Primer probe for detecting mycoplasma pneumoniae 23S rRNA gene mutation and application thereof

Abstract

The invention discloses a primer probe for detecting mycoplasma pneumoniae 23S rRNA (ribosomal Ribonucleic Acid) gene mutation and application of the primer probe. The nucleic acid sequence of the primer comprises any one group or a combination of at least two groups of the sequences as shown in SEQ ID NO. 3 to SEQ ID NO. 4, SEQ ID NO. 7 to SEQ ID NO. 8, SEQ ID NO. 11 to SEQ ID NO. 12 or SEQ ID NO. 13 to SEQ ID NO. 14; the nucleic acid sequence of the fluorescent probe comprises any one or a combination of at least two of the sequences as shown in SEQ ID NO. 20, SEQ ID NO. 24, SEQ ID NO. 28 or SEQ ID NO. 30. The primer probe and the digital PCR method can specifically detect mycoplasma pneumoniae 23S rRNA gene mutation, and are high in sensitivity, good in repeatability, excellent in stability and good in application performance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a primer probe for detecting Mycoplasma pneumoniae 23S rRNA gene mutation and an application thereof. Background Art

[0002] Mycoplasma pneumoniae (MP), one of 16 mycoplasma species isolated from human respiratory and urogenital specimens, belongs to the mollicutes family and is one of the smallest known free-living single-celled organisms. MP can infect both the upper and lower respiratory tracts and is prevalent worldwide in individuals of all ages, with children being the most susceptible. Key symptoms include headache, sore throat, fever, cough, and other common respiratory symptoms. MP can be transmitted via droplets, often within families or communities, with an incubation period of 2 to 4 weeks. MP is one of the leading pathogens causing community-acquired respiratory infections in children and adults, and is the primary etiology of community-acquired pneumonia (CAP) in childhood. Epidemiological studies have shown that MP infection occurs sporadically year-round, but incidence is higher in late autumn or winter, with peaks occurring every 3-7 years. Therefore, MP is a respiratory pathogen worthy of close attention.

[0003] Due to its lack of a cell wall, Mycoplasma pneumoniae naturally develops resistance to antibiotics that act on the cell wall, such as β-lactam antibiotics and other antibiotics that inhibit cell wall synthesis. Macrolides, tetracyclines, and fluoroquinolones offer better clinical efficacy in the treatment of Mycoplasma pneumoniae infections. However, tetracyclines and fluoroquinolones may affect development in children and are not recommended for use in children. Therefore, macrolides, such as erythromycin (14-membered ring) and azithromycin (15-membered ring), are the preferred drugs for the clinical treatment of Mycoplasma pneumoniae pneumonia in children. They inhibit bacterial growth by binding to 23S rRNA and inhibiting protein synthesis. Due to their high oral bioavailability and once-daily dosage form, macrolide antibiotics are widely used in outpatient settings. However, over the past decade, macrolide-resistant Mycoplasma pneumoniae (MRMP) have emerged worldwide.

[0004] Macrolide antibiotics act on the 50S ribosomal subunit to inhibit protein synthesis, targeting the central loop of domain V of the 23S rRNA. Nucleotides within this central loop are essential components of the macrolide binding site. Mutations in the 23S rRNA cause conformational changes in the ribosome, altering the macrolide binding site and potentially contributing to resistance. Currently identified 23S rRNA mutations include 2063, 2064, 2617, and 2067. Among these, the most common mutation, A2063G, confers resistance to 14- and 16-membered macrolides; A2064G confers resistance to 14- and 15-membered macrolides; C2617G confers resistance to 14- and 15-membered macrolides; and A2067G confers resistance to yosamycin. Commonly used clinical methods for detecting Mycoplasma pneumoniae infection include culture of the pathogen, direct detection of the antigen, serum antibody testing, and nucleic acid testing. Each method has its own advantages and limitations.

