Primer group for detecting mycoplasma pneumoniae and drug-resistant mutation of mycoplasma pneumoniae and application of primer group

Abstract

The invention discloses a primer group for detecting mycoplasma pneumoniae and drug-resistant mutation thereof and application of the primer group, and belongs to the technical field of gene detection. The primer group is designed aiming at the A / G variation site of the 2063rd site of an MP 23S rRNA conserved region, and specifically comprises an MP primer pair and a drug-resistant mutation primer pair thereof. The kit prepared by the invention comprises the primer group, and further comprises heat-resistant RNase H2 enzyme and in-vitro immune nucleic acid lateral chromatography test paper. According to the application, the kit is combined with an rhPCR-NALF technology, high-resolution detection of MP and drug-resistant mutation thereof can be realized through two-tube reaction and one-time detection, then visualization of the detection result is realized by combining a lateral chromatography technology, and result interpretation and analysis are more efficient and convenient. The invention aims to provide a rapid detection method for MP and drug-resistant mutation thereof with high sensitivity and specificity, so as to promote clinical precise diagnosis and treatment.

Description

Technical Field

[0001] This application belongs to the technical field of gene detection, and specifically relates to a primer set for detecting Mycoplasma pneumoniae and its drug-resistant mutations and its application. Background Art

[0002] Mycoplasma pneumoniae (M. pneumoniae, MP) is a pathogenic microorganism that can survive independently between bacteria and viruses. It is one of the most common pathogens causing community-acquired pneumonia (Mycoplasma pneumoniae pneumonia) in preschool children, with severe cough and repeated fever as the main clinical symptoms. The infection of MP has now become a prominent factor affecting children's respiratory health. With the development and widespread use of antibiotics, the infection of MP resistant to macrolide antibiotics in the domestic children's population has become more common. The infection rate of resistant strains is as high as over 90% in regions such as Beijing and Shanghai, bringing great inconvenience to the corresponding diagnosis and treatment process. Therefore, it is urgent to strengthen the synchronous detection of MP and its drug-resistant mutations.

[0003] In current clinical diagnosis and treatment, most use the fluorescence PCR method. By adding different primers and fluorescent probes to the reaction system, multiplex fluorescence detection of MP and its drug-resistant mutations can be achieved. For example, CN118792429A, CN112458194A, and CN113462794A have successively disclosed the use of the fluorescence PCR method to achieve rapid and accurate detection of MP and its drug-resistant mutations by designing specific primers and probes. Therefore, the fluorescence PCR method has always been recognized as the "gold standard" for clinical diagnosis. However, this method has high requirements for reagent research and development, the detection process is highly dependent on precision instruments, and it also has high requirements for the result interpretation ability of the detection personnel. Therefore, it is difficult to be widely carried out clinically. Another example is that CN118600050A discloses a method for detecting MP drug-resistant mutations by combining multiplex PCR and sequencing. By connecting the Barcode primer with the specific primer, the PCR amplification products can be directly mixed and sequenced for multiple samples, and then the mutation situation of all sequence information in a specific region of MP 23sRNA is analyzed to achieve accurate and comprehensive detection of macrolide-resistant MP. Although this method has a large detection throughput and comprehensive coverage, the cost of sequencing all samples is relatively high and it is not suitable for the detection in primary medical care and emergency departments. Another example is that CN118792429A discloses a method for amplifying a gene fragment containing a drug-resistant mutation site by using the LAMP method. In the first step, the isothermal amplification is used to confirm that the sample to be detected is MP positive, and then in the second step, a laser instrument is used to detect the fluorescence of the DNA silver nanoclusters formed by the positive amplification products. According to the quenching and weakening conditions, the MP wild type and mutant types are distinguished. This method gets rid of the dependence on expensive instruments such as PCR instruments, but the detection process is relatively cumbersome and time-consuming, and the detection cycle for multiple samples is relatively long, which may cause delays in diagnosis and treatment.

[0004] Therefore, in view of the above deficiencies in the existing detection methods for MP and its drug-resistant mutations, there is an urgent need for a detection method for MP and its drug-resistant mutations that is accurate, rapid, low-cost, easy to operate, and has low operation requirements, so as to achieve the purpose of early diagnosis, early treatment, and early recovery of MP and its drug-resistant mutations. Summary of the Invention

[0005] 1. Problems to be Solved

[0006] In view of the deficiencies in the existing detection of MP and its drug-resistant mutations, the present application provides a precise and rapid detection method with high specificity, strong sensitivity, and simple operation based on the rhPCR-NALF technology. Through only two-tube reactions and one detection, the visual interpretation and analysis of the conditions of MP and its drug-resistant mutations can be synchronously achieved.

