Method, primer combination and kit for detecting p53 gene mutation based on nucleic acid flight mass spectrometry and application

Abstract

The invention discloses a method for detecting p53 gene mutation based on nucleic acid flight mass spectrometry, a primer combination, a kit and application, the primer combination comprises an amplification primer group and an extension primer group for p53 gene mutation detection, the amplification primer group comprises primer sequences as shown in SEQ ID NO.1-SEQ ID NO.14, and the extension primer group comprises primer sequences as shown in SEQ ID NO.15-SEQ ID NO.39. The invention further discloses a kit for detecting p53 gene mutation based on nucleic acid flight mass spectrometry. According to the primer combination disclosed by the invention, accurate detection on p53 gene mutation can be realized, meanwhile, the requirements of a mass spectrometric detection technology are met, and rapid and effective detection on p53 gene mutation of a to-be-detected sample by applying a MassARRAY system is realized. Meanwhile, the primer combination covers 47 mutation types in 25 SNP sites of the p53 gene, and the detection sites are more comprehensive. The method for detecting the p53 gene mutation by using the primer combination for detecting the p53 gene mutation, provided by the invention, also has the advantages of high accuracy, high sensitivity, good repeatability, low cost, short detection period, more comprehensive detection sites, intuitive result, no need of signal analysis and the like.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a method, primers, a kit and application for detecting p53 gene mutation based on nucleic acid flight mass spectrometry. Background Art

[0002] The human p53 gene is located at 17p13.1 on the short arm of chromosome 17. It is 20kb in length and consists of 11 exons and 10 introns. The transcription product of the p53 gene is 2.5kb mRNA, encoding a protein of 393 amino acids with a molecular weight of about 53kDa. The p53 gene is the most frequently mutated gene in human cancer (>50%), and p53 mutations can greatly increase the risk of cancer development. p53 plays a role in the regulation or progression of the cell cycle, apoptosis, and genomic stability through a variety of mechanisms. Wild-type p53 can promote the elimination of new cancer cells by inducing various forms of cell death or senescence, and these reactions help limit the ability of tumor development. p53 can also induce reversible cell cycle arrest when cells are damaged or stressed. Once the damage or stress is resolved, the cells can resume proliferation. When p53 mutations lead to functional loss, it not only loses its original tumor suppressor functions such as cell cycle arrest, induction of apoptosis, maintenance of genomic stability, and mismatch DNA repair. Moreover, p53 mutants may also play an important role in tumorigenesis and metastasis, promoting tumorigenesis.

[0003] The types of p53 mutations include gene fragment deletion, insertion, missense mutations caused by point mutations, and loss of heterozygosity. About 80% of the mutations are missense mutations caused by point mutations, mainly involving the part encoding the DBD domain of the p53 protein. In this region, 8 amino acid mutations (R175H, G245S, R248Q, R248W, R249S, R273H, R273S, and R282W) account for 27% of all mutant p53 proteins identified in human cancers and are called hotspot mutations. The mutated p53 can cause regulation disorders of the encoded product, cell differentiation disorders, uncontrolled growth, and even cancer.

[0004] Abnormal expression of p53 is associated with tumor metastasis, recurrence and poor prognosis, so the detection of p53 gene mutations in tumor patients is of great significance. At present, there are many methods for detecting p53 mutations, such as Sanger sequencing, PCR, high-throughput sequencing, etc. Among them, Sanger sequencing has high sample requirements, limited detection range, relatively low detection sensitivity, low throughput, and long detection time; PCR-based detection methods, such as ARMS-PCR, fluorescent quantitative PCR, digital PCR, etc., are simple and fast to operate, and data analysis is simple, but due to the limitation of throughput, they cannot meet the clinical detection needs of p53 multi-sites; and although high-throughput sequencing solves the limitation of throughput and can comprehensively detect p53 mutations, the experimental operation is cumbersome, the detection cycle is long, the cost is high, and the technical requirements for experimental operation and data analysis personnel are high. Therefore, there is an urgent need for an economical, fast, and easy-to-operate detection method. Summary of the invention

[0005] In view of this, the object of the present invention is to provide a method, primers, kit and application for detecting p53 gene mutation based on nucleic acid flight mass spectrometry.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a primer combination for detecting p53 gene mutation based on nucleic acid flight mass spectrometry, the primer combination comprising an amplification primer set and an extension primer set for detecting p53 gene mutation, the amplification primer set comprising primer sequences shown in SEQ ID NO.1-SEQ ID NO.14, and the extension primer set comprising primer sequences shown in SEQ ID NO.15-SEQ ID NO.39.

