Nucleic acid composition and kit for mycoplasma pneumoniae detection, typing and / or drug resistance gene detection and application of nucleic acid composition and kit

Abstract

The invention relates to a nucleic acid composition for mycoplasma pneumoniae detection, typing and / or drug resistance gene detection, a kit and application thereof. The nucleic acid composition and the kit not only can be used for efficiently, sensitively and accurately qualitatively / quantitatively detecting mycoplasma pneumoniae infection, but also can be used for synchronously determining the subtype and drug-resistant gene conditions of the infected mycoplasma pneumoniae, so that comprehensive and accurate mycoplasma pneumoniae infection related information is obtained; when the kit is applied to a clinical scene, the infection state, the typing result and the drug resistance condition of the mycoplasma pneumoniae can be efficiently and accurately obtained, and the kit has important value and significance for rapid detection and prevention and control of the mycoplasma pneumoniae, reasonable selection of antibiotics for treatment and clinical prognosis judgment.

Description

Technical Field

[0001] This invention relates to the field of Mycoplasma pneumoniae detection technology, specifically to nucleic acid compositions, kits, and their applications for Mycoplasma pneumoniae detection, typing, and / or drug resistance gene detection. Background Technology

[0002] Mycoplasma pneumoniae (MP) is a major pathogen causing community-acquired pneumonia. It is primarily transmitted through respiratory droplets and is prone to causing outbreaks in enclosed environments. Children and adolescents are particularly susceptible. Since September 2023, several countries worldwide have experienced outbreaks of Mycoplasma pneumoniae, and the incidence of severe Mycoplasma pneumoniae pneumonia in my country has also been gradually increasing.

[0003] MP lacks a cell wall structure and is naturally resistant to β-lactam antibiotics. While macrolide antibiotics remain the first-line treatment for pediatric MP infections, resistance rates during epidemics have reached over 99%, and this resistance phenotype is primarily caused by the AG mutation at position 2063 in the 23S rRNA V region. Furthermore, MP can be classified into P1 genotype I and P1 genotype II strains based on P1 gene polymorphism. Previous studies have shown that some P1 genotype II strains are more virulent and associated with more severe clinical symptoms. Since 2015, the prevalence of P1 genotype II strains has been observed to gradually increase in several countries, while P1 genotype I strains are more often associated with higher drug resistance rates.

[0004] Therefore, during MP epidemics and clinical testing, it is crucial to establish a method that can simultaneously determine MP positivity / positivity, genotyping, and detect drug resistance gene mutations. This method can provide key references for clinicians to rationally select antibiotics and is also of great significance for the control of pathogens during epidemics. Currently, there is no technical solution that can simultaneously complete MP positivity / positivity detection, P1 genotyping, and drug resistance mutation identification in a single reaction. Existing detection methods are all separate tests for the above three aspects—including methods for determining MP positivity / positivity separately, methods for P1 gene polymorphism typing separately, and methods for detecting mutations at the 23S rRNA V region 2063 site separately. Among these, except for MP positivity / positivity determination, which often uses the simple-to-operate quantitative real-time PCR technology, the operation procedures for P1 gene polymorphism typing and 23S rRNA V region 2063 site mutation detection are relatively complex: P1 gene polymorphism detection requires first amplifying the full-length P1 gene, then digesting it with HaeIII restriction enzymes and observing fragment differences to complete the typing; 23S rRNA V region 2063 site mutation detection requires amplifying the target fragment and then performing sequencing analysis. These methods are not only time-consuming and labor-intensive, but also unable to simultaneously obtain the MP genotype and drug resistance information of infected patients, and are difficult to meet the timeliness requirements of rapid clinical testing.

[0005] In summary, there is an urgent clinical need for a detection system that can efficiently and specifically detect MP infection and simultaneously perform genotyping and drug resistance mutation identification. Summary of the Invention

[0006] Purpose of the invention In view of the above-mentioned problems or needs in the existing technology, the purpose of this invention is to provide a nucleic acid composition, kit and its application that can efficiently, sensitively and accurately detect whether a person has Mycoplasma pneumoniae, and can also simultaneously identify the subtype of Mycoplasma pneumoniae and its drug resistance genes.