[0005] MP pathogen culture: MP grows slowly, has strict requirements on the culture environment, has a long culture cycle and low sensitivity. It can be used for strain identification, typing and drug sensitivity testing, but the culture cycle is longer. Direct detection of MP antigens: high specificity but low sensitivity, prone to false negative results. MP serum antibody detection: simple detection, rapid results, suitable for rapid screening, but the sampling method is invasive and has poor sensitivity. MP nucleic acid detection: has the characteristics of high sensitivity and specificity, but different nucleic acid detection methods also have their own advantages and disadvantages. Sequencing technology: has many detection targets and good specificity, but the operation is cumbersome, the detection cycle is long, and the cost is high; ordinary PCR technology: low cost, wide application, simple operation, but easy to contaminate and unable to distinguish single nucleotide polymorphisms; fluorescent quantitative PCR technology: has the characteristics of simple operation, fast speed, low cost, high sensitivity, good specificity, etc., and is widely used in clinical practice, but well-designed primers are required for single nucleotide polymorphism detection.

[0006] CN106566874A discloses a specific primer set for detecting drug-resistant mutant genes of Mycoplasma pneumoniae. The primer set is designed using the wild-type sequence and the A2063G and A2064G mutant sequences of the Mycoplasma pneumoniae 23S rRNA V region as templates. The primer set has high sensitivity and specificity, can quantify mutant DNA and wild template DNA, effectively distinguish various genotypes, and calculate the corresponding proportions of various genotypes. It can realize dynamic monitoring of bacterial genotypes and content changes in patients with clinical Mycoplasma pneumoniae infection, facilitate clinical research on the mechanism and progression of Mycoplasma pneumoniae resistance, and facilitate observation of clinical treatment efficacy.

[0007] Therefore, there is an urgent need to provide a digital PCR method that can specifically detect Mycoplasma pneumoniae 23S rRNA gene mutations. Summary of the Invention

[0008] In response to the deficiencies of the existing technology and actual needs, the present invention provides a primer probe for detecting Mycoplasma pneumoniae 23S rRNA gene mutations and its application. The primer probe and digital PCR method of the present invention can specifically detect Mycoplasma pneumoniae 23S rRNA gene mutations with high sensitivity, good repeatability, excellent stability and good application performance.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a primer and a fluorescent probe for detecting mutations in the 23S rRNA gene of Mycoplasma pneumoniae, wherein the nucleic acid sequence of the primer includes any one or a combination of at least two of the sequences shown in SEQ ID NO.3-SEQ ID NO.4, SEQ ID NO.7-SEQ ID NO.8, SEQ ID NO.11-SEQ ID NO.12, or SEQ ID NO.13-SEQ ID NO.14; the nucleic acid sequence of the fluorescent probe includes any one or a combination of at least two of the sequences shown in SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.28, or SEQ ID NO.30.

[0011] The primers and fluorescent probes designed in the present invention can specifically detect Mycoplasma pneumoniae 23S rRNA gene mutations with high sensitivity, good repeatability, excellent stability and good application performance.

[0012] SEQ ID NO. 3: ATCCAGGTACGGGTGAAGAC.

[0013] SEQ ID NO. 4: CGCATCAACAAGTCCTAGCGAA.

[0014] SEQ ID NO. 7: ATCCAGGTACGGGTGAAGAC.

[0015] SEQ ID NO. 8: CGCATCAACAAGTCCTAGCGAA.

[0016] SEQ ID NO. 11: ATCCAGGTACGGGTGAAGAC.

[0017] SEQ ID NO. 12: CGCATCAACAAGTCCTAGCGAA.

[0018] SEQ ID NO. 13: GATACGTGAGTGTGGGTTCAAACC.

[0019] SEQ ID NO. 14: TCGTACTAGAAGCAACACTCTTCAATC.

[0020] SEQ ID NO. 20: CGGGACGGGAAGAC.

[0021] SEQ ID NO. 24: GACGGAGAGACCC.

[0022] SEQ ID NO. 28: GGAAAGGCCCCGTG.

[0023] SEQ ID NO. 30: AGGTTGGTCGCTATC.

[0024] Preferably, the 5' end of the fluorescent probe contains a fluorescent group, and the 3' end contains a quenching group.

[0025] Preferably, the fluorescent group includes any one of FAM, VIC or ROX, or a combination of at least two of them.

[0026] Preferably, the quenching group includes any one of BHQ1, BHQ2 or MGB, or a combination of at least two of them.

[0027] In a second aspect, the present invention provides the use of the primers and fluorescent probes for detecting Mycoplasma pneumoniae 23S rRNA gene mutations described in the first aspect in the preparation of a product for detecting Mycoplasma pneumoniae 23S rRNA gene mutations.