[0008] Preferably, the amplification primer set includes W1 Primer MIX, W2 Primer MIX and W3 Primer MIX, wherein W1 Primer MIX includes the primer sequence shown in SEQ ID NO.1-SEQ ID NO.10, W2 Primer MIX includes the primer sequences shown in SEQ ID NO.1-SEQ ID NO.6 and SEQ ID NO.11-SEQ ID NO.14, and W3PrimerMIX includes the sequence shown in SEQ ID NO.1-SEQ ID NO.6; the extension primer set includes W1 UEP MIX, W2 UEPMIX and W3 UEP MIX, wherein W1 UEP MIX includes the primer sequence shown in SEQ ID NO.15-SEQ ID NO.25, W2UEP MIX includes the primer sequence shown in SEQ ID NO.26-SEQ ID NO.34, and W3 UEP MIX includes the sequence shown in SEQ IDNO.35-SEQ ID NO.39.

[0009] The present invention also provides the use of the primer combination for detecting p53 gene mutation in preparing a p53 gene mutation detection product.

[0010] The present invention also provides a p53 gene mutation detection kit, which comprises the above-mentioned primer combination for detecting p53 gene mutation.

[0011] The present invention also provides a method for detecting p53 gene mutation using the above primer combination for detecting p53 gene mutation, the method comprising the following steps:

[0012] S1. Primer configuration: configure an amplification primer set and an extension primer set, wherein the amplification primer combination includes W1 PrimerMIX, W2 Primer MIX, and W3 Primer MIX, and the extension primer set includes W1 UEP MIX, W2 UEP MIX, and W3 UEPMIX; the specific information of the amplification primer set and the extension primer set is:

[0013]

[0014]

[0015]

[0016] S2. Multiplex PCR amplification reaction: prepare PCR mixture according to the number of test copies, add W1 Primer MIX, W2 Primer MIX and W3 Primer MIX to prepare W1, W2 and W3 reaction systems respectively, and dispense into 96-well plates; add the sample DNA to be tested into W1, W2 and W3 reaction systems respectively; seal the 96-well plate with a membrane, vortex and centrifuge, and then place the 96-well plate on a PCR instrument for PCR amplification reaction;

[0017] S3, SAP reaction: Use SAP enzyme to remove residual dNTPs in the PCR reaction system;

[0018] S4, extension reaction: prepare iPLEX extension mixture, add W1 UEP MIX, W2 UEP MIX and W3 UEPMIX to prepare iPLEX extension mixture of W1, W2 and W3 reaction systems respectively; add corresponding iPLEX extension mixture to each reaction well and mix; seal the 96-well plate with a membrane, vortex and centrifuge, and then place the 96-well plate on a PCR instrument for thermal cycling reaction to perform single base extension;

[0019] S5. Time-of-flight mass spectrometry detection: Add 41 μl ddH2O to each well of a 96-well plate with a sample, and then centrifuge. Use CPM to desalt the sample, spot it onto a chip and mass spectrometer for time-of-flight mass spectrometry detection to obtain data. Determine the mutation status of the corresponding site based on the peak position corresponding to the molecular weight of the product.

[0020] Preferably, in the S2 multiplex PCR amplification reaction, the PCR mixture is prepared as follows:

[0021]

[0022] The aliquot volume of the W1, W2, and W3 reaction systems was 3 μL per well, the concentration of the sample DNA to be tested was 5 ng / μL, and the added volume of the sample DNA to be tested was 2 μL;

[0023] The PCR amplification reaction conditions are:

[0024]

[0025] Preferably, in the S3 SAP reaction, the SAP reaction solution is prepared as follows:

[0026]