[0007] Solution To achieve the above objectives, the present invention provides the following technical solution.

[0008] In a first aspect, the present invention provides a nucleic acid composition for detecting Mycoplasma pneumoniae infection and / or its typing, said nucleic acid composition comprising: (1) Primer pairs and their detection probe MP-I-Probe for specifically amplifying the Mycoplasma pneumoniae type I specific fragment MP-I, wherein MP-I is a gene fragment whose nucleotide position relative to the genome of the MP standard strain M129 is between positions 177739 and 178529; and / or, (2) Primer pairs and detection probes for specifically amplifying the MP-II specific fragment of Mycoplasma pneumoniae type II, wherein the MP-II is a gene fragment whose nucleotide position relative to the FH genome of the MP standard strain is between positions 703624 and 709505.

[0009] When the nucleic acid composition includes only item (1), it can be used to specifically detect Mycoplasma pneumoniae type I; when the nucleic acid composition includes only item (2), it can be used to specifically detect Mycoplasma pneumoniae type II; when the nucleic acid composition includes both items (1) and (2), it can be used to detect all types of Mycoplasma pneumoniae and can simultaneously identify the specific type of Mycoplasma pneumoniae.

[0010] In a feasible implementation, the primer pair for specifically amplifying MP-I includes an upstream primer MP-IF and a downstream primer MP-IR, wherein the nucleotide sequence of the upstream primer MP-IF is shown in SEQ ID NO:1, the nucleotide sequence of the downstream primer MP-IR is shown in SEQ ID NO:2, and the nucleotide sequence of its detection probe MP-I-Probe is shown in SEQ ID NO:3.

[0011] In a feasible implementation, the primer pair for specifically amplifying MP-II includes an upstream primer MP-II-F and a downstream primer MP-II-R, wherein the nucleotide sequence of the upstream primer MP-II-F is shown in SEQ ID NO:4, the nucleotide sequence of the downstream primer MP-II-R is shown in SEQ ID NO:5, and the nucleotide sequence of its detection probe MP-II-Probe is shown in SEQ ID NO:6.

[0012] In a further feasible embodiment, the nucleic acid composition further includes: (3) Primer pairs 2063-F and 2063-R and detection probes 2063-Probe-W and 2063-Probe-M for specific amplification and detection of the A2063G mutation in the 23S rRNA gene of Mycoplasma pneumoniae, wherein the nucleotide sequence of primer 2063-F is shown in SEQ ID NO:7, the nucleotide sequence of primer 2063-R is shown in SEQ ID NO:8, the nucleotide sequence of detection probe 2063-Probe-W is shown in SEQ ID NO:9, and the nucleotide sequence of detection probe 2063-Probe-M is shown in SEQ ID NO:10.

[0013] In the above feasible implementation, preferably, each detection probe has a different fluorescent reporter group labeled at its 5' end and a fluorescent quencher group labeled at its 3' end.

[0014] More preferably, the fluorescent reporter group is selected from: FAM, VIC, Cy5, ROX, JOE, TET, HEX and Cy3; more preferably, the fluorescent quencher group is selected from: TAMRA, MGB, BHQ1 and BHQ2.

[0015] In a second aspect, the present invention provides the use of the nucleic acid composition described in the first aspect above in the preparation of kits for any of the following applications: (1) To detect whether the subject is infected with Mycoplasma pneumoniae; (2) Detecting the type of Mycoplasma pneumoniae infecting the subjects; and, (3) Detect whether the Mycoplasma pneumoniae infected by the subject has a drug resistance mutation, wherein the drug resistance mutation is the A2063G mutation of the Mycoplasma pneumoniae 23s rRNA gene.

[0016] Thirdly, the present invention provides a kit for detecting Mycoplasma pneumoniae infection, Mycoplasma pneumoniae typing and / or its drug resistance mutations, the kit comprising the nucleic acid composition as described in the first aspect above.

[0017] In some feasible implementations, the kit is a real-time quantitative PCR kit, which includes the reagents required for real-time quantitative PCR detection.

[0018] In some other feasible implementations, the kit is a digital PCR kit, which includes the reagents required for digital PCR detection.