[0028] In a third aspect, the present invention provides a kit for detecting Mycoplasma pneumoniae 23S rRNA gene mutations, the kit comprising the primers and fluorescent probes for detecting Mycoplasma pneumoniae 23S rRNA gene mutations described in the first aspect.

[0029] In a fourth aspect, the present invention provides use of the primers and fluorescent probes for detecting Mycoplasma pneumoniae 23S rRNA gene mutations described in the first aspect in detecting Mycoplasma pneumoniae 23S rRNA gene mutations.

[0030] In a fifth aspect, the present invention provides a device for detecting 23SrRNA gene mutations of Mycoplasma pneumoniae for purposes other than disease diagnosis and / or treatment, the device comprising: an extraction module and a detection module;

[0031] The extraction module is used to perform the following operations: extracting DNA from the sample to be tested;

[0032] The detection module is configured to perform the following steps: performing digital PCR amplification using the primers and fluorescent probes for detecting Mycoplasma pneumoniae 23S rRNA gene mutations described in the first aspect, and making judgments based on the digital PCR amplification results.

[0033] Preferably, the sample to be tested includes any one of nasopharyngeal swab, throat swab or sputum, or a combination of at least two of them.

[0034] Preferably, the amplification system of the digital PCR amplification includes the primers and fluorescent probes for detecting the Mycoplasma pneumoniae 23S rRNA gene mutation described in the first aspect, a DNA template and enzyme-free water.

[0035] Preferably, the final concentration of the primer is 500-700 nM (eg, 500 nM, 600 nM, or 700 nM), and the final concentration of the fluorescent probe is 50-150 nM (eg, 50 nM, 100 nM, or 150 nM).

[0036] Preferably, the amplification program of the digital PCR amplification includes: (1) denaturation: 93-95°C (e.g., 93°C, 94°C, or 95°C), 8-10 min (e.g., 8 min, 9 min, or 10 min), 1-2 cycles; (2) annealing: 92-94°C (e.g., 92°C, 93°C, or 94°C), 20-30s (e.g., 20s, 25s, or 30s), 58-60°C (e.g., 58°C, 59°C, or 60°C), 1-2 min (e.g., 1 min or 2 min), 1-2 cycles; (3) extension: 96-98°C (e.g., 96°C, 97°C, or 98°C), 8-10 min, (e.g., 8 min, 9 min, or 10 min) 1-2 cycles.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The primer-fluorescent probe and digital PCR method of the present invention can specifically detect the Mycoplasma pneumoniae 23S rRNA gene mutations A2063G, A2064G, A2067G and C2617G, with high sensitivity, good repeatability, excellent stability and good application performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1A is the scanning electron micrograph of WT pseudovirus;

[0040] Figure 1B This is a scanning electron micrograph of the A2063G pseudovirus;

[0041] Figure 1C This is a scanning electron micrograph of the A2064G pseudovirus;

[0042] Figure 1DThis is a scanning electron micrograph of the A2067G pseudovirus;

[0043] Figure 1E is a scanning electron micrograph of the C2617G pseudovirus;

[0044] Figure 2A This is a graph showing the optimization results for primer and probe types targeting A2063G;

[0045] Figure 2B This is a graph showing the optimization results for primer and probe types targeting A2064G;

[0046] Figure 2C This is a graph showing the optimization results for primer and probe types targeting A2067G;

[0047] Figure 2D This is a graph showing the optimization results for primer and probe types targeting C2617G;

[0048] Figure 3A This is the result diagram of primer-probe concentration optimization for A2063G;

[0049] Figure 3B This is a graph showing the optimization results of the primer and probe concentration for A2064G;

[0050] Figure 3C This is the result diagram of primer-probe concentration optimization for A2067G;

[0051] Figure 3D This is a graph showing the optimization results of primer and probe concentrations for C2617G;

[0052] Figure 4A This is the annealing temperature optimization result diagram for A2063G;

[0053] Figure 4B This is the annealing temperature optimization result diagram for A2064G;

[0054] Figure 4C This is the annealing temperature optimization result diagram for A2067G;

[0055] Figure 4D This is the annealing temperature optimization result diagram for C2617G;

[0056] Figure 5A This is the annealing time optimization result diagram for A2063G;