[0007] 2. Technical Solutions

[0008] To solve the above problems, the technical solutions adopted in the present application are as follows:

[0009] The present application provides a primer set for detecting MP and its drug-resistant mutations. The primer set includes an MP primer pair and a drug-resistant mutation primer pair, where:

[0010] The MP primer pair includes Primer-F-wild-FAM and Primer-R-Biotin, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.3 respectively;

[0011] The drug-resistant mutation primer pair includes Primer-F-mutant-FAM and Primer-R-Biotin, and their nucleotide sequences are shown in SEQ ID NO.2 and SEQ ID NO.3 respectively.

[0012] Furthermore, the 5'-end of the above-mentioned Primer-F-wild-FAM is modified with a FAM small molecule antigen, and an adenosine ribonucleotide / rA / is inserted at a position 6 bases away from the 3'-end, and the 3'-end of the primer is modified with a C3 Spacer for blocking; the 5'-end of the above-mentioned Primer-F-mutant-FAM is modified with a FAM small molecule antigen, and a guanosine ribonucleotide / rG / is inserted at a position 6 bases away from the 3'-end, and the 3'-end is modified with a C3 Spacer for blocking; the 5'-end of the above-mentioned Primer-R-Biotin is modified with a Biotin small molecule antigen, and a guanosine ribonucleotide / rG / is inserted at the 6th base position away from the 3'-end, and the 3'-end is modified with a C3 Spacer for blocking. In this application, the inserted adenosine ribonucleotide / rA / corresponds to the base at the MP 23S rRNA A2063G mutation site, and the inserted guanosine ribonucleotide / rG / is homologous to the target gene sequence. In the sequence, / rA / is different from A. The former represents adenosine ribonucleotide, and the latter represents adenosine deoxyribonucleotide. The analysis of / rG / different from G is the same.

[0013] The present application also provides the use of the above primer set in the preparation of a reagent for detecting MP and its drug-resistant mutations.

[0014] The present application also provides a reagent for detecting MP and its drug-resistant mutations, and the reagent includes the above primer set.

[0015] The present application also provides a kit for detecting MP and its drug-resistant mutations, and the kit includes the above primer set or reagent.

[0016] Furthermore, the above kit also includes RNase H2 enzyme and an in vitro immune nucleic acid lateral flow test strip.

[0017] The present application also provides the use of the above reagent or kit in detecting MP and its drug-resistant mutations, and the use includes the following steps:

[0018] S1: Obtain the DNA of the sample to be tested;

[0019] S2: Using the above DNA as a template, perform rhPCR amplification respectively with the MP primer pair and the drug-resistant mutation primer pair in the above primer set to obtain rhPCR amplification products;

[0020] S3: Perform visual detection of the above amplification products on an in vitro immune nucleic acid lateral flow test strip;

[0021] S4: Judge whether there is MP or its drug-resistant mutation according to the visual result;

[0022] In the above S2, the rhPCR amplification is divided into two groups: one group uses the above MP primer pair for amplifying MP; the other group uses the above drug-resistant mutation primer pair for amplifying MP drug resistance mutation; the two amplifications are carried out in two tubes. The components in the tubes are the same except that the forward primers Primer-F-wild-FAM and Primer-F-mutant-FAM are different. After the amplification reaction is initiated, only when the forward modified primer accurately recognizes the MP 23S rRNA 2063 mutation site, the RNase H2 enzyme can cleave the corresponding modified RNA base, enabling the forward and reverse primers to break away from the blocking effect of the C3 Spacer and obtain the ability to extend under the action of the hot-start Taq enzyme, thus initiating the rhPCR amplification reaction. Finally, the ends of the nucleic acid amplification product target DNA will be respectively labeled with FAM and Biotin small molecule antigens;

[0023] In the visual detection of the nucleic acid test strip in the above S3, Biotin-BSA is coated on the test strip quality control line (C line), and anti-FAM antibody is coated on the test line (T line); the amplification product is mixed with streptavidin (SA)-labeled red polystyrene microspheres (SA-red microspheres). The target DNA with Biotin label will bind to the SA-red microspheres in large quantities and stably, forming a FAM-target DNA-Biotin-SA-red microsphere complex; when the mixed solution is added for detection, the anti-FAM antibody pre-coated on the test line can intercept and bind the FAM-target DNA-Biotin-SA-red microsphere complex in the mixed solution and show red; the Biotin-BSA pre-coated on the quality control line can intercept and bind the free SA-red microspheres in the mixed solution and show red; if the Primer-F-wild-FAM tube shows a positive reaction and Primer-F-mutant-FAM is negative, it indicates that there is a positive MP pathogen and no mutation occurs at the 23S rRNA 2063 site; if the Primer-F-wild-FAM (wild) tube shows a negative reaction and Primer-F-mutant-FAM is positive, it indicates that there is a positive MP pathogen and a mutation occurs at the 23S rRNA 2063 site; if the Primer-F-wild-FAM tube shows a negative reaction and Primer-F-mutant-FAM is also negative, it indicates that the MP pathogen is negative.

