33),

the specific targeted fluorescent probe is characterized by being designed to be composed of a base sequence which is continuous and has a length of 16 to 23 bases in the base sequence shown by 2040 to 2081 of the Mycoplasma pneumoniae nucleic acid 23SrRNA gene (SEQ ID NO: 33) or in a base sequence complementary to the base sequence, and a fluorescent dye is marked on any terminal base.

The primer MPN-R, the primer MPN-F and the specific targeting fluorescent probe are not particularly limited as long as they are composed of the base sequence as described above, and from the viewpoint of enabling detection of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene with higher sensitivity,

the primer MPN-F is preferably a forward primer composed of a base sequence having a length of 25 to 36 bases which is continuous in a base sequence represented by the base sequences 1988 to 2030 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or in a base sequence complementary thereto;

the primer MPN-R is preferably a reverse primer composed of a base sequence having a length of 25 to 36 continuous bases in the base sequence represented by the nucleotide sequence 2101 to 2140 times the M.pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or in a base sequence complementary thereto;

the specific targeting fluorescent probe is preferably designed to have a base sequence of 16 to 22 bases in length and continuous from No. 2050 to No. 2071 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33), and a fluorescent dye is labeled on an arbitrary terminal base.

In a particularly preferred embodiment of the present invention,

the primer MPN-F is a forward primer composed of a base sequence represented by any one of SEQ ID Nos. 1 to 10 or a base sequence complementary thereto;

the primer MPN-R is a reverse primer composed of a base sequence represented by any one of SEQ ID Nos. 11 to 20 or a base sequence complementary thereto;

the specific targeted fluorescent probe is designed to be composed of the base sequence shown in any one of SEQ ID Nos. 21 to 30 or a base sequence complementary thereto, and a fluorescent dye is labeled on any terminal base, whereby the detection of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene can be more reliably achieved with high sensitivity.

Here, the primer MPN-F composed of the base sequence shown in any one of SEQ ID Nos. 1 to 10 corresponds to a base sequence of 25 to 36 bases which is continuous in length among the base sequences shown from 1910 to 2039 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33).

The primer MPN-R consisting of the base sequence shown in any one of SEQ ID Nos. 11 to 20 corresponds to a base sequence having a length of 25 to 36 bases which is continuous in a base sequence complementary to the base sequence shown in SEQ ID Nos. 2091 to 2257 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO. 33).

The specific targeted fluorescent probe composed of the base sequence represented by any one of SEQ ID Nos. 21 to 30 corresponds to a base sequence having a length of 16 to 23 bases which is continuous among the base sequences represented by 2040 to 2081 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO. 33).

In one embodiment, the kit of the present invention may further include 2 positive control reagents (positive control reagent 1 and positive control reagent 2). By including such a positive control reagent, it is easy to verify whether or not the detection result of the 23S rRNA gene of the mycoplasma pneumoniae nucleic acid is contained in the test sample accurately, and the problem of quality detection in the 23S rRNA gene test of the mycoplasma pneumoniae nucleic acid can be solved.

In a specific embodiment, the positive control reagent 1 is a DNA sequence designed using the wild-type Mycoplasma pneumoniae nucleic acid 23S rRNA gene as a template, and the sequence thereof may be 5'-CCATCTCTTGACTGTCTCGGCTATAGACTCGGTGAAATCCAGGTACGGGTGAAGACACCCGTTAGGCGCAACGGGACGGAAAGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCAATCGATGCAAGTTCGCTAGGACTTGTTGATGCGAAAGGTGGAATACT-3' (SEQ ID NO: 38).

In a specific embodiment, the positive control reagent 2 is a DNA sequence designed using the Mycoplasma pneumoniae nucleic acid 23S rRNA gene having a variation at the 2063 locus as a template, and the sequence thereof may be 5'-CCATCTCTTGACTGTCTCGGCTATAGACTCGGTGAAATCCAGGTACGGGTGAAGACACCCGTTAGGCGCAACGGGACGGGAAGACCCCGTGAAGCTTTACTGTAGCTTAATATTGATCAGGACATTATCATGTAGAGAATAGGTAGGAGCAATCGATGCAAGTTCGCTAGGACTTGTTGATGCGAAAGGTGGAATACT-3' (SEQ ID NO: 39).

The kit can also comprise an internal control primer IC-F, an internal control primer IC-R, an internal control probe and an internal control template.