[0019] Preferably, the kit also includes reagents selected from the following: positive control, negative control and dUTP / UNG (to prevent contamination and avoid false positive results).

[0020] More preferably, the positive control is the genomic DNA or specific fragment MP-I of Mycoplasma pneumoniae type I strain, the genomic DNA or specific fragment MP-II of Mycoplasma pneumoniae type II strain, and / or a gene sequence containing a mutation or no mutation at the 2063 site of the Mycoplasma pneumoniae 23S rRNA gene; more preferably, the negative control is an amplification system that does not contain any specific sequence of Mycoplasma pneumoniae.

[0021] In feasible implementations, the test samples for the kit include, but are not limited to, throat swabs, nasal swabs, nasopharyngeal swabs, sputum, bronchoalveolar lavage fluid, or puncture fluid.

[0022] Fourthly, the present invention provides a detection device for detecting Mycoplasma pneumoniae infection, Mycoplasma pneumoniae typing, and / or its drug resistance mutations, said detection device comprising the nucleic acid composition described in the first aspect above.

[0023] Fifthly, the present invention provides a method for detecting whether a sample contains Mycoplasma pneumoniae, the typing of the contained Mycoplasma pneumoniae, and / or its drug-resistant mutations for non-disease diagnostic purposes, the method comprising: detecting the DNA of the sample to be tested using the nucleic acid composition described in the first aspect above, the kit described in the third aspect above, or the detection device described in the fourth aspect above.

[0024] In a feasible implementation, the detection is performed by real-time quantitative PCR or digital PCR; preferably, the amplification curve is used to determine whether the sample contains Mycoplasma pneumoniae, the type of Mycoplasma pneumoniae contained therein, and / or whether it has a drug-resistant mutation (i.e., 23S rRNA gene A2063G mutation).

[0025] In feasible implementations, the samples to be tested include, but are not limited to, throat swabs, nasal swabs, nasopharyngeal swabs, sputum or bronchoalveolar lavage fluid samples.

[0026] In a feasible implementation, the method includes: (1) Establish a multiplex real-time fluorescence PCR or digital PCR detection system and perform the corresponding detection; For example, a 20 μL real-time fluorescence PCR reaction system is prepared as follows: 2 μL of DNA template; Super Real PreMix 10μL; A primer-probe mixture of appropriate concentration; Add ultrapure water to a final volume of 20 μL.

[0027] Preferably, the primer-probe mixture comprises: primer pairs MP-IF and MP-IR for specific amplification of MP-I and their detection probe MP-I-Probe; primer pairs MP-II-F and MP-II-R for specific amplification of MP-II and their detection probe MP-II-Probe; and primer pairs 2063-F and 2063-R for specific amplification of the A2063G mutation in the Mycoplasma pneumoniae 23S rRNA gene and detection probes 2063-Probe-1 and 2063-Probe-2. Preferably, the final concentrations of each primer and probe in the primer-probe mixture are as follows: For specific amplification and detection of MP-I, the upstream and downstream primers and their detection probe are each 0.1-0.3 μM, preferably 0.2 μM; For specific amplification and detection of MP-II, the upstream and downstream primers and their detection probe are each 0.1-0.3 μM, preferably 0.2 μM; For the specific amplification of the A2063G mutation in the 23S rRNA gene of Mycoplasma pneumoniae, the upstream and downstream primers are 0.1-0.3 μM, preferably 0.1-0.2 μM, and the probes 2063-Probe-1 and 2063-Probe-2 are 0.1-0.2 μM.

[0028] The PCR reaction conditions are as follows: Initial denaturation at 95℃ for 20 seconds; followed by 40 cycles, each cycle consisting of denaturation at 95℃ for 1 second and annealing / extending at 58-62℃ for 20 seconds.