[0027] The SAP reaction conditions are as follows: add 2 μl of SAP mixture to the reaction wells of the 96-well plate, and the total volume after adding the SAP mixture is 7 μL; seal the 96-well plate with a membrane, vortex and centrifuge, and the centrifugation condition is: 4000 rpm for 5 seconds; put the 96-well plate on the PCR instrument for reaction, and the reaction conditions are:

[0028] temperature time Stage 1 37℃ 40min Stage 2 85℃ 5min Stage 3 4℃ Hold

[0029] Preferably, in the S4 extension reaction, the iPLEX extension mixture is prepared as follows:

[0030]

[0031] The volume of iPLEX extension mix added was 2 μl;

[0032] The extension reaction conditions are:

[0033]

[0034] The beneficial effects of the present invention are as follows: the present invention discloses a method, primers, kit and application for detecting p53 gene mutation based on nucleic acid flight mass spectrometry, wherein the primer combination includes an amplification primer set and an extension primer set for detecting p53 gene mutation, wherein the amplification primer set includes the primer sequence shown in SEQ ID NO.1-SEQ ID NO.14, and the extension primer set includes the primer sequence shown in SEQ ID NO.15-SEQ ID NO.39. The primer combination for detecting p53 gene mutation disclosed in the present invention can realize accurate detection of p53 gene mutation, and at the same time meet the requirements of mass spectrometry detection technology, and realize rapid and effective detection of p53 gene mutation of the sample to be tested using the MassARRAY system. At the same time, the primer combination for detecting p53 gene mutation disclosed in the present invention covers a total of 47 mutation types in 25 SNP sites of the p53 gene, which is more comprehensive than the detection sites of the traditional PCR method. The method for detecting p53 gene mutation using a primer combination for detecting p53 gene mutation provided by the present invention has the advantages of high accuracy, high sensitivity, good repeatability, low cost, short detection cycle, more comprehensive detection sites, intuitive results and no need for bioinformatics analysis, etc. compared with the detection methods currently on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the test result diagram of lung cancer tissue sample LC506.

[0036] Figure 2 This is the test result diagram of lung cancer tissue sample LC509.

[0037] Figure 3 This is the test result diagram of lung cancer tissue sample LC511.

[0038] Figure 4 This is the test result diagram of lung cancer tissue sample LC514.

[0039] Figure 5 This is the test result of LC520 in lung cancer tissue sample. DETAILED DESCRIPTION

[0040] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary illustrations and explanations of the present invention, and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are covered within the scope that the present invention is intended to protect. It should be noted that the experimental materials whose sources are not indicated in the embodiments of the present invention can all be obtained commercially, and the experimental methods in the embodiments of the present invention whose specific conditions are not indicated are usually carried out according to conventional experimental methods or according to the methods recommended by the experimental material manufacturers.

[0041] The present invention is based on nucleic acid flight mass spectrometry, and designs a primer combination for detecting 47 mutation types in 25 SNP sites of the p53 gene (Table 1), the primer combination includes an amplification primer set and an extension primer set, the 3' end of the amplification primer matches the target gene sequence, and the 5' end has a universal tag sequence of 10 bases (ACGTTGGATG). Specifically, the amplification primer set includes the primer sequence shown in SEQ ID NO: 1-SEQ ID NO: 14, and the extension primer set includes the primer sequence shown in SEQ ID NO: 15-SEQ ID NO: 39.

[0042] Table 1: 47 mutation types in 25 SNP sites of p53 gene

[0043]

[0044] The present invention also discloses a method for detecting p53 gene mutations using a designed primer combination for detecting 47 mutation types in 25 SNP sites of the p53 gene, the method comprising the following steps:

[0045] S1. Primer configuration: configure an amplification primer set and an extension primer set, wherein the amplification primer combination includes W1 PrimerMIX, W2 Primer MIX, and W3 Primer MIX, and the extension primer set includes W1 UEP MIX, W2 UEP MIX, and W3 UEPMIX; the specific information of the amplification primer set and the extension primer set is:

[0046]

[0047]

[0048] S2. Multiplex PCR amplification reaction: prepare PCR mixture according to the number of test copies, add W1 Primer MIX, W2 Primer MIX and W3 Primer MIX to prepare W1, W2 and W3 reaction systems respectively, and dispense into 96-well plates; add the sample DNA to be tested into W1, W2 and W3 reaction systems respectively; seal the 96-well plate with a membrane, vortex and centrifuge, and then place the 96-well plate on a PCR instrument for PCR amplification reaction;