In a specific embodiment, the internal control primer IC-F is, for example, a forward primer designed for the matK gene of Grateloupia macrantha and having a sequence of 5'-CCCGGTTATTGTAGAAATTCCTTTCTCCCGTC-3' (SEQ ID NO: 34),

the internal control probe is designed, for example, using the matK gene of Grateloupia matsutake as a template, and has a sequence of 5'-GATCTATTCATTCGATATTCC-3' (SEQ ID NO: 36),

the internal control template may be, for example, 1 fragment of matK gene of pteris madagassita, and the sequence may be 5'-GCGGTTATTGTAGAAATTCCTTTCTCCCGTCCATTTTTTCTTGAAGAAAAAAAAGAAATACCAAAATATCAAAATTTACGATCTATTCATTCGATATTCCCTTTTTTAGAGGACAAATTTTTACATTTAAATTATGTGTCTGATATAGTAATACCTTATCCTATTCATCTCGAAATCTTGATTCAAATTCTACAATCCTGGAT-3' (seq id No. 37).

As shown in FIG. 5, the internal control primer IC-F, the internal control primer IC-R, the internal control probe and the internal control template are used to verify whether the detection of the mutation in the drug resistance gene of the Mycoplasma pneumoniae nucleic acid by the kit is true and effective.

In actual production, the detection kit for a mycoplasma pneumoniae nucleic acid and a drug-resistant gene mutation thereof may be produced as a kit containing the following components. The names, liquid amounts, and counts of the respective reagents and samples listed below are examples, and the reagent kit may be configured by reagent or sample names, liquid amounts, and counts other than those described below.

(1) Polymerase reagent [ KOD Mix ]: comprises KOD DNA polymerase and dNTPs, and has a specification of 140. mu.L.times.1, 140. mu.L.times.2, 140. mu.L.times.3, or 140. mu.L.times.6.

(2) Primer Probe reagent [ MPN Mix ]: that is, the mixed reagent containing the primer MPN-F, the primer MPN-R and the specific targeting fluorescent probe has a specification of 140. mu.L.times.1, 140. mu.L.times.2, 140. mu.L.times.3 or 140. mu.L.times.6.

(3) Positive control sample 1[ MPN PC1 ]: that is, the standard was 300. mu.L.times.1 counts for positive control reagent 1.

(4) Positive control sample 2[ MPN PC2 ]: that is, the standard was 300. mu.L.times.1 counts for positive control reagent 2.

(5) Negative control sample [ MPN NC ]: that is, the specification was 300. mu.L.times.1 in Tris-HCl buffer containing no DNA.

The positive control reagent 1 and the positive control reagent 2 are both constructed on a carrier plasmid for storage, and the original plasmid is purchased by the company of Biotechnology engineering (Shanghai). The plasmid was constructed as follows.

Primer design-PCR amplification-DNA fragment recovery from gel-vector construction-transformation culture-positive clone identification-37 ℃ shake cultivation-plasmid extraction-sequencing confirmation base sequence.

The extracted plasmid was prepared as the positive control reagent 1 by the following method.

(1) Preparation of a diluent: adding 50% of the compounding amount of purified water → adding Tris-HCl buffer (pH7.6) → adding the remaining purified water (vortex shaking 3 times, 5 seconds each time) → obtaining 0.002mol/L of Tris-HCl (pH7.6) dilution;

(2) pre-dilution of wild-type mycoplasma pneumoniae vector plasmid: adding the aforementioned diluent in a 50% compounding amount → adding the wild type mycoplasma pneumoniae carrier plasmid solution → adding the remaining aforementioned diluent (vortex shaking 3 times, 5 seconds each) → obtaining X copies/microliter of the wild type mycoplasma pneumoniae carrier plasmid solution (X represents a specific numerical value of the plasmid concentration);

(3) acquisition of positive control reagent 1: addition of 50% of the compounded amount of the dilution solution → addition of X copies/microliter of the wild-type mycoplasma pneumoniae carrier plasmid solution → addition of the remaining dilution solution (vortex shaking 3 times, 5 seconds each) → obtaining a positive control reagent 1.

The extracted plasmid was prepared as the positive control reagent 2 by the following method.

(2) pre-dilution of mycoplasma pneumoniae vector plasmid (variation at 2063 locus of 23S rRNA gene): adding 50% of the diluted solution → adding the solution of mycoplasma pneumoniae vector plasmid (having variation at 2063 locus of 23S rRNA gene) → adding the rest of the diluted solution (vortex shaking 3 times, 5 seconds each) to obtain X copies/microliter of the solution of mycoplasma pneumoniae vector plasmid (having variation at 2063 locus of 23S rRNA gene);

(3) obtaining a positive control reagent 2: addition of 50% of the compounded amount of dilution → addition of X copies/μ l of a solution of mycoplasma pneumoniae vector plasmid (having a variation at 2063 locus of 23S rRNA gene) → addition of the remaining dilution (3 vortexing, 5 seconds each) → obtaining a positive control reagent 2.

The negative control reagent and the positive control reagent are injected together with the sample to be tested for detection, and are used for evaluating the effectiveness of the detection process and monitoring the environment.

The detection method comprises the following steps.

In step 1, a target substance is prepared as a test sample collected via sputum, a pharyngeal swab, or a nasal swab and subjected to pretreatment as a test object;

the conditions for sample collection were as follows.