[0029] For example, the digital PCR detection method is as follows: The digital PCR kit from Beijing Xinyi Biotechnology Co., Ltd. was used for detection in a 30 μL reaction system. The polymerase chain reaction (PCR) mixture contained: 6 μL PCR reaction mixture A; 1.5 μL PCR reaction mixture B; for each primer pair and probe, 1.5 μL each of the forward and reverse primer solutions (final primer concentration 500 nmol / L), 0.75 μL of probe (final probe concentration 250 nmol / L); and 2 μL template DNA. The prepared reaction mixture was transferred to a chip, and droplets were generated using a Drop Maker M1 device. The droplet emulsion was pre-denatured at 95 °C for 10 min, followed by 40 cycles, each cycle consisting of denaturation at 94 °C for 30 sec and annealing / extension at 55–66 °C for 1 min, and finally cooling at 12 °C for 5 min. Finally, the fluorescence signals of positive and negative droplets were analyzed using a Chip Reader TD2 device (Beijing Xinyi Biotechnology Co., Ltd.).

[0030] Based on the fluorescence signal detection results of the fluorescence detection instrument, an analysis and judgment are made: if the fluorescence detection instrument detects the target fluorescence signal within the set reaction time, it is judged as positive; otherwise, it is judged as negative.

[0031] Beneficial effects The nucleic acid composition and kit provided by this invention can not only efficiently, sensitively and accurately detect whether there is Mycoplasma pneumoniae infection, but also simultaneously identify the subtype and drug resistance genes of the infected Mycoplasma pneumoniae, thereby obtaining comprehensive and accurate information related to Mycoplasma pneumoniae infection. When applied in clinical settings, users can efficiently and accurately obtain the Mycoplasma pneumoniae infection status, subtyping results and drug resistance of subjects, which has important value and significance for the rapid detection and prevention of Mycoplasma pneumoniae, the rational selection of therapeutic antibiotics and the judgment of clinical prognosis. Attached Figure Description

[0032] Figure 1 This diagram shows the experimental workflow of multiplex ddPCR detection; Figure A shows sample processing and DNA extraction, and Figure B shows ddPCR detection and analysis.

[0033] Figure 2 This shows the specificity test results of the multiplex ddPCR detection of Mycoplasma pneumoniae.

[0034] Figure 3 This shows the sensitivity test results for the multiplex ddPCR detection of Mycoplasma pneumoniae.

[0035] Figure 4 This shows the regression analysis curves of DNA copy number versus serial dilution concentration in multiplex ddPCR detection of Mycoplasma pneumoniae.

[0036] Figure 5 This displays the specificity test results of multiplex real-time fluorescent PCR detection of Mycoplasma pneumoniae.

[0037] Figure 6 The results show the sensitivity test results of multiplex real-time fluorescence PCR detection of Mycoplasma pneumoniae; Figure A shows the sensitivity test results of real-time fluorescence quantitative PCR detection of type I MP strains, and Figure B shows the sensitivity test results of real-time fluorescence quantitative PCR detection of type II MP strains. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. The described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components; the term "and / or" shall be understood to mean any one of the options or any combination of two or more of the options.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available; unless otherwise specified, the examples are conducted under conventional experimental conditions or according to the manufacturer's instructions.

[0041] Example 1: Discovery of a novel MP type-specific gene fragment Although the current MP typing is based on variations in the P1 gene among strains, the inventors have found in long-term clinical studies that other genomic regions also show consistent differences between type I and type II MP strains.

[0042] To systematically identify conserved type-specific gene fragments, the inventors performed whole-genome sequencing on 17 clinical isolates of type I MP and 12 clinical isolates of type II MP, and conducted multiple sequence alignment analyses on their whole-genome data. This analysis identified two type-specific genomic regions: (1) A 791-bp gene fragment unique to type I MP strains (hereinafter referred to as MP-I fragment) has a genomic nucleotide position of 177739-178529 relative to type I MP standard strain M129; (2) A 5882-bp gene fragment unique to type II MP strains (hereinafter referred to as MP-II fragment) has a genomic nucleotide position of 703624-709505 relative to the type II MP standard strain FH.

[0043] Example 2: Design of primers and probes for newly discovered MP type-specific gene fragments For the two newly discovered type-specific gene fragments in Example 1, the inventors designed their PCR amplification primers and detection probes, the specific sequences of which are shown in Table 1 below.

[0044] In addition, given the ample literature documentation linking the A2063G mutation in the 23s rRNA gene of MP strains to macrolide resistance (reportedly, over 90% of macrolide-resistant strains or clinical samples are caused by the AG mutation at the 2063 site), we also designed PCR amplification primers and MGB probes targeting this resistance mutation for simultaneous detection with the aforementioned MP type-specific detection system. The specific sequences of the primers and probes are shown in Table 1 below.