[0049] S3, SAP reaction: Use SAP enzyme to remove residual dNTPs in the PCR reaction system;

[0050] S4, extension reaction: prepare iPLEX extension mixture, add W1 UEP MIX, W2 UEP MIX and W3 UEPMIX to prepare iPLEX extension mixture of W1, W2 and W3 reaction systems respectively; add corresponding iPLEX extension mixture to each reaction well and mix; seal the 96-well plate with a membrane, vortex and centrifuge, and then place the 96-well plate on a PCR instrument for thermal cycling reaction to perform single base extension;

[0051] S5. Time-of-flight mass spectrometry detection: Add 41 μl ddH2O to each well of a 96-well plate with a sample, and then centrifuge. Use CPM to desalt the sample, spot it onto a chip and mass spectrometer for time-of-flight mass spectrometry detection to obtain data. Determine the mutation status of the corresponding site based on the peak position corresponding to the molecular weight of the product.

[0052] Preferably, in the S2 multiplex PCR amplification reaction, the PCR mixture is prepared as follows:

[0053]

[0054] The aliquot volume of the W1, W2, and W3 reaction systems was 3 μL per well, the concentration of the sample DNA to be tested was 5 ng / μL, and the added volume of the sample DNA to be tested was 2 μL;

[0055] The PCR amplification reaction conditions are:

[0056]

[0057] Preferably, in the S3 SAP reaction, the SAP reaction solution is prepared as follows:

[0058]

[0059] The SAP reaction conditions are as follows: add 2 μl of SAP mixture to the reaction wells of the 96-well plate, and the total volume after adding the SAP mixture is 7 μL; seal the 96-well plate with a membrane, vortex and centrifuge, and the centrifugation condition is: 4000 rpm for 5 seconds; put the 96-well plate on the PCR instrument for reaction, and the reaction conditions are:

[0060] temperature time Stage 1 37℃ 40min Stage 2 85℃ 5min Stage 3 4℃ Hold

[0061] Preferably, in the S4 extension reaction, the iPLEX extension mixture is prepared as follows:

[0062]

[0063] The volume of iPLEX extension mix added was 2 μl;

[0064] The extension reaction conditions are:

[0065]

[0066] The present invention will be further described below with reference to specific embodiments.

[0067] Example 1 Detection of p53 mutation in human cell lines

[0068] (1) Extraction of DNA from human cell lines

[0069] QIAamp kit was used to extract DNA from human cell lines. The specific extraction method is as follows:

[0070] (1.1) Wash the cells with 1 mL of PBS and digest with 1 mL of trypsin for 5 min;

[0071] (1.2) Centrifuge at 800 rpm for 5 min and discard the supernatant;

[0072] (1.3) Add 180 μL Buffer ATL, mix well, and pipette into an EP tube;

[0073] (1.4) Add 20 μL proteinase K, mix well, and incubate at 55°C for 30 min until the cells are completely lysed; (1.5) Add 20 μL 20 mg / μL RNase A, shake and mix well for 15 s, and incubate at room temperature for 2-5 min;

[0074] (1.6) Add 200 μL Buffer AL, mix for 15 seconds, and incubate at 70°C for 10 minutes;

[0075] (1.7) Add 200 μL of anhydrous ethanol and mix for 15 seconds;

[0076] (1.8) The liquid was aspirated into the centrifugal column sleeve and centrifuged at 8000 rpm for 1 min;

[0077] (1.9) Discard the liquid in the collection tube, add 500 μL Buffer AW1, and centrifuge at 8000 rpm for 1 min;

[0078] (1.10) Discard the liquid in the collection tube, add 500 μL Buffer AW2, and centrifuge at 12000 rpm for 3 min; (1.11) Discard the liquid in the collection tube, centrifuge at 12000 rpm for 1 min;

[0079] (1.12) Transfer the column to a new EP tube, add 30 μL Buffer AE / ddH2O, and let stand at room temperature for 3 min;

[0080] (1.13) Centrifuge at 8000 rpm for 1 min. The DNA in the EP tube is extracted and the concentration is measured.