[0057] Figure 5B This is the annealing time optimization result diagram for A2064G;

[0058] Figure 5C This is the annealing time optimization result diagram for A2067G;

[0059] Figure 5D This is the result diagram of annealing time optimization for C2617G;

[0060] Figure 6 is the repeatability result graph;

[0061] Figure 7A is a graph showing specific results for A2063G;

[0062] Figure 7B is a graph showing specific results for A2064G;

[0063] Figure 7C is a graph showing specific results for A2067G;

[0064] Figure 7D is a graph showing specific results for C2617G;

[0065] Figure 8A This is the result diagram of the uniformity of pseudovirus standard material;

[0066] Figure 8B This is the short-term stability result of pseudovirus standard material. DETAILED DESCRIPTION

[0067] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0068] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0069] Primer and probe synthesis (Shanghai Bio-Tech Co., Ltd.); virus preservation solution (Guangzhou Medical Device Preparation No. 20160299, Guangzhou Bondsheng Biotechnology Co., Ltd.); viral nucleic acid extraction kit (Takara Biotechnology); RNase contamination removal reagent (RNaseZap TM , Thermofisher); nuclease-free water (Thermofisher); digital PCR premix (2×ddPCR Supermix for Probes, Bio-Rad); other biochemical reagents were imported or domestically produced analytical grade.

[0070] Nanodrop 2000 micro-volume nucleic acid and protein quantification instrument (Thermo Corporation), droplet digital PCR instrument (QX200, Bio-Rad).

[0071] Example 1

[0072] This embodiment provides a digital PCR method for detecting Mycoplasma pneumoniae 23S rRNA gene mutations.

[0073] 1. Preparation of pseudovirus standard material:

[0074] The wild type (WT), A2063G, A2064G, C2617G and A2067G DNA fragments of the Mycoplasma pneumoniae (MP) 23S rRNA gene were cloned into adeno-associated virus (AAV) vectors to construct five recombinant pseudovirus plasmids. Each plasmid was then extracted and endotoxins were removed to ensure the safety and purity of the subsequent virus packaging process. The pseudovirus was then packaged and purified. In addition, the content of residual plasmid DNA in the virus liquid was evaluated to ensure that the proportion of plasmid DNA remaining outside the AAV particles was less than 5%. Finally, the obtained pseudovirus particles were morphologically observed using a scanning electron microscope (SEM) to evaluate their structural integrity and particle uniformity. SEM images are shown in Figure 2. Figure 1A-1E shown.

[0075] Wild-type pseudovirus and pseudovirus samples carrying the A2063G, A2064G, A2067G, and C2617G mutations were diluted to 1000 copies / μL and 100 copies / μL, respectively. These samples were then mixed with the WT pseudovirus to prepare pseudovirus standards with approximately 10% mutation abundance. The resulting mutation abundance reference materials were aliquoted into 1.5 mL cryovials, 230 μL per tube, and stored at -80°C for later use.

[0076] 2. DNA Extraction and Purification: DNA was extracted from four pseudovirus standards using the MiniBEST Viral RNA / DNA Extraction Kit Ver. 5.0 (TaKaRa, Cat. No. 9766) according to the manufacturer's instructions. The purity and concentration of the extracts were determined using a NanoDrop microspectrophotometer (Thermo Fisher Scientific, USA). A260 / 280 ratios between 1.8 and 2.0, and A260 / 230 ratios greater than 2.0, indicated high purity and minimal contamination of the extracted DNA.

[0077] 3. Optimization of digital PCR primer and probe sequences: Based on the Mycoplasma pneumoniae MP 23S rRNA gene reference sequence obtained from the NCBI database, four site mutations (A2063G, A2064G, A2067G, and C2617G) located in the conserved region were selected as detection targets. Using NCBI's Primer-BLAST tool, two sets of primers and dual-labeled hydrolysis probes were designed for each site for specific detection of wild-type and mutant alleles. The 5' end of the probe is labeled with a FAM fluorescent group (for identifying wild-type sequences) and a VIC fluorescent group (for identifying mutant sequences), and the 3' end is labeled with a microgroove binder (MGB) quenching group to improve the specificity of detection. All primers and probes are synthesized by Bio-Bio, and the specific sequences are shown in Table 1.