[0024] Further, in the above S2, the concentration of RNase H2 enzyme in the rhPCR amplification system is 0 - 1U.

[0025] Further, in the above S2, the concentration of RNase H2 enzyme in the rhPCR amplification system is 0.75U.

[0026] Further, in the above S2, the annealing temperature of the rhPCR amplification reaction is 50°C - 60°C.

[0027] Further, the annealing temperature of the rhPCR amplification reaction in S2 above is 55.5 °C.

[0028] Further, the above rhPCR amplification system includes: 2 μL of 10×Reaction Buffer, 0.5 μL of dNTP Mix (25 mM each), 1 μL of Primer-F-FAM (10 μM), 1 μL of Primer-R-Biotin (10 μM), 0.5 μL of Hotstart J-Taq DNA polymerase (5 U / μL), 0.375 μL of RNase H2 (2 U / μL), 1 μL of EveGreen (20× in water), H 2 O 11.625 μL, 2 μL of DNA Template, and the total volume is 20 μL.

[0029] Further, the above rhPCR amplification program is set as follows: Preheating: 95 °C, 5 min; Denaturation: 95 °C, 15 sec; Annealing and Extension: 55.5 °C, 40 sec, for a total of 40 cycles.

[0030] This application also provides a method for detecting MP and its drug-resistant mutations, and this method includes the following steps:

[0031] S1: Obtain the DNA of the sample to be tested;

[0032] S2: Using the above DNA as a template, perform rhPCR amplification respectively with the MP primer pair and the drug-resistant mutation primer pair in the above primer group to obtain rhPCR amplification products;

[0033] S3: Perform visual detection of the above amplification products on an in vitro immune nucleic acid lateral flow test strip;

[0034] S4: Judge whether there is MP or its drug-resistant mutation according to the visual result;

[0035] In the above S2, the rhPCR amplification is divided into two groups: one group uses the above MP primer pair for amplifying MP; the other group uses the above drug-resistant mutation primer pair for amplifying MP drug resistance mutation. The two groups of amplification are carried out in two tubes. Except that the forward primers Primer-F-wild-FAM and Primer-F-mutant-FAM are different, the other components in the tubes are the same. After the amplification reaction is initiated, only when the forward modified primer accurately recognizes the MP 23S rRNA 2063 mutation site, the RNase H2 enzyme can cleave the corresponding modified RNA base, enabling the forward and reverse primers to break away from the blocking effect of the C3 Spacer and obtain the ability to extend under the action of the hot-start Taq enzyme, thus initiating the rhPCR amplification reaction. Finally, the nucleic acid amplification product, the target DNA, will be respectively labeled with FAM and Biotin small molecule antigens at both ends;

[0036] In the visual detection of the nucleic acid test strip in the above S3, Biotin-BSA is coated on the quality control line (C line) of the test strip, and the anti-FAM antibody is coated on the test line (T line); the amplification product is mixed with streptavidin (SA)-labeled red polystyrene microspheres (SA-red microspheres). The target DNA with Biotin label will bind to the SA-red microspheres in large quantities and stably, forming a FAM-target DNA-Biotin-SA-red microsphere complex; when the mixed solution is added for detection, the anti-FAM antibody pre-coated on the test line can intercept and bind the FAM-target DNA-Biotin-SA-red microsphere complex in the mixed solution and show red; the Biotin-BSA pre-coated on the quality control line can intercept and bind the free SA-red microspheres in the mixed solution and show red; if the Primer-F-FAM(wild) tube shows a positive reaction and Primer-F-FAM(mutant) is negative, it indicates that there is a positive MP pathogen and no mutation occurs at the 23SrRNA2063 site; if the Primer-F-FAM(wild) tube shows a negative reaction and Primer-F-FAM(mutant) is positive, it indicates that there is a positive MP pathogen and a mutation occurs at the 23S rRNA 2063 site; if the Primer-F-FAM(wild) tube shows a negative reaction and Primer-F-FAM(mutant) is also negative, it indicates that the MP pathogen is negative.

[0037] Furthermore, in the above S2, the concentration of RNase H2 enzyme in the rhPCR amplification system is 0 - 1U.

[0038] Furthermore, in the above S2, the concentration of RNase H2 enzyme in the rhPCR amplification system is 0.75U.