[0081] (2) Primer configuration

[0082] An amplification primer set and an extension primer set are configured, wherein the amplification primer combination includes W1 Primer MIX, W2 Primer MIX, and W3 Primer MIX, and the extension primer set includes W1 UEP MIX, W2 UEP MIX, and W3 UEP MIX; the specific configuration of the amplification primer set and the extension primer set is:

[0083]

[0084]

[0085] (3) Multiplex PCR amplification reaction

[0086] (3.1) Prepare PCR mixture (single test amount) according to the number of test copies, add W1 Primer MIX, W2 Primer MIX and W3 Primer MIX respectively to configure three tubes of W1, W2 and W3 reaction systems, and dispense 3 μL per well into a 96-well plate. The PCR mixture is prepared as follows:

[0087]

[0088] (3.2) Dilute the sample DNA to be tested to a concentration of 5 ng / μL and add 2 μL of DNA to the W1, W2 and W3 reaction systems respectively.

[0089] (3.3) Seal the 96-well plate with membrane, vortex and centrifuge.

[0090] (3.4) Place the 96-well plate on a PCR instrument to perform a PCR amplification reaction, wherein the PCR amplification reaction conditions are:

[0091]

[0092] (4) SAP reaction: Use SAP enzyme to remove residual dNTPs in the PCR reaction system

[0093] (4.1) Prepare SAP reaction solution (single test amount), wherein the SAP reaction solution is configured as follows:

[0094]

[0095] (4.2) Add 2 μl of SAP mixture to the reaction wells of the 96-well plate. The total volume after adding the mixture is 7 μL.

[0096] (4.3) Seal the 96-well plate with a membrane, vortex and centrifuge at 4000 rpm for 5 seconds.

[0097] (4.4) Place the 96-well plate on a PCR instrument and perform the following procedure: 37°C, 40 min; 85°C, 5 min; 4°C, hold.

[0098] (5) Extension reaction

[0099] (5.1) Prepare iPLEX extension mixed solution, add W1 UEP MIX, W2 UEP MIX and W3 UEP MIX to prepare iPLEX extension mixed solution of W1, W2 and W3 reaction systems respectively, wherein the iPLEX extension mixed solution is prepared as follows:

[0100]

[0101] (5.2) Add the corresponding iPLEX extension mixture to each reaction well and mix.

[0102] (5.3) Seal the 96-well plate with a membrane, vortex and centrifuge at 4000 rpm for 5 seconds.

[0103] (5.4) Place the 96-well plate on a PCR instrument to perform thermal cycling reaction for single base extension; the thermal cycling reaction conditions are:

[0104]

[0105] (6) Time-of-flight mass spectrometry

[0106] (6.1) Add 41 μl of water to each well of a 96-well plate containing samples and centrifuge.

[0107] (6.2) Use CPM to desalt the sample.

[0108] (6.3) Spot the sample onto a chip and mass spectrometer for time-of-flight mass spectrometry to obtain data, and determine the mutation status of the corresponding site based on the peak position corresponding to the molecular weight of the product.

[0109] The above method was used to detect the p53 mutation in p53 mutant cell lines CAL12T, SK-BR-3, H1651, H2122, H596, KYSE150, PC-9, BT549, MB468, MB231, HaCaT, BxPC-3, KYSE410, MCF7, and A549 with known mutation types. The test results are shown in the following table:

[0110]

[0111]

[0112] Note: “ / ” indicates no mutation was detected

[0113] The results showed that the detected p53 mutations were consistent with the mutation types of the corresponding cell lines, indicating that in cell lines, this method can accurately detect the mutations of 25 SNP sites of the p53 gene within the detection range.

[0114] Example 2 Detection of p53 mutation in lung cancer tissue samples

[0115] (1) Tissue DNA extraction

[0116] DNA was extracted and purified from 27 lung cancer tissues according to the QIAamp DNA Mini Kit (Qiagen, USA) instructions, and the DNA concentration was measured.