[0078] Table 1

[0079]

[0080]

[0081] The extracted DNA was used as a template and digital PCR amplification was performed using the above primers and probes. The specific amplification results are shown in the figure. Figure 2A-2D Based on the digital PCR amplification results obtained with the two designed primer-probe combinations, the signal cluster separation between the FAM channel (representing the wild type) and the VIC channel (representing the mutant type) was compared. Specifically, the second set of primer-probe combinations targeting the A2063G, A2064G, and A2067G mutation sites, and the first set targeting the C2617G site, showed more defined and well-defined oil droplet clusters, with almost no overlap between the negative, single-positive, and double-positive groups ( Figure 2A-2D These results demonstrate that this combination offers superior discrimination between target and non-target signals, enabling more accurate quantification. Therefore, this primer-probe combination was selected as the optimized solution for subsequent experiments.

[0082] 4. Optimization of digital PCR primer and probe concentrations: Three combinations were set up: the first was 600nM primer + 100nM probe, the second was 600nM primer + 200nM probe, and the third was 600nM primer + 400nM probe. Digital PCR amplification was performed separately. By comparing the performance of each combination in terms of fluorescence signal cluster resolution and signal clarity, the optimal concentration conditions were determined to ensure high specificity of the reaction and accuracy of oil droplet classification. The results showed that when the primer concentration was 600nM and the probe concentration was 100nM, the four clusters in the figure were clearly distributed, with clear boundaries and no obvious overlap ( Figures 3A-3D ), indicating that the primer-probe binding efficiency under this condition is high and the specificity is excellent.

[0083] 5. Optimization of digital PCR annealing temperature and time: Set the gradient temperature (55℃~65℃) as the annealing temperature for digital PCR and compare the amplification results of each annealing temperature. According to the separation and aggregation effect of positive and negative oil droplets, 60℃ was determined to be the optimal annealing temperature ( Figures 4A-4D In addition, different annealing times (30s, 60s, and 120s) were evaluated, of which 120s performed best in terms of oil droplet resolution and was therefore selected as the final condition ( Figures 5A-5D ).

[0084] 6. Establish an optimized digital PCR detection protocol as follows:

[0085] 6.1. Pseudovirus nucleic acid extraction: Thaw the standard material and equilibrate it to room temperature. After brief centrifugation, aspirate 200 μL of the standard material and perform nucleic acid extraction according to the following protocol.

[0086] (1) Add 200 μL of Buffer VGB, 20 μL of Proteinase K, and 1 μL of Carrier RNA to 200 μL of standard material, mix thoroughly, and incubate at 56°C for 10 min;

[0087] (2) Add 200 μL of anhydrous ethanol to the lysate and mix thoroughly by pipetting;

[0088] (3) Place the centrifuge column in the collection tube, transfer the solution to the centrifuge column, and centrifuge at 12,000 rpm for 2 min;

[0089] (4) Add 500 μL of Buffer RWA to the centrifuge column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate;

[0090] (5) Add 700 μL of Buffer RWB to the spin column, centrifuge at 12,000 rpm for 1 min, and discard the filtrate (add Buffer RWB along the sides of the spin column to help completely wash away the salt adhering to the column wall);

[0091] (6) Repeat step (5);

[0092] (7) Place the spin column in the collection tube and centrifuge at 12,000 rpm for 2 min;

[0093] (8) Place the spin column in a new 1.5 mL enzyme-free centrifuge tube, add 100 μL of RNase-free water to the center of the spin column membrane, and let it stand at room temperature for 5 min;

[0094] (9) Centrifuge at 12,000 rpm for 2 min to elute nucleic acids;

[0095] (10) Add 100 μL of RNase-free water to the center of the spin column membrane and let it stand at room temperature for 5 min.

[0096] (11) Centrifuge at 12,000 rpm for 2 min to elute nucleic acid;

[0097] (12) Mix the nucleic acids from the two elutions, totaling 200 μL, and store them at -80°C to 70°C until use, avoiding repeated freezing and thawing.

[0098] 6.2. Digital PCR Amplification of Mycoplasma pneumoniae 23S rRNA Gene Mutations (A2063G, A2064G, A2067G, C2617G) The primer and probe information for pseudovirus standards is shown in Table 2. Before preparing the reaction system, the primer and probe solutions must be fully dissolved.