[0045] Table 1. Multiplex PCR primer and probe sequence information for MP type-specific gene fragments and A2063G drug resistance mutations.

[0046] The primers and probes shown in Table 1 are assembled into a kit as a representative example of the kit of the present invention (hereinafter referred to as the kit of the present invention).

[0047] Example 3: Establishment of a digital PCR (ddPCR) detection method based on the kit of the present invention In this embodiment, using the kit of the present invention, a multiplex ddPCR detection method for simultaneously performing typing of Mycoplasma pneumoniae and macrolide resistance analysis was established. The detection procedure is as follows: Figure 1 As shown, the specific detection method is as follows: 1. Sample processing and DNA extraction Clinical samples such as bronchoalveolar lavage fluid, sputum, or pharyngeal swabs were collected, and genomic DNA was extracted by centrifugation lysis and column purification.

[0048] 2. ddPCR detection and analysis The detection was performed using a digital PCR kit from Beijing Xinyi Biotechnology Co., Ltd., in a 30 μL reaction system. The polymerase chain reaction (PCR) mixture contained: 6 μL PCR reaction mixture A; 1.5 μL PCR reaction mixture B; for each primer pair and probe, 1.5 μL each of the forward and reverse primer solutions (final primer concentration 500 nmol / L), 0.75 μL of probe (final probe concentration 250 nmol / L); and 2 μL template DNA. The prepared reaction mixture was transferred to a chip, and droplets of approximately 40,000 nanoliters were first generated using a fully automated digital PCR instrument (D20, Beijing Xinyi Biotechnology Co., Ltd.). The droplet emulsion was pre-denatured at 95 °C for 10 min, followed by 40 cycles, each cycle consisting of denaturation at 94 °C for 30 sec and annealing / extension at 55–66 °C for 1 min, and finally cooling at 12 °C for 5 min. Finally, the fluorescence signals of positive and negative droplets were analyzed.

[0049] Using the above reaction system, the detection of multiple targets was achieved, and the specific determination method is as follows: (1) When FAM and / or VIC signals are detected, mycoplasma pneumoniae infection can be determined; (2) Furthermore, when the FAM signal is detected, it indicates type I MP infection; when the VIC signal is detected, it indicates type II MP infection. Therefore, the genotype of Mycoplasma pneumoniae can be identified simultaneously; and, (3) When Cy5 signal is detected, it can be determined that there is no macrolide resistance-related mutation at position 2063 in the 23S rRNA gene; when ROX fluorescence signal is detected, it can be determined that there is macrolide resistance-related mutation at position 2063 in the 23S rRNA gene.

[0050] Therefore, by comprehensively analyzing fluorescence signals, it is possible to quickly and economically identify whether there is Mycoplasma pneumoniae infection, the type of Mycoplasma pneumoniae, and the macrolide resistance status directly from clinical samples. This not only improves efficiency but also greatly simplifies the diagnostic process.

[0051] Example 4: Sensitivity and Specificity Tests for Digital PCR Detection To evaluate the specificity of the multiplex ddPCR detection method established in Example 3, we tested a group of common clinical respiratory pathogens: Haemophilus influenzae, Mycobacterium tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Chlamydia pneumoniae, Streptococcus pneumoniae, Legionella pneumophila, genital mycobacteria, adenovirus, and Candida auris. The results are as follows: Figure 2As shown, only type I MP strain M129 and type II MP strain FH exhibited high-intensity positive fluorescent signals; specifically, the FAM signal copy number of M129 was 77,283.3, and the VIC signal copy number of FH was 172,949.9; all other strains showed negative results. Furthermore, for strains M129 and FH, over 30,000 droplets and over 3,000 positive threshold events were generated, effectively excluding interference from non-target bacterial species and environmental factors. These results demonstrate that the multiplex ddPCR detection using the kit of this invention has high detection specificity.