[0039] Furthermore, in the above S2, the annealing temperature of the rhPCR amplification reaction is 50°C - 60°C.

[0040] Further, the annealing temperature of the rhPCR amplification reaction in S2 above is 55.5 °C.

[0041] Further, the above rhPCR amplification system includes: 2 μL of 10×Reaction Buffer, 0.5 μL of dNTP Mix (25 mM each), 1 μL of Primer-F-FAM (10 μM), 1 μL of Primer-R-Biotin (10 μM), 0.5 μL of Hotstart J-Taq DNA polymerase (5 U / μL), 0.375 μL of RNase H2 (2 U / μL), 1 μL of EveGreen (20× in water), 11.625 μL of H2O, and 2 μL of DNA Template, with a total volume of 20 μL.

[0042] Further, the above rhPCR amplification program is set as follows: preheating: 95 °C, 5 min; denaturation: 95 °C, 15 sec; annealing and extension: 55.5 °C, 40 sec, for a total of 40 cycles.

[0043] 3. Beneficial effects

[0044] Compared with the prior art, the beneficial effects of this application are as follows:

[0045] (1) This application provides a primer set for detecting MP and its drug-resistant mutations and its application. For the first time, in combination with the rhPCR amplification technology, the primer set is modified such as RNA bases and C3 Spacer blocking according to its amplification principle. Through precise complementary pairing with the target sequence, the RNase H2 enzyme cuts the RNA base away from the C3 Spacer blocking, starting the amplification reaction. The modified primer set adopted in this application improves the specificity and sensitivity of detecting MP and its drug-resistant mutations.

[0046] (2) This application provides a primer set for detecting MP and its drug-resistant mutations and its application. In this application, primer pairs for the wild type and mutant type of MP are respectively designed for the A / G mutation site at the 2063rd position in the conserved region of MP23S rRNA. The two use a common reverse primer. The detection of MP can be achieved with two-tube reactions, and at the same time, it can be judged whether it is a drug-resistant MP infection. The whole operation is convenient and efficient, and it can promote the accurate diagnosis and treatment of MP and its drug-resistant mutant types clinically.

[0047] (3) This application provides a primer set for detecting MP and its drug-resistant mutations and its application. In this application, combined with the NALF technology (nucleic acid lateral flow chromatography technology), the rhPCR amplification results of MP and its drug-resistant mutations are interpreted and analyzed on a lateral flow test strip, realizing the visualization of the detection results. The whole process does not rely on other precision instruments, has low technical operation requirements and low cost investment, and is suitable for outpatient and emergency departments and primary medical units. Description of the Drawings

[0048] Figure 1 It is an amplification curve graph of DNA amplification with wild-type and mutant plasmids at the 2063 site of MP 23S rRNA as the template.

[0049] Figure 2 It is a melting curve graph of DNA amplification with wild-type and mutant plasmids at the 2063 site of MP 23S rRNA as the template.

[0050] Figure 3 It is the specificity verification of the detection method for the 2063 site of MP 23S rRNA.

[0051] Figure 4 It is an rhPCR amplification curve graph at an annealing temperature of 52.5°C.

[0052] Figure 5 It is an rhPCR amplification curve graph at an annealing temperature of 54.0°C.

[0053] Figure 6 It is an rhPCR amplification curve graph at an annealing temperature of 55.5°C.

[0054] Figure 7 It is an rhPCR amplification curve graph at an annealing temperature of 57.0°C.

[0055] Figure 8 It is an rhPCR amplification curve graph at an annealing temperature of 58.5°C.

[0056] Figure 9 It is an rhPCR amplification curve graph at an annealing temperature of 60.0°C.

[0057] Figure 10 It is an rhPCR amplification curve graph under different concentrations of RNase H enzyme.

[0058] Figure 11It is a schematic structural diagram of an in vitro immune nucleic acid lateral flow test strip (the upper figure is a three-dimensional structural diagram: from left to right are the sample application hole, the test line T line, and the control line C line; the lower figure is a main view sectional structural diagram: the bottom layer is a PVC bottom plate, and the upper layer from left to right is a glass fiber membrane, an NC membrane, and a blotting paper. The FAM antibody is coated at the test line position of the NC membrane, and biotinylated bovine serum albumin is coated at the control line position).

[0059] Figure 12 It is a visualization test strip diagram of the amplification results of wild-type and mutant plasmids at the MP 23S rRNA 2063 site. Detailed implementation manners

[0060] The following further describes the present application in combination with specific embodiments.