[0099] Table 2

[0100]

[0101]

[0102] 6.3. Preparation of digital PCR reaction system: The digital PCR reaction system was prepared by referring to Table 3 for the pseudovirus standard substances enriched with Mycoplasma pneumoniae 23S rRNA gene mutations (A2063G, A2064G, A2067G, C2617G).

[0103] Table 3

[0104] Components Volume (μL) 2×ddPCR Supermix for Probes 10 Upstream primer (10 μM) 1.2 Downstream primer (10 μM) 1.2 Probe (10 μM) 0.2 DNA extraction 5 Enzyme-free water 2.4 Total 20 μL

[0105] 6.4. Amplification program: Refer to Table 4, annealing temperature is 60℃.

[0106] Table 4

[0107]

[0108] Example 2

[0109] Repeatability test.

[0110] Eight tubes of standard material samples were taken for each test. After nucleic acid extraction, the nucleic acid was added to the optimal digital PCR amplification system determined in Example 1. The results were repeated 8 times independently, and the relative standard deviation (RSD0) of the 8 test data was recorded, which is the repeatability of the determination method. The repeatability test data of the Mycoplasma pneumoniae 23S rRNA gene mutation (A2063G, A2064G, A2067G, C2617G) pseudovirus standard material are shown in Table 5 and Figure 6 .

[0111] Table 5

[0112] Mutation abundance Repeat 1 Repeat 2 Repeat 3 Repeat 4 Repeat 5 Repeat 6 Repeat 7 Repeat 8 average value RSD A2063G 8.32％ 8.03％ 8.52％ 8.30％ 7.92％ 8.26％ 8.13％ 8.39％ 8.23％ 2.38％ A2064G 7.13％ 7.17％ 6.97％ 6.61％ 7.30％ 7.09％ 7.38％ 7.20％ 7.11％ 3.32％ A2067G 7.33％ 7.18％ 7.15％ 7.23％ 7.31％ 7.04％ 7.22％ 7.33％ 7.22％ 1.39％ C2617G 8.13％ 7.57％ 7.84％ 7.90％ 8.06％ 8.16％ 8.14％ 7.87％ 7.96％ 2.56％

[0113] From Table 5 and Figure 6 The results show that the method of the present invention has good repeatability.

[0114] Example 3

[0115] Specificity verification.

[0116] The allele specificity and quantitative discrimination ability of the digital PCR detection method for the A2063G, A2064G, A2067G and C2617G mutation sites were evaluated using pseudovirus DNA samples containing different mutant allele ratios (0%, 10% and 100%). In the reaction system containing 100% mutant pseudovirus DNA, only the VIC fluorescence signal (corresponding to the mutation-specific probe) was detected, while there was no signal in the FAM channel, indicating that the wild-type probe could not bind to or amplify the mutant sequence. In contrast, in the reaction containing 100% wild-type pseudovirus DNA, only the FAM signal was detected, and no VIC fluorescence was seen, further confirming that the mutant probe did not cross-react with the wild-type sequence. In the sample with a mutant allele ratio of 10%, the droplet diagram ( Figures 7A-7D ) showed four clearly separated clusters with no crosstalk. These results demonstrate the high specificity of this assay, enabling accurate identification and quantification of low-frequency mutations in the presence of a high background of wild-type DNA without generating false positive signals.

[0117] Example 4

[0118] Uniformity testing.

[0119] In the uniformity evaluation, 11 bottles of standard material samples were randomly selected, and 50 μL of each sample was sampled three times for testing. A total of 33 samples were extracted from the samples, and the standard materials were tested using the digital PCR method to obtain mutation ratio data. The uniformity results are shown in Table 6 and Figure 8A The data were then analyzed by F test, and the calculated F value was less than the critical value Fa. At the 95% confidence level, there was no significant difference between the samples, indicating that the units were well homogeneous.

[0120] Table 6

[0121]

[0122] From the results in Table 6, we can see that there is no significant difference between the samples, indicating that the units are well uniform.

[0123] Example 5

[0124] Stability verification.