[0117] (2) Primer configuration

[0118] An amplification primer set and an extension primer set are configured, wherein the amplification primer combination includes W1 Primer MIX, W2 Primer MIX, and W3 Primer MIX, and the extension primer set includes W1 UEP MIX, W2 UEP MIX, and W3 UEP MIX; the specific configuration of the amplification primer set and the extension primer set is:

[0119]

[0120]

[0121] (3) Multiplex PCR amplification reaction (3.1) Prepare PCR mixture (single test amount) according to the number of test copies, add W1 Primer MIX, W2 Primer MIX and W3 Primer MIX respectively to configure three tubes of W1, W2 and W3 reaction systems, and dispense 3 μL per well into a 96-well plate. The PCR mixture is prepared as follows:

[0122]

[0123] (3.2) Dilute the sample DNA to be tested to a concentration of 5 ng / μL and add 2 μL of DNA to the W1, W2 and W3 reaction systems respectively.

[0124] (3.3) Seal the 96-well plate with membrane, vortex and centrifuge.

[0125] (3.4) Place the 96-well plate on a PCR instrument to perform a PCR amplification reaction, wherein the PCR amplification reaction conditions are:

[0126]

[0127] (4) SAP reaction: Use SAP enzyme to remove residual dNTPs in the PCR reaction system

[0128] (4.1) Prepare SAP reaction solution (single test amount), wherein the SAP reaction solution is configured as follows:

[0129]

[0130] (4.2) Add 2 μl of SAP mixture to the reaction wells of the 96-well plate. The total volume after adding the mixture is 7 μL.

[0131] (4.3) Seal the 96-well plate with a membrane, vortex and centrifuge at 4000 rpm for 5 seconds.

[0132] (4.4) Place the 96-well plate on a PCR instrument and perform the following procedure: 37°C, 40 min; 85°C, 5 min; 4°C, hold.

[0133] (5) Extension reaction

[0134] (5.1) Prepare iPLEX extension mixed solution, add W1 UEP MIX, W2 UEP MIX and W3 UEP MIX to prepare iPLEX extension mixed solution of W1, W2 and W3 reaction systems respectively, wherein the iPLEX extension mixed solution is prepared as follows:

[0135]

[0136] (5.2) Add the corresponding iPLEX extension mixture to each reaction well and mix.

[0137] (5.3) Seal the 96-well plate with a membrane, vortex and centrifuge at 4000 rpm for 5 seconds.

[0138] (5.4) Place the 96-well plate on a PCR instrument to perform thermal cycling reaction for single base extension; the thermal cycling reaction conditions are:

[0139]

[0140] (6) Time-of-flight mass spectrometry

[0141] (6.1) Add 41 μl of water to each well of a 96-well plate containing samples and centrifuge.

[0142] (6.2) Use CPM to desalt the sample.

[0143] (6.3) Spot the sample onto a chip and mass spectrometer for time-of-flight mass spectrometry to obtain data, and determine the mutation status of the corresponding site based on the peak position corresponding to the molecular weight of the product.

[0144] The p53 mutation was detected in 27 lung cancer tissues, and the results were compared with the NGS results. The experimental results are shown in Table 2 and Table 3. The detection spectrum of the corresponding p53 gene mutation is shown in Figure 1-5 As shown. The experimental results show that the test results are consistent with the NGS results. In 27 lung cancer tissues, the mutation of 25 SNP sites of the p53 gene within the detection range can be accurately detected. Compared with the NGS method, this method has lower cost and faster detection time than NGS, which can be completed within 8 hours. The data analysis is simple and no bioinformatics analysis is required.

[0145] Table 2 p53 mutation detection results in lung cancer tissues 1

[0146]

[0147] Note: “ / ” indicates no mutation was detected

[0148] Table 3 p53 mutation detection results in lung cancer tissues 2

[0149]

[0150]

[0151] Note: “ / ” indicates no mutation was detected

[0152] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. All modifications, equivalent substitutions, improvements, etc. within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A primer combination for detecting p53 gene mutation based on nucleic acid flight mass spectrometry, characterized in that: The primer combination includes an amplification primer set and an extension primer set for detecting p53 gene mutations, the amplification primer set includes primer sequences shown in SEQ ID NO.1-SEQ ID NO.14, and the extension primer set includes primer sequences shown in SEQ ID NO.15-SEQ ID NO.

39.