[0061] It should be noted that the terms such as "upper", "lower", "left", "right", "middle", etc. cited in this specification are only for the convenience of clear narration and are not used to limit the scope of implementation. The change or adjustment of their relative relationship, without substantial change in the technical content, should also be regarded as the scope where the present application can be implemented.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which this application belongs; the term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0063] For those not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0064] As used herein, the term "about" is used to provide flexibility and imprecision associated with a given term, measurement, or value. Those skilled in the art can easily determine the degree of flexibility of a specific variable.

[0065] As used herein, the term "at least one of..." is intended to be synonymous with "one or more of...". For example, "at least one of A, B, and C" clearly includes only A, only B, only C, and their respective combinations.

[0066] Concentrations, amounts, and other numerical data may be presented herein in a range format. It should be understood that such range formats are used merely for convenience and brevity and should be interpreted flexibly as including not only the numerical values explicitly recited as the limits of the range, but also all individual numerical values or sub-ranges subsumed within that range as if each numerical value and sub-range were explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted as including not only the recited limits of 1 to about 4.5, but also the individual numbers (such as 2, 3, 4) and sub-ranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5", which should be interpreted as including all of the above values and ranges. In addition, this interpretation should apply regardless of the breadth of the range or feature being described.

[0067] Example 1

[0068] This example provides the primer pair design for MP and its drug-resistant mutations and the construction of wild-type and mutant plasmids at the 2063 site of MP 23S rRNA.

[0069] (1) Primer pair design for MP and its drug-resistant mutations

[0070] In this example, based on the conserved sequence of MP 23S rRNA in the GenBank database of the National Center for Biotechnology Information (NCBI) in the United States, a specific primer set was designed for the A / G mutation site at the 2063 site. This primer set includes two pairs of primer pairs, namely the MP primer pair Primer-F-wild-FAM, Primer-R-Biotin and its drug-resistant mutation primer pair Primer-F-mutant-FAM, Primer-R-Biotin; the forward and reverse primers in the above primer pairs were subjected to a closed and cleavable modification, and the specific modification is as follows:

[0071] Modification of the forward primer: The 5' end of the primer was modified with a FAM small molecule antigen, a ribonucleotide corresponding to the MP23S rRNA A2063G mutation site was inserted at a position 6 bases away from the 3' end, and the 3' end of the primer was modified with a C3 Spacer for blocking; specifically, an adenine ribonucleotide / rA / was inserted at a position 6 bases away from the 3' end of Primer-F-wild-FAM, and a guanine ribonucleotide / rG / was inserted at a position 6 bases away from the 3' end of Primer-F-mutant-FAM. The nucleotide sequences of the modified forward primers are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively;

[0072] Modification of reverse primer: The 5'-end of the primer is modified with a small molecule antigen Biotin, and ribonucleotides homologous to the target gene sequence are inserted at a position 6 bases away from the 3'-end. Similarly, the 3'-end of the primer is modified with C3 Spacer for blocking; specifically, the reverse primer Primer-R-Biotin in the above two primer pairs is a common primer with the same nucleotide sequence, and guanine ribonucleotide / rG / is inserted at a position 6 bases away from the 3'-end. The nucleotide sequence of the modified reverse primer is shown in SEQ ID NO.3. The above primers were synthesized by Suzhou Hongxun Biotechnology Co., Ltd. The specific nucleotide sequences of the primers involved in this application are shown in Table 1 below.

[0073] Table 1

[0074]

[0075] (2) Construction of wild-type and mutant plasmids at the 2063 site of MP 23S rRNA.

[0076] In this example, the construction of wild-type and mutant plasmids at the 2063 site of MP 23S rRNA was completed by Suzhou Hongxun Biotechnology Co., Ltd., and specifically included the following steps:

[0077] S1. Construction of wild-type and mutant MP plasmids: According to the conserved sequence of MP 23S rRNA in the GenBank database of the National Center for Biotechnology Information (NCBI) in the United States, Suzhou Hongxun Biotechnology Co., Ltd. chemically synthesized and ligated it to the pMD8-T vector and stored it at -20°C.

[0078] S2. PCR verification: The composition of the PCR system is as follows: 10 μL of qPCR SYBR Green Master Mix, 1 μL of Primer-F-fragment (10 μM), 1 μL of Primer-R-fragment (10 μM), H 2 O 6 μL, 2 μL of DNA Template; The PCR reaction program is set as follows: Preheating: 95°C, 5 min; Denaturation: 95°C, 10 sec; Annealing: 56°C, 20 sec; Extension: 72°C, 20 sec, 40 cycles. After the amplification is completed, it is the melting curve reaction stage.