The natural expectoration method: preferably morning sputum, which is expectorated forcefully to the deep part of respiratory tract, is directly put into a sputum box, and a sample volume of 1ml or more is taken.

A throat swab-based sputum extraction method: pressing the back of the tongue with a bent tongue plate, extending the swab into the pharynx, rotating the swab to take out mucus, placing in a sterile glass tube, and sealing for inspection.

Nasal swab extraction based on nasal swab: the specialist inserts the bent swab into the nasal cavity, rotates the swab to take out mucus, places the mucus into a sterile glass tube, and seals the tube for inspection.

Regarding the effect caused by human gene contamination, it was confirmed that 10 ten thousand copies/test of human gene contamination had no effect on the detection of the present kit.

Sample shelf life: can be stored for 24 hours at the temperature of 2-8 ℃, can be stored for 3 months at the temperature below-20 ℃ and can be stored for a long time at the temperature below-70 ℃. Repeated freezing and thawing should be avoided when using cryopreserved samples, and the samples should be returned to room temperature for reuse. The transport is carried out using an iced cold box or an iced foam box.

In step 2, the target is used as a template gene strand, and the fluorescence PCR amplification reaction is carried out in the following reaction system as a preferred example using the above kit.

The reaction system for the fluorescent PCR amplification reaction can be such that the amount of each component used is 2 times or less of the above-mentioned value without changing the concentration of each component used, i.e., the volume of the primer MPN-F used is 0.03 to 0.3. mu.L, the amount of the primer MPN-R used is 0.03 to 0.3. mu.L, the amount of the specific targeting fluorescent probe used is 0.03 to 0.3. mu.L, the amount of the internal control primer IC-F used is 0.01 to 0.2. mu.L, the amount of the internal control primer IC-R used is 0.01 to 0.2. mu.L, the amount of the internal control probe used is 0.01 to 0.2. mu.L, and the amount of the internal control template used is 0.005 to 0.05. mu. L, MgSO₄The amount of the buffer solution (B) is 1 to 3. mu.L, the amount of the buffer solution (B) is 1 to 5. mu.L, the amount of the buffer solution (B) is 0.1 to 1. mu. L, KOD, and the amount of the template is 3 to 10. mu.L。

For the reaction process of the fluorescent PCR amplification reaction, the following is a preferred example:

(1) pre-denatured at 94.0 deg.c for 30.0 sec to 2.0 min,

(2) denaturating for 1.0-10.0 seconds at 97.0-98.0 ℃,

(3) annealing at 58.0-60.0 ℃ for 3.0-30.0 seconds,

In step 3, as a preferable example, the detection is performed by a high-resolution melting curve method under the following detection conditions.

(1)94.0 ℃, 30.0 seconds,

(2)39.0 ℃, 30.0 seconds,

(3) 40.0-75.0 deg.C, 0.09 deg.C/sec.

The entire fluorescent quantitative PCR amplification reaction was carried out directly by using a full-automatic nucleic acid purification/fluorescent PCR analysis apparatus (GENECUBE (registered trademark), manufactured by Toyo chemical Co., Ltd.). Reagents, samples, control samples and corresponding consumables required for the test are placed in the fully automatic nucleic acid purification/fluorescence PCR analysis device, which is specifically shown below.

(1) The polymerase reagent and the primer probe reagent were mixed by vortex oscillation for 5 to 6 seconds, centrifuged at 8000rpm for several seconds, and placed at the designated position of the apparatus.

(2) The treated sample, the positive control reagent and 10 mul of the negative control reagent are respectively added into a plurality of 0.5mL centrifuge tubes, mixed for 5-6 seconds by vortex oscillation, centrifuged for a plurality of seconds at 8000rpm, and placed at the designated position of the instrument.

(3) The pipette tip with filter, plastic capillary cup and tube (e.g., octal or dodecal) corresponding to the amount of test reagent are placed in the designated position of the instrument.

(4) Running a full-automatic nucleic acid purification/fluorescence PCR analysis device to perform PCR reaction. The full-automatic nucleic acid purification/fluorescence PCR analysis device automatically displays the detection result on a display screen after the reaction is finished.

In step 4, the detection result is judged by the positive judgment value, and the judgment of 3 types of results of the positive judgment value is as follows.

(1) As shown in FIG. 1, the differential fluorescence value was 7.5 or more, and when the melting temperature at which the peak of the melting curve was located was 56.1 to 64.1 ℃, it was judged as positive for Mycoplasma pneumoniae and no variation was observed at the loci of 23S rRNA 2063 and 2064.

(2) As shown in FIGS. 2 and 3, when the fluorescence differential value was 7.5 or more and the melting temperature at the peak of the melting curve was 46.8 to 54.8 ℃, it was judged that Mycoplasma pneumoniae was positive and that there was a variation at the 23S rRNA gene 2063 or 2064 locus.