[0052] To evaluate the sensitivity of the above-mentioned multiplex ddPCR detection method, we performed 10-fold serial dilution tests on the DNA of type I MP strains, type II MP strains, macrolide-sensitive MP strains, and macrolide-resistant MP strains under previously optimized experimental conditions. The original DNA concentration was approximately 1 ng / μL. The results are as follows: Figure 3 As shown, the lowest detectable concentrations are as follows: 11.3 copies / reaction for type I, 482.5 copies / reaction for type II, 20.7 copies / reaction for macrolide-sensitive strains, and 35.2 copies / reaction for macrolide-resistant strains. This result indicates that the detection method has extremely high sensitivity, with a lowest detectable concentration of only about 11 copies / reaction.

[0053] Furthermore, regression analysis was performed on the above test results, and the results are as follows: Figure 4 As shown in the figure; this figure shows that the detection method is effective in various dilution series at 10... -6 A strong linear relationship was observed in the DNA concentration range up to 1 ng / μL, which confirms the reliability of the detection method of the present invention (i.e., it can guarantee the stability and reproducibility of quantitative results within this concentration range).

[0054] Furthermore, the regression analysis results show that the detection method exhibits high correlation coefficients (R² values) for all the aforementioned strains: 0.9938 for type I, 0.9979 for type II, 0.9979 for susceptible strains, and 0.9958 for drug-resistant strains (see...). Figure 4 The correlation coefficients mentioned above are all close to 1, which indicates that the correspondence between the content of the target substance in the sample and the detection signal is quite stable, which further supports the reliability of the detection method.

[0055] Example 5: Establishment of a real-time quantitative PCR detection method based on the kit of the present invention In this embodiment, using the kit of the present invention, a multiplex real-time quantitative PCR detection method for simultaneously performing typing of Mycoplasma pneumoniae and macrolide resistance analysis was established. The specific detection method is as follows: 1. Sample processing and DNA extraction, refer to Example 3; 2. Real-time fluorescence PCR detection and analysis The 20 μL real-time fluorescence PCR reaction system is prepared as follows: 2 μL of DNA template; Super Real PreMix 10μL; Primer-probe mixture (with an initial concentration of 10 μM for each primer and probe solution, the amounts of each primer and probe added are as follows: 0.4 μL of primer MP-IF, 0.4 μL of primer MP-II-F, 0.4 μL of primer MP-IR, 0.4 μL of primer MP-II-R, 0.4 μL of probe MP-I-Probe, 0.4 μL of probe MP-II-Probe; 0.28 μL of primer 2063-F, 0.28 μL of primer 2063-R, 0.2 μL of probe 2063-Probe-1, and 0.36 μL of probe 2063-Probe-2). Add ultrapure water to a final volume of 20 μL.

[0056] The PCR reaction conditions are as follows: Initial denaturation at 95℃ for 20 seconds; followed by 40 cycles, each cycle consisting of denaturation at 95℃ for 1 second and annealing / extending at 58-62℃ for 20 seconds.

[0057] Example 6: Sensitivity and Specificity of Real-Time Fluorescent PCR Detection Method To evaluate the specificity of the real-time fluorescence PCR detection method established in Example 5, we tested a group of common clinical respiratory pathogens: Haemophilus influenzae, Mycobacterium tuberculosis, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Chlamydia pneumoniae, Streptococcus pneumoniae, Legionella pneumophila, genital mycobacteria, adenovirus, and Candida auris. The results are as follows: Figure 5 As shown in the figure, only type I MP strain M129 and type II MP strain FH exhibited high-intensity positive fluorescence signals, while all other strains showed negative results. These results demonstrate that the real-time fluorescent PCR detection using the kit of this invention has high detection specificity.