[0079] S3. Result analysis: Using the diluted wild-type and mutant plasmids as templates, amplification was carried out using the conditions in S2 and the specific primers shown in SEQ ID NO.4 and SEQ ID NO.5. The results are as Figure 1 shown, and typical positive amplification curves can be observed. At Figure 2Subtle differences between the wild-type and mutant plasmids can also be observed in the melting curve, indicating that the wild-type and mutant plasmids targeting the MP 23S rRNA 2063 site were successfully constructed and can be used in subsequent related experiments.

[0080] Example 2

[0081] This example provides the specificity verification of the rapid detection method for MP and its drug-resistant mutations, including the following steps.

[0082] (1) Obtaining template DNA

[0083] Select the wild-type plasmid and mutant plasmid at the MP 23S rRNA 2063 site constructed in Example 1. After diluting 100-fold with DEPC water, they are used as templates for the rhPCR amplification reaction respectively.

[0084] (2) Designing primer pairs with different RNA base modifications

[0085] In this example, based on the two forward primers Primer-F-wild-FAM and Primer-F-mutant-FAM in Example 1, two additional forward primers are designed. The difference is that cytosine ribonucleotide / rC / and thymine ribonucleotide / rT / are inserted and modified at the 6-base position at the 3' end of the forward primers in Example 1. The four forward primers are all removed the modification of the FAM small molecule antigen at the 5' end; the reverse primer is different from the reverse primer in Example 1 by removing the modification of the Biotin small molecule antigen at the 5' end, and the others remain unchanged. The specific nucleotide sequences of the primers used are as follows:

[0086] Forward primers:

[0087] rA: 5'-GTTAGGCGCAACGGGACGG / rA / AAGAT-C3spacer-3' (SEQ ID NO.1 without FAM);

[0088] rG: 5'-GTTAGGCGCAACGGGACGG / rG / AAGAT-C3spacer-3' (SEQ ID NO.2 without FAM);

[0089] rC: 5'-GTTAGGCGCAACGGGACGG / rC / AAGAT-C3spacer-3' (SEQ ID NO.6);

[0090] rT: 5'-GTTAGGCGCAACGGGACGG / rT / AAGAT-C3spacer-3' (SEQ ID NO.7).

[0091] Reverse primer:

[0092] 5’-GTCCTAGCGAACTTGCATC / rG / ATTGT-C3 spacer-3’ (SEQ ID NO.3 without Biotin).

[0093] (3) rhPCR amplification reaction

[0094] Use the above 4 groups of primers to amplify the wild-type and mutant plasmid templates of MP 23S rRNA 2063 DNA respectively. The plasmid template DNA used was diluted 100-fold with DEPC water before amplification.

[0095] The composition of the rhPCR amplification reaction system is as follows: 10×Reaction Buffer 2 μL, dNTP Mix (25 mM each) 0.5 μL, Primer-F-FAM (10 μM) 1 μL, Primer-R-Biotin (10 μM) 1 μL, Hotstart J-Taq DNA polymerase 0.5 μL, RNase H2 (2 U / μL) 1 μL, EveGreen (20× in water) 1 μL, H 2 O 11 μL, DNA Template 2 μL, and the total volume is 20 μL. The relevant reaction reagents were all purchased from Yeasen Biotechnology (Shanghai) Co., Ltd.

[0096] The rhPCR amplification reaction program is set as follows:

[0097] Preheat: 95°C, 5 min; Denaturation: 95°C, 15 sec; Annealing and Extension: 55°C, 40 sec, for a total of 40 cycles.

[0098] (4) Result analysis

[0099] Perform agarose gel electrophoresis on the above rhPCR amplification products. The results are as Figure 3 shown. The MP primer pair and its drug-resistant mutant primer pair provided in this application successfully amplified the corresponding bands of MP and its drug-resistant mutants respectively. When the RNA base modification type in the primer was changed, no corresponding electrophoresis band was observed, that is, specific amplification was not achieved. This result indicates that the primer group designed in this application can detect MP and its drug-resistant mutants and has good specificity.

[0100] Example 3

[0101] This example provides the optimization of the annealing temperature in the rhPCR amplification reaction program.

[0102] In this embodiment, since the composition of the rhPCR amplification reaction system for MP and its drug-resistant mutants is the same for all components except that the forward primers Primer-F-wild-FAM and Primer-F-mutant-FAM are slightly different, the annealing temperature optimization experiment in the rhPCR reaction procedure was carried out using the amplification of MP as an example, which specifically includes the following steps:

[0103] (1) Template DNA acquisition

[0104] Select the wild-type plasmid at the 2063 site of MP 23S rRNA constructed in Example 1, and dilute it 100 times with DEPC water as the template for the rhPCR amplification reaction.