[0125] The short-term stability of the standard substance was investigated by digital PCR. The mutation ratio of the standard substance was measured after the standard substance was placed at -20℃, 4℃ and 20℃ for 0, 1, 3, 7, 10 and 14 days to see if there was any change in the value with the extension of storage time. The data are shown in Table 7 and Figure 8B The results showed that there was no statistically significant difference between the time points within 14 days of storage at -20°C, 4°C and 20°C, indicating that the A2063G mutant pseudovirus standard material has good short-term stability under the above transportation conditions.

[0126] Table 7

[0127]

[0128]

[0129] Example 6

[0130] Application performance verification.

[0131] The analytical performance and clinical applicability of the digital PCR detection method developed in this invention and its accompanying 23S rRNA gene mutation-enriched pseudovirus reference material were evaluated using five clinical MP-positive samples (including nasopharyngeal swabs, throat swabs, and sputum). The test results showed that the corresponding mutant genes were detected in all samples (Table 8), demonstrating the good performance of the detection method.

[0132] Table 8

[0133]

[0134] In summary, the primer-fluorescent probe and digital PCR method of the present invention can specifically detect the 23S rRNA gene mutation of Mycoplasma pneumoniae with high sensitivity, good repeatability, excellent stability and good application performance.

[0135] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A primer and a fluorescent probe for detecting Mycoplasma pneumoniae 23S rRNA gene mutations, characterized in that: The nucleic acid sequence of the primer includes any one or a combination of at least two of the sequences shown in SEQ ID NO.3-SEQ ID NO.4, SEQ ID NO.7-SEQ ID NO.8, SEQ ID NO.11-SEQ ID NO.12 or SEQ ID NO.13-SEQ ID NO.14; the nucleic acid sequence of the fluorescent probe includes any one or a combination of at least two of the sequences shown in SEQ ID NO.20, SEQ ID NO.24, SEQ ID NO.28 or SEQID NO.

30.

2. The primers and fluorescent probe for detecting Mycoplasma pneumoniae 23S rRNA gene mutation according to claim 1, characterized in that: The 5' end of the fluorescent probe contains a fluorescent group, and the 3' end contains a quenching group; Preferably, the fluorescent group includes any one of FAM, VIC or ROX, or a combination of at least two; Preferably, the quenching group includes any one of BHQ1, BHQ2 or MGB, or a combination of at least two of them.

3. Use of the primers and fluorescent probe for detecting Mycoplasma pneumoniae 23S rRNA gene mutations according to claim 1 or 2 in the preparation of a product for detecting Mycoplasma pneumoniae 23S rRNA gene mutations.

4. A kit for detecting Mycoplasma pneumoniae 23S rRNA gene mutations, characterized in that: The kit comprises the primers and fluorescent probe for detecting Mycoplasma pneumoniae 23S rRNA gene mutations according to claim 1 or 2.

5. Use of the primers and fluorescent probe for detecting Mycoplasma pneumoniae 23S rRNA gene mutations according to claim 1 or 2 in detecting Mycoplasma pneumoniae 23S rRNA gene mutations.

6. A device for detecting Mycoplasma pneumoniae 23S rRNA gene mutations for purposes other than disease diagnosis and / or treatment, characterized in that: The device comprises: an extraction module and a detection module; The extraction module is used to perform the following operations: extracting DNA from the sample to be tested; The detection module is configured to perform the following steps: performing digital PCR amplification using the primers and fluorescent probes for detecting Mycoplasma pneumoniae 23S rRNA gene mutations according to claim 1 or 2, and making judgments based on the digital PCR amplification results.

7. The device according to claim 6, characterized in that The sample to be tested includes any one of nasopharyngeal swab, throat swab or sputum, or a combination of at least two of them.

8. The device according to claim 6 or 7, characterized in that The digital PCR amplification system includes the primers and fluorescent probes for detecting Mycoplasma pneumoniae 23S rRNA gene mutations according to claim 1 or 2, a DNA template, and enzyme-free water.

9. The device according to claim 8, characterized in that The final concentration of the primer is 500-700 nM, and the final concentration of the fluorescent probe is 50-150 nM.

10. The device according to any one of claims 6 to 9, characterized in that The amplification procedure of the digital PCR amplification includes: (1) denaturation: 93-95°C, 8-10 min, 1-2 cycles; (2) annealing: 92-94°C, 20-30 s, 58-60°C, 1-2 min, 1-2 cycles; (3) extension: 96-98°C, 8-10 min, 1-2 cycles.

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