(3) As shown in FIG. 4, when the differential fluorescence value is less than 7.5, the differential fluorescence value of the internal control is not less than 1.5, and the melting temperature at which the peak of the melting curve is located is between 52.0 ℃ and 62.0 ℃, it is judged that Mycoplasma pneumoniae is negative.

The fluorescence differential value is obtained by differentiating a value of the change in the fluorescence intensity of the target object with temperature.

Only when the internal control fluorescence differential value in the melting curve of FIG. 5 is not less than 1.0 and the melting temperature at which the peak of the melting curve is located is between 42 ℃ and 68 ℃, it is indicated that the whole detection system is normal and effective, and this is particularly important when the result of the determination is negative.

(evaluation test 1 of primer and Probe)

Further, the following tests were carried out to evaluate the primers and probes having specific sequences of the present invention.

First, primers MPN-F and MPN-R shown in Table 1 below were synthesized by a conventional method.

[ Table 1]

It was confirmed whether or not amplification of the genome of Mycoplasma pneumoniae could be performed by using the above primers. The PCR conditions were as follows.

< PCR Mix composition (volume of each sample) >)

(1: use of boiled inactivated Mycoplasma pneumoniae)

< PCR cycling conditions >

2 minutes at 94℃,

10 seconds at 98 ℃ to 30 seconds at 60 ℃ to 30 seconds at 68 ℃ (30 cycles)

4 ℃ (reaction stop)

The product obtained by the PCR reaction was subjected to agarose electrophoresis, and the amount of the amplified product was evaluated by the depth of the band. The evaluation criteria are as follows: o: deep bands can be seen, Δ: a shallow band can be seen, x: the band is not visible. The results are shown in table 2 below.

[ Table 2]

	MPN-F1	MPN-F2	MPN-F3
				MPN-R1	○	○	○
MPN-R2	△	△	○
				MPN-R3	△	△	△

As shown in the above results, it was confirmed that: the combination of all of the primers MPN-F and MPN-R was able to amplify the genomic sequence of Mycoplasma pneumoniae. In particular, the following tendency can be observed: under the present test conditions, it was found that a high amplification amount was easily obtained by using the primer MPN-F3 as the primer MPN-F and the primer MPN-R1 as the primer MPN-R, and that the amplification amount varied depending on the kind of the primer used.

(evaluation test 2 of primers and probes)

Then, the detection by the Q-probe method was evaluated using a combination of primers (combination of MPN-F3 and MPN-R1, combination of MPN-F3 and MPN-R2) which was confirmed from the above test results to be effective for amplification of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene. The following probe was used as a specific labeled fluorescent probe used in the Q-probe method.

[ Table 3]

A Mycoplasma pneumoniae 23S rRNA gene detection reagent having the following composition was prepared, and detection by the Q probe method was attempted using GENECUBE (registered trademark). The PCR cycling conditions and the melting curve analysis conditions were as follows.

< 23S rRNA Gene detection reagent composition (volume per 1 sample) >

(2: use of boiled Mycoplasma pneumoniae inactivated material, use of 4 kinds in total, dilution ratio 10 times, 10 times ²10 times of³In addition, purified water was used as a comparative example. )

< PCR cycling conditions >

94 ℃ for 30 seconds

1 second at 98 ℃ to 3 seconds at 60 ℃ to 5 seconds at 63 ℃ (cycle number 50 times)

< melting curve analysis Condition >

94 ℃ for 30 seconds

39 ℃ for 30 seconds

40.0-75.0 deg.C, 0.09 deg.C/sec

The results are shown in fig. 6. The upper graph of FIG. 6 shows the result of probe QP-1, and the lower graph of FIG. 6 shows the result of probe QP-2. As shown by the results, any combination of probes can detect Mycoplasma pneumoniae. In particular, a combination of primers MPN-F3 and MPN-R2 was used, and it was confirmed that the fluorescence intensity was stronger.

(evaluation test of primers and probes 3)

Using a combination of primers MPN-F3 and MPN-R2, which was confirmed to have strong fluorescence intensity in test example 2, it was evaluated whether the wild type and the mutant type of Mycoplasma pneumoniae nucleic acid 23S rRNA could be recognized (A2063G). In this test example, QP-1 used in test example 2 was used as a specifically labeled fluorescent probe.

Specifically, a reagent for detecting the Mycoplasma pneumoniae 23S rRNA gene having the following composition was prepared, and detection by the Q probe method was attempted using GENECUBE (registered trademark). The PCR cycling conditions and melting curve analysis conditions were the same as in test example 2.