[0105] (2) Set the annealing temperature of the rhPCR amplification reaction

[0106] To ensure the best amplification result of rhPCR, in this embodiment, 52.5°C, 54°C, 55.5°C, 57°C, 58.5°C, and 60°C were selected respectively at 50°C - 60°C to explore the optimal annealing temperature.

[0107] (3) rhPCR amplification reaction

[0108] The composition of the rhPCR amplification reaction system is the same as that in Example 2;

[0109] The rhPCR amplification reaction procedure is set as follows:

[0110] Preheating: 95°C, 5 min; Denaturation: 95°C, 15 sec; Annealing and extension: 40 sec at the above-set temperature; A total of 40 cycles.

[0111] (4) Result analysis

[0112] The rhPCR amplification curves at different annealing temperatures are as Figures 4 to 9 shown. At the same time, combined with the CT values in Table 2 for joint analysis, it can be concluded that when the annealing temperature is 55.5°C, the amplification efficiency is the highest. Therefore, the annealing temperature in the rhPCR amplification reaction procedure is set to 55.5°C.

[0113] Table 2

[0114]

[0115]

[0116] Example 4

[0117] This embodiment provides the optimization of the RNase H2 enzyme concentration in the rhPCR amplification reaction system.

[0118] Similar to Example 3, taking the amplification of MP as an example, an optimization experiment on the concentration of RNase H2 enzyme in the rhPCR reaction system was carried out, which specifically included the following steps:

[0119] (1) The acquisition of template DNA was the same as in Example 3.

[0120] (2) Set the concentration of RNase H2 enzyme

[0121] To ensure the best amplification result of rhPCR, in this example, 0U, 0.25U, 0.5U, 0.75U, and 1U were respectively selected from 0 to 1U to explore the optimal concentration of RNase H2 enzyme.

[0122] (3) rhPCR amplification reaction

[0123] The composition of the rhPCR amplification reaction system was as follows:

[0124] 10×Reaction Buffer 2μL, dNTP Mix (25mM each) 0.5μL, Primer-F-FAM (10μM) 1μL, Primer-R-Biotin (10μM) 1μL, Hotstart J-Taq DNApolymerase 0.5μL, corresponding concentration of RNaseH2 (2U / μL), EveGreen (20× in water) 1μL, DNA Template 2μL, and H 2 O was used to supplement the total volume of the system to 20μL. The relevant reaction reagents were all purchased from Yeasen Biotechnology (Shanghai) Co., Ltd.

[0125] The reaction program was set as follows:

[0126] Preheating: 95°C, 5 min; Denaturation: 95°C, 15 sec; Annealing and extension: 55.5°C, 40 sec, for a total of 40 cycles.

[0127] (4) Result analysis

[0128] The amplification curves at different concentrations of RNase H2 enzyme were as Figure 10 shown. When the concentration of RNase H2 enzyme in the reaction system was 0.75U, the amplification efficiency was the highest. Therefore, 0.75U RNase H2 enzyme was used in the rhPCR amplification reaction system.

[0129] Example 5

[0130] This example provides a visual interpretation and analysis of the rhPCR amplification result by an in vitro immune nucleic acid lateral flow test strip.

[0131] This example combines the nucleic acid lateral flow technology, such as Figure 11As shown in the schematic diagram of the front view sectional structure of the test strip, Biotin-BSA is coated on the control line Cline, and the anti-FAM antibody is coated on the test line T line. After the rhPCR nucleic acid amplification is completed, the amplification product is mixed with red polystyrene microspheres labeled with streptavidin (SA) (SA-red microspheres). The target DNA with Biotin label will bind to the SA-red microspheres in large quantities and stably, forming a FAM-target DNA-Biotin-SA-red microsphere complex. The mixed solution is dropped onto the chromatographic test strip. The anti-FAM antibody pre-coated on the test line can intercept and bind the FAM-target DNA-Biotin-SA-red microsphere complex in the mixed solution and show red; the Biotin-BSA pre-coated on the control line can intercept and bind the free SA-red microspheres in the mixed solution and show red. Therefore, under normal circumstances, the control line will show color, and only if there is an amplification of the target DNA fragment in the amplification product, the test line will show color.