[0058] To evaluate the sensitivity of the above-mentioned real-time quantitative PCR method, we performed a 10-fold serial dilution test on the DNA of type I and type II MP strains (both sensitive to macrolide drugs) under previously optimized experimental conditions, with the original DNA concentration being approximately 1 ng / μL. The results are as follows: Figure 6As shown in the figure, the limit of detection (LOD) can reach 70 copies / μL for type I MP strains and 50 copies / μL for type II MP strains. This result demonstrates that the detection method has extremely high sensitivity, with a minimum detection concentration of only about 50 copies / μL.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nucleic acid composition for detecting Mycoplasma pneumoniae infection and / or its typing, characterized in that, The nucleic acid composition includes: (1) Primer pairs and their detection probe MP-I-Probe for specifically amplifying the Mycoplasma pneumoniae type I specific fragment MP-I, wherein MP-I is a gene fragment whose nucleotide position relative to the genome of the MP standard strain M129 is between positions 177739 and 178529; and / or, (2) Primer pairs and detection probes for specifically amplifying the MP-II specific fragment of Mycoplasma pneumoniae type II, wherein the MP-II is a gene fragment whose nucleotide position relative to the FH genome of the MP standard strain is between positions 703624 and 709505.

2. The nucleic acid composition according to claim 1, characterized in that, The primer pair for specific amplification of MP-I includes an upstream primer MP-IF and a downstream primer MP-IR, wherein the nucleotide sequence of the upstream primer MP-IF is shown in SEQ ID NO:1, the nucleotide sequence of the downstream primer MP-IR is shown in SEQ ID NO:2, and the nucleotide sequence of its detection probe MP-I-Probe is shown in SEQ ID NO:

3. And / or, the primer pair for specific amplification of MP-II includes an upstream primer MP-II-F and a downstream primer MP-II-R, wherein the nucleotide sequence of the upstream primer MP-II-F is shown in SEQ ID NO:4, the nucleotide sequence of the downstream primer MP-II-R is shown in SEQ ID NO:5, and the nucleotide sequence of its detection probe MP-II-Probe is shown in SEQ ID NO:

6.

3. The nucleic acid composition according to claim 2, characterized in that, The nucleic acid composition further includes: (3) Primer pairs 2063-F and 2063-R and detection probes 2063-Probe-W and 2063-Probe-M for specific amplification and detection of the A2063G mutation in the 23S rRNA gene of Mycoplasma pneumoniae, wherein the nucleotide sequence of primer 2063-F is shown in SEQ ID NO:7, the nucleotide sequence of primer 2063-R is shown in SEQ ID NO:8, the nucleotide sequence of detection probe 2063-Probe-W is shown in SEQ ID NO:9, and the nucleotide sequence of detection probe 2063-Probe-M is shown in SEQ ID NO:

10.

4. The nucleic acid composition according to any one of claims 1-3, characterized in that, Each detection probe has a different fluorescent reporter group labeled at its 5' end and a fluorescent quencher group labeled at its 3' end.

5. The nucleic acid composition according to claim 4, characterized in that, The fluorescent reporter group is selected from: FAM, VIC, Cy5, ROX, JOE, TET, HEX and Cy3; And / or, the fluorescence quenching group is selected from: TAMRA, MGB, BHQ1 and BHQ2.

6. Use of the nucleic acid composition according to any one of claims 1-5 in the preparation of a kit for any of the following applications: (1) To detect whether the subject is infected with Mycoplasma pneumoniae; (2) Detecting the type of Mycoplasma pneumoniae infecting the subjects; and, (3) Detecting whether the Mycoplasma pneumoniae infected in the subjects has drug-resistant mutations, among which, The drug resistance mutation is the A2063G mutation in the 23S rRNA gene.

7. A kit for detecting Mycoplasma pneumoniae infection, Mycoplasma pneumoniae typing, and / or its drug resistance mutations, characterized in that, The kit comprises the nucleic acid composition as described in any one of claims 1-5.

8. The reagent kit according to claim 7, characterized in that, The kit also includes positive control, negative control, and reaction reagents and buffers required for RT-PCR and / or digital PCR.

9. A detection device for detecting Mycoplasma pneumoniae infection, Mycoplasma pneumoniae typing, and / or its drug resistance mutations, characterized in that, The detection device comprises the nucleic acid composition as described in any one of claims 1-5.

10. A method for detecting whether a sample contains Mycoplasma pneumoniae, the typing of the contained Mycoplasma pneumoniae, and / or its drug resistance mutations, for non-disease diagnostic purposes, characterized in that, The method includes: detecting the DNA of the sample to be tested using the nucleic acid composition as described in any one of claims 1-5, the kit as described in claim 7 or 8, or the detection device as described in claim 9.

Images