< 23S rRNA Gene detection reagent composition (volume per 1 sample) >

(3: use of Mycoplasma pneumoniae specimen in which A → G mutation occurred at position 2063 of 23S rRNA gene by another evaluation system or wild-type Mycoplasma pneumoniae nucleic acid 23S rRNA specimen)

The results are shown in FIG. 7. As shown by the results, it was confirmed that: by using the combination of the primer and the probe of the present invention, it is possible to detect a wild-type and a variant mycoplasma pneumoniae nucleic acid by distinguishing them from each other based on the difference in peak temperature. The mutant (A2063G) used in this test was known as a drug-resistant gene mutant. Thus, it was found that the present invention allows the detection of a wild-type Mycoplasma pneumoniae nucleic acid and a drug-resistant mutant thereof with discrimination.

(evaluation test of primers and probes 4)

For the purpose of detection with higher sensitivity, new primers MPN-R5 and R6 were prepared, and detection of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene by the Q probe method was attempted in combination with the primers MPN-F3 and F4.

[ Table 4]

A Mycoplasma pneumoniae 23S rRNA gene detection reagent having the following composition was prepared, and detection by the Q probe method was attempted using GENECUBE (registered trademark). In this test example, QP-1, the probe used in test example 2, was used as a specific labeled fluorescent probe. The PCR cycling conditions were the same as in test example 1.

< 23S rRNA Gene detection reagent composition (volume per 1 sample) >

(3: Mycoplasma pneumoniae specimen in which A → G mutation occurred at position 2063 of 23S rRNA gene was determined by using another evaluation system or wild-type Mycoplasma pneumoniae nucleic acid 23S rRNA specimen)

< PCR cycling conditions >

94 ℃ for 30 seconds

97 ℃ for 1 second to 60 ℃ for 3 seconds to 63 ℃ for 5 seconds (cycle number 50 times)

< melting curve analysis Condition >

94 ℃ for 30 seconds

39 ℃ for 30 seconds

40.0-75.0 deg.C, 0.09 deg.C/sec

As a representative example of the results of this test, the results obtained when the primer MPN-F4 and the primer MPN-R6 were combined are shown in FIG. 8. As shown by the results, it was confirmed that: by using the combination of the primer and the probe of the present invention, it is possible to detect a wild-type and a variant Mycoplasma pneumoniae nucleic acid in a distinguishable manner. The same results were confirmed when other primer combinations were used. If the results of this test are compared with the results of the above-described evaluation test 3, it can be confirmed that: the case of using the primer combination of this evaluation test has higher fluorescence intensity than the case of using the primer combination of evaluation test 3, and enables the wild type and the mutant type of Mycoplasma pneumoniae nucleic acid 23S rRNA to be detected separately with higher sensitivity.

Here, the positions where the primers MPN-F3 and MPN-F4 and MPN-R5 and MPN-R6 anneal to the base sequence of the 23R rRNA of Mycoplasma pneumoniae nucleic acid are shifted by 3 to 7 bases. From these results, it was found that the annealing of the Mycoplasma pneumoniae nucleic acid 23S rRNA can be performed at a position where several bases (for example, 2 to 8 bases, preferably 3 to 7 bases) are added or deleted to the 5 '-end or 3' -end of each primer MPN-F or MPN-R.

(evaluation test of primers and probes 5)

Further, primers MPN-F11 and F12 were newly prepared, and detection of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene by the Q probe method was attempted in combination with the primer MPN-R5 used in the above-mentioned evaluation test 4.

[ Table 5]

A Mycoplasma pneumoniae 23S rRNA gene detection reagent having the following composition was prepared, and detection by the Q probe method was attempted using GENECUBE (registered trademark). In this test example, QP-1, the probe used in test example 2, was used as a specific labeled fluorescent probe. The PCR cycling conditions and melting curve analysis conditions were the same as in test example 2.

< 23S rRNA Gene detection reagent composition (volume per 1 sample) >

(1: use of boiled inactivated Mycoplasma pneumoniae)

The evaluation results are shown in fig. 9. Here, the results obtained by combining the primer MPN-F11 and the primer MPN-R5 are shown in the upper part of FIG. 9, and the results obtained by combining the primer MPN-F12 and the primer MPN-R5 are shown in the lower part of FIG. 9. As shown in the results, it was found that: when the primers MPN-F11 and F12 were used, the fluorescence intensity was low and the detection sensitivity was low even when they were combined with the primer MPN-R5. The primers MPN-F11 and MPN-F12 are primers MPN-F designed in the following way: the Mycoplasma pneumoniae nucleic acid 23S rRNA was annealed at a position shifted by about 25 residues from the 5' -end side of the primer MPN-F2 used in the above evaluation test 1. As shown in the results, it was found that: when designing a primer at a position shifted from the base sequence of the Mycoplasma pneumoniae nucleic acid 23SrRNA corresponding to the primer of the present invention, the Mycoplasma pneumoniae nucleic acid 23S rRNA cannot be detected with high sensitivity.