[0132] Specifically, take 5 μL of the rhPCR amplification product and 5 μL of red polystyrene microspheres labeled with streptavidin (SA), mix them evenly and add 2 90 μL of H

[0133] O. Drop the mixed solution at the sample pad position of the nucleic acid lateral flow test strip and interpret the result within about 5 minutes. In this application, the rhPCR amplification reaction is carried out in two tubes. One tube uses the MP primer pair to amplify the wild type of MP 23S rRNA 2063; the other tube uses the drug-resistant mutant primer pair to amplify the mutant type of MP 23S rRNA 2063. The reaction components of the two tubes are the same except for the forward primers Primer-F-wild-FAM and Primer-F-mutant-FAM. If the Primer-F-wild-FAM tube shows a positive reaction and the Primer-F-mutant-FAM is negative, it indicates that there is a positive MP pathogen, and there is no mutation at the 23SrRNA 2063 site, and the patient is infected with non-macrolide-resistant Mycoplasma pneumoniae; if the Primer-F-wild-FAM tube shows a negative reaction and the Primer-F-mutant-FAM is positive, it indicates that there is a positive MP pathogen, and there is a mutation at the 23S rRNA2063 site, and the patient is infected with macrolide-resistant Mycoplasma pneumoniae; if the Primer-F-wild-FAM tube shows a negative reaction and the Primer-F-mutant-FAM is also negative, it indicates that the MP pathogen is negative and the patient is not infected with Mycoplasma pneumoniae. Figure 12As shown, the color development results of the test line of the test strip are analyzed accordingly according to the above situation, accurately corresponding to the determined wild-type and mutant plasmid standard samples of MP 23SrRNA 2063, indicating that the in vitro immune nucleic acid lateral flow test strip provided by this application can accurately and conveniently interpret the amplification results of MP and its drug-resistant mutations, and is very suitable for outpatient and emergency departments and primary medical units.

Claims

1. A primer set for detecting MP and its drug-resistant mutation, characterized in that: The primer set includes an MP primer pair and a drug-resistant mutation primer pair thereof, wherein: The MP primer pair includes Primer-F-wild-FAM and Primer-R-Biotin, and the nucleotide sequences thereof are shown in SEQ ID NO.1 and SEQ ID NO.3 respectively; The drug-resistant mutation primer pair includes Primer-F-mutant-FAM and Primer-R-Biotin, and the nucleotide sequences thereof are shown in SEQ ID NO.2 and SEQ ID NO.3, respectively.

2. The primer set according to claim 1, characterized in that The 5' end of the Primer-F-wild-FAM is modified with a FAM small molecule antigen, an adenine ribonucleotide / rA / is inserted at the 6 base positions away from the 3' end, and the 3' end is modified with a C3 Spacer for blocking; the 5' end of the Primer-F-mutant-FAM is modified with a FAM small molecule antigen, a guanine ribonucleotide / rG / is inserted at the 6 base positions away from the 3' end, and the 3' end is modified with a C3 Spacer for blocking; the 5' end of the Primer-R-Biotin is modified with a Biotin small molecule antigen, a guanine ribonucleotide / rG / is inserted at the 6th base position away from the 3' end, and the 3' end is modified with a C3 Spacer for blocking.

3. Use of the primer set according to claim 1 or 2 in the preparation of a reagent for detecting MP and its drug-resistant mutation.

4. A reagent for detecting MP and its drug-resistant mutation, characterized in that: The reagent comprises the primer set according to claim 1 or 2.

5. A kit for detecting MP and its drug-resistant mutation, characterized in that: The kit comprises the primer set according to claim 1 or 2, or the reagent according to claim 4.

6. The kit according to claim 5, characterized in that The kit also includes RNase H2 enzyme and in vitro immune nucleic acid lateral flow chromatography test paper.

7. Use of the reagent according to claim 4 and / or the kit according to claim 5 or 6 in detecting MP and its drug-resistant mutation, characterized in that: The following steps are involved: S1: Obtain the DNA of the sample to be tested; S2: using the DNA as a template, respectively using the MP primer pair and the drug-resistant mutation primer pair in the primer set to perform rhPCR amplification to obtain rhPCR amplification products; S3: visually detecting the amplified product on an in vitro immune nucleic acid lateral flow test paper; S4: Determine whether MP or its drug-resistant mutation exists based on the visualization results.

8. A method for detecting MP and its drug-resistant mutation, characterized in that: The method comprises the following steps: S1: Obtain the DNA of the sample to be tested; S2: using the DNA as a template, respectively using the MP primer pair and the drug-resistant mutation primer pair in the primer set to perform rhPCR amplification to obtain rhPCR amplification products; S3: visually detecting the amplified product on an in vitro immune nucleic acid lateral flow test paper; S4: Determine whether MP or its drug-resistant mutation exists based on the visualization results.

9. The use according to claim 7 or the detection method according to claim 8, characterized in that: In the reaction system of rhPCR amplification: The annealing temperature is 50°C to 60°C; and / or The concentration of RNase H2 enzyme is 0~1U.

10. The use according to claim 7 or the detection method according to claim 8, characterized in that: In the reaction system of rhPCR amplification: The annealing temperature is 55.5°C; and / or The concentration of RNase H2 enzyme was 0.75U.

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