2. The primer combination for detecting p53 gene mutation according to claim 1, characterized in that: The amplification primer set includes W1 Primer MIX, W2 Primer MIX and W3 Primer MIX, wherein W1 Primer MIX includes the primer sequence shown in SEQ ID NO.1-SEQ ID NO.10, W2 Primer MIX includes the primer sequences shown in SEQ ID NO.1-SEQ ID NO.6 and SEQID NO.11-SEQ ID NO.14, and W3 Primer MIX includes the sequence shown in SEQ ID NO.1-SEQ ID NO.6; the extension primer set includes W1 UEP MIX, W2 UEP MIX and W3 UEP MIX, wherein W1 UEPMIX includes the primer sequence shown in SEQ ID NO.15-SEQ ID NO.25, W2 UEP MIX includes the primer sequence shown in SEQ ID NO.26-SEQID NO.34, and W3 UEP MIX includes the sequence shown in SEQ ID NO.35-SEQ ID NO.

39.

3. Use of the primer combination for detecting p53 gene mutation according to any one of claims 1 to 2 in preparing a product for detecting p53 gene mutation.

4. A p53 gene mutation detection kit, characterized in that: The p53 gene mutation detection kit comprises the primer combination for detecting p53 gene mutation according to any one of claims 1 to 2.

5. A method for detecting p53 gene mutation using the primer combination for detecting p53 gene mutation according to any one of claims 1 to 2, wherein: The method comprises the following: S1. Primer configuration: configure an amplification primer set and an extension primer set, wherein the amplification primer combination includes W1 PrimerMIX, W2 Primer MIX, and W3 Primer MIX, and the extension primer set includes W1 UEP MIX, W2 UEP MIX, and W3 UEPMIX; the specific information of the amplification primer set and the extension primer set is: S2. Multiplex PCR amplification reaction: prepare PCR mixture according to the number of test copies, add W1 Primer MIX, W2 Primer MIX and W3 Primer MIX to prepare W1, W2 and W3 reaction systems respectively, and dispense into 96-well plates; add the sample DNA to be tested into W1, W2 and W3 reaction systems respectively; seal the 96-well plate with a membrane, vortex and centrifuge, and then place the 96-well plate on a PCR instrument for PCR amplification reaction; S3, SAP reaction: Use SAP enzyme to remove residual dNTPs in the PCR reaction system; S4, extension reaction: prepare iPLEX extension mixture, add W1 UEP MIX, W2 UEP MIX and W3 UEP MIX to prepare iPLEX extension mixture of W1, W2 and W3 reaction systems respectively; add corresponding iPLEX extension mixture to each reaction well and mix; seal the 96-well plate with a membrane, vortex and centrifuge, and then place the 96-well plate on a PCR instrument for thermal cycling reaction to perform single base extension; S5. Time-of-flight mass spectrometry detection: Add 41 μl ddH2O to each well of a 96-well plate with a sample, and then centrifuge. Use CPM to desalt the sample, spot it onto a chip and mass spectrometer for time-of-flight mass spectrometry detection to obtain data. Determine the mutation status of the corresponding site based on the peak position corresponding to the molecular weight of the product.

6. The method according to claim 5, characterized in that In the S2 multiplex PCR amplification reaction, the PCR mixture is prepared as follows: The aliquot volume of the W1, W2, and W3 reaction systems was 3 μL per well, the concentration of the sample DNA to be tested was 5 ng / μL, and the added volume of the sample DNA to be tested was 2 μL; The PCR amplification reaction conditions are:

7. The method according to claim 5, characterized in that In the S3 SAP reaction, the SAP reaction solution is prepared as follows: The SAP reaction conditions are as follows: add 2 μl of SAP mixture to the reaction wells of the 96-well plate, and the total volume after adding the SAP mixture is 7 μL; seal the 96-well plate with a membrane, vortex and centrifuge, and the centrifugation condition is: 4000 rpm for 5 seconds; put the 96-well plate on the PCR instrument for reaction, and the reaction conditions are: 。 8. The method according to claim 5, characterized in that In the S4 extension reaction, the iPLEX extension mixture is prepared as follows: The volume of iPLEX extension mix added was 2 μl; The extension reaction conditions are:

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