Based on the results of the above evaluation results 1 to 5, examples of the nucleotide sequences of the primer MPN-F, the primer MPN-R and the specifically labeled fluorescent probe of the present invention, which are preferable for detecting the wild type and the drug-resistant mutant type of Mycoplasma pneumoniae nucleic acid 23SrRNA, are shown in the following tables.

[ Table 6]

[ Table 7]

[ Table 8]

Any combination of the above primers MPN-F, MPN-R and specific targeting fluorescent probe can be used in the present invention. In addition, a combination of a primer and a probe comprising a base sequence complementary to each of the base sequences shown in tables 6 to 8 may be used.

The above embodiments are merely illustrative of the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the scope of the present invention.

Sequence listing

<110> Toyo Boseki Kabushiki Kaisha (TOYOBO CO., LTD)

<120> kit and detection method for detecting mycoplasma pneumoniae nucleic acid and presence or absence of drug-resistant gene variation thereof

<130> 200142JP01

<160> 39

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F1

<400> 1

cgggtaaatt ccgtcccgct tgaat 25

<210> 2

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F2

<400> 2

cggccgtaac tataacggtc ctaagg 26

<210> 3

<211> 36

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F3

<400> 3

ctcttgactg tctcggctat agactcggtg aaatcc 36

<210> 4

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F4

<400> 4

actgtctcgg ctatagactc ggtgaaatcc 30

<210> 5

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-5

<400> 5

cttgactgtc tcggctatag actcggtgaa atcc 34

<210> 6

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F6

<400> 6

gactgtctcg gctatagact cggtgaaatc c 31

<210> 7

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F7

<400> 7

gtctcggcta tagactcggt gaaatccagg 30

<210> 8

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F8

<400> 8

cggctataga ctcggtgaaa tccaggtacg 30

<210> 9

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F9

<400> 9

ctcggtgaaa tccaggtacg ggtgaag 27

<210> 10

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F10

<400> 10

gtaaattccg tcccgcttga atggtg 26

<210> 11

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R1

<400> 11

ccaattagaa cagcacacaa ccaagggta 29

<210> 12

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R2

<400> 12

caagtcctag cgaacttgca tcgattgct 29

<210> 13

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R3

<400> 13

ccagtcaaac tgcccaccta acactgtctt 30

<210> 14

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R4

<400> 14

cgattgctcc tacctattct ctacatg 27

<210> 15

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R5

<400> 15

atcgattgct cctacctatt ctctacatga taatg 35

<210> 16

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R6

<400> 16

gctcctacct attctctaca tgataatgtc c 31

<210> 17

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R7

<400> 17

gattgctcct acctattctc tacatg 26

<210> 18

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R8

<400> 18

caggacatta tcatgtagag aataggtagg 30

<210> 19

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R9

<400> 19

cctattctct acatgataat gtcctgatc 29

<210> 20

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-R10

<400> 20

cctacctatt ctctacatga taatgtcctg 30

<210> 21

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-1

<400> 21

caacgggacg gaaagac 17

<210> 22

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-2

<400> 22

acgggacgga aagacc 16

<210> 23

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-3

<400> 23

aacgggacgg aaagacc 17

<210> 24

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-4

<400> 24

cgggacggaa agacccc 17

<210> 25

<211> 16

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-5

<400> 25

cgggacggaa agaccc 16

<210> 26

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-6

<400> 26

caacgggacg gaaagaccc 19

<210> 27

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-7

<400> 27

cgcaacggga cggaaagacc c 21

<210> 28

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-8

<400> 28

cgcaacggga cggaaagac 19

<210> 29

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-9

<400> 29

cgttaggcgc aacgggacgg aaa 23

<210> 30

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> QP-10

<400> 30

cggaaagacc ccgtgaagc 19

<210> 31

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F11

<400> 31

agaaggaggt tagcgcaagc gaag 24

<210> 32

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> MPN-F12

<400> 32

aaggaggtta gcgcaagcga ag 22

<210> 33

<211> 2905

<212> DNA

<213> Mycoplasma pneumoniae 23S rRNA sequence (Mycoplasma pneumoniae 23S rRNA sequence)

<400> 33

caataagtta ctaagggctt atggtggatg ccttggcact aataggcgat gaaggacgtg 60

ttaacctgcg ataagcttcg ggtaggtggt aagaacctca gatccggaga tttccgaatg 120

gagcaatccg gtagttggaa acagctatca ttaattgatg aataaatagt caattaaagc 180

aatacgtggt gaagtgaaac atctcagtag ccacaggaaa agaaaacgaa tgtgattccg 240

tgtgtagtgg cgagcgaaag cggaacaggc caaacttatc attagatagg ggttgtaggg 300

cttgcaatgt ggacttgaaa acgatagaag aagctgttgg aaagcagcgc gcaaaagggt 360

gatagccccg tatttgaaat tgttttcata cctagcgaga tccctgagta gctcggaaaa 420

cgttattttg agtgaatctg cccagaccat tgggtaagcc taaatactaa ttagtgaccg 480

atagcgaaac agtaccgtga gggaaaggtg aaaagaaccc agagatggga gtgaaataga 540

ttctgaaacc atatgcctac aacgtgtcag agcacattaa tgtgtgatgg cgtgcgtttt 600

gaagtatgag ccggcgagtt atgatagcaa gcgttagtta accaggagat ggggagctgt 660

agcgaaagcg agttttaaaa gagcgtttgt ttgttattat agacccgaaa cgggttgagc 720

tagtcatgag caggttgaag gttgagtaac atcaactgga ggaccgaacc gactctcgtt 780

gaaacgatag cggatgactt gtgattaggg gtgaaattcc aatcgaaatc cgtgatagct 840

ggttctcgtc gaaatagctt taaggctagc gtgagatcac aaataagtgg aggtaaagct 900

actgaatgta tgatggcgcc acctaggcgt actgaataca attaaactct gaatgccatt 960

tattttattc tcgcagtcag acagtggggg ataagcttca ttgtcaagag gggaagagcc 1020

cagatcatta aataaggtcc ccaaaatata ctaagtggaa aaggatgtga aagtgctaaa 1080

acagcaagga tgttggctta gaagcagcca tcgtttaaag agtgcgtaac agctcacttg 1140

tcgagtgttt ttgcgccgaa gatgtaacgg ggctaagtat attaccgaat ttatggataa 1200

gatttatatc ttgtggtaga cgagcgttgt attggagttg aagtcaaagc gtgagcattg 1260

gtggatccaa tacaagtgag aatgccggca tgagtaacgc ttgggagtga gaatctccca 1320

aaccgattga ctaaggtttc ctggaccagg gtcgtccttc cagggttagt ctggacctaa 1380

gctgaggctg aaaagcgtag gcgatggaca acaggttaat attcctgtac ttacagttag 1440

actgatggag tgacaaagaa ggttttccac ccccataatt ggatttgggg ataaatcata 1500

aggtggtaca ataggcaaat ccgttgtgca taacattgag tgatgatgtc gagtgaatga 1560

gtgatcaagt agcgaaggtg gtattaatca tgctttcaag aaaagcttct agggttaatc 1620

tagctgtaac cagtaccgag aacgaacaca cgtagtcaag gagaggatcc taaggttagc 1680

gagtgaacta tagccaagga actctgcaaa ttaaccccgt aagttagcga gaaggggtgc 1740

ttatgtaaaa gtaagccgca gtgaagaacg aggggggact gtttaactaa aacacaactc 1800

tatgccaaac cgtaaggtga tgtatatggg gtgacacctg cccagtgctg gaaggttaaa 1860

gaaggaggtt agcgcaagcg aagcttttaa ctgaagcccc agtgaacggc ggccgtaact 1920

ataacggtcc taaggtagcg aaattcctag tcgggtaaat tccgtcccgc ttgaatggtg 1980

taaccatctc ttgactgtct cggctataga ctcggtgaaa tccaggtacg ggtgaagaca 2040

cccgttaggc gcaacgggac ggaaagaccc cgtgaagctt tactgtagct taatattgat 2100

caggacatta tcatgtagag aataggtagg agcaatcgat gcaagttcgc taggacttgt 2160

tgatgcgaaa ggtggaatac tacccttggt tgtgtgctgt tctaattggt aactgttatc 2220

cagtttcaag acagtgttag gtgggcagtt tgactggggc ggtcgcctcc taaaaggtaa 2280

cggaggcgta caaaggtacc ttcagtacgg ttggaaatcg tatgtagagt gtaatggtgt 2340

aagggtgctt gactgtgaga catacaggtc gaacaggtga gaaatcaggt catagtgatc 2400

cggtggtcca gtatggaatg gccatcgctc aacggataaa agctactccg gggataacag 2460

gctgatactg cccaagagtt catatcgacg gcagtgtttg gcacctcgat gtcgactcat 2520

ctcatcctcg agctgaagca ggttcgaagg gttcggctgt tcgccgatta aagagatacg 2580

tgagttgggt tcaaaccgtc gtgagacagg ttggtcccta tctattgtgc ccgtaggaag 2640

attgaagagt gttgcttcta gtacgagagg accgaagcga ggacacctct tatgctccag 2700

ttgtagcgcc agctgcaccg ctgggtagta acgtgtctat tagataaacg ctgaaagcat 2760

ctaagtgtga aactatctca aagattaatc ttcccatttc gcaagaaagt aagagccgtc 2820

aaagacgatg acgttgatag gttacaggtg taagcatagt gatatgttga gctgagtaat 2880

actaattgct cgaggactta ttgga 2905

<210> 34

<211> 32

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IC-F

<400> 34

cccggttatt gtagaaattc ctttctcccg tc 32

<210> 35

<211> 31

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IC-R

<400> 35

ccccatccag gattgtagaa tttgaatcaa g 31

<210> 36

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> IC Probe

<400> 36

gatctattca ttcgatattc c 21

<210> 37

<211> 203

<212> DNA

<213> matK of Madarussa ciliate (Lagarosiphon madagascariensis matK)

<400> 37

gcggttattg tagaaattcc tttctcccgt ccattttttc ttgaagaaaa aaaagaaata 60

ccaaaatatc aaaatttacg atctattcat tcgatattcc cttttttaga ggacaaattt 120

ttacatttaa attatgtgtc tgatatagta ataccttatc ctattcatct cgaaatcttg 180

attcaaattc tacaatcctg gat 203

<210> 38

<211> 198

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Positive control-1

<400> 38

ccatctcttg actgtctcgg ctatagactc ggtgaaatcc aggtacgggt gaagacaccc 60

gttaggcgca acgggacgga aagaccccgt gaagctttac tgtagcttaa tattgatcag 120

gacattatca tgtagagaat aggtaggagc aatcgatgca agttcgctag gacttgttga 180

tgcgaaaggt ggaatact 198

<210> 39

<211> 198

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Positive control-2

<400> 39

ccatctcttg actgtctcgg ctatagactc ggtgaaatcc aggtacgggt gaagacaccc 60

gttaggcgca acgggacggg aagaccccgt gaagctttac tgtagcttaa tattgatcag 120

gacattatca tgtagagaat aggtaggagc aatcgatgca agttcgctag gacttgttga 180

tgcgaaaggt ggaatact 198

Claims

1. A detection kit for mycoplasma pneumoniae nucleic acid and drug-resistant gene variation thereof is characterized by comprising KOD DNA polymerase, a primer MPN-F, a primer MPN-R and a specific targeted fluorescent probe;

the primer MPN-F is a forward primer composed of a continuous base sequence with the length of 25-36 bases in the base sequence from No. 1910 to No. 2039 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or in the base sequence complementary to the base sequence;

the primer MPN-R is a reverse primer composed of a continuous base sequence with the length of 25-36 bases in the base sequence shown in No. 2091 to No. 2257 of Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or in a base sequence complementary to the base sequence;

the specific targeted fluorescent probe is designed to be composed of a base sequence which is continuous and has a length of 16-23 bases in a base sequence shown from 2040 th to 2081 th of a Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or a base sequence complementary to the base sequence, and any terminal base is marked with a fluorescent pigment.

2. The kit for assaying Mycoplasma pneumoniae nucleic acid and drug-resistant gene variation according to claim 1,

the primer MPN-F is a forward primer composed of a base sequence which is continuous and has a length of 25 to 36 bases in the base sequence shown in the base sequences from No. 1988 to No. 2030 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or in the base sequence complementary to the base sequence;

the primer MPN-R is a reverse primer composed of a base sequence which is continuous and 25-36 bases in length in the base sequence from No. 2101 to No. 2140 of the Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33) or in a base sequence complementary to the base sequence;

the specific targeting fluorescent probe is designed to be composed of a continuous base sequence with the length of 16-22 bases in No. 2050 to No. 2071 of Mycoplasma pneumoniae nucleic acid 23S rRNA gene (SEQ ID NO: 33), and any terminal base is marked with fluorescent pigment.

3. The kit for assaying Mycoplasma pneumoniae nucleic acid and drug-resistant gene variation according to claim 1,

the primer MPN-R is a reverse primer composed of a base sequence shown by any one of the sequence numbers 11-20 or a base sequence complementary to the base sequence;

the specific targeting fluorescent probe is designed to be composed of a base sequence represented by any one of SEQ ID Nos. 21 to 30 or a base sequence complementary thereto, and a fluorescent dye is labeled at any terminal base.

4. The kit for assaying mycoplasma pneumoniae nucleic acid and drug-resistant gene mutation according to claim 1, further comprising an internal control primer IC-F, an internal control primer IC-R, an internal control probe, and an internal control template.

5. A detection method of mycoplasma pneumoniae nucleic acid and drug-resistant gene variation thereof comprises the following steps:

step 1: preparing a test sample which is collected by any of sputum, a pharyngeal swab or a nasal swab and is subjected to pretreatment;

step 2: performing a PCR amplification reaction using the kit according to any one of claims 1 to 4, using a nucleic acid contained in the test sample as a template gene strand; and

and step 3: the amplification product obtained in step 2 was detected by a melting curve analysis method, and the presence or absence of the drug-resistant gene mutation in the amplification product was confirmed by using the melting temperatures of the amplification product having no drug-resistant gene mutation and the probe, and the difference between the melting temperatures of the amplification product having the drug-resistant gene mutation and the probe.