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

Abstract

The present invention discloses a method, primer combination, kit and application for detecting p53 gene mutations based on nucleic acid flight mass spectrometry, the primer combination including an amplification primer set and an extension primer set for detecting p53 gene mutations, the amplification primer set including the primer sequence shown in SEQ ID NO.1-SEQ ID NO.14, and the extension primer set including the primer sequence shown in SEQ ID NO.15-SEQ ID NO.39. The primer combination disclosed in the present invention can accurately detect p53 gene mutations, while meeting the requirements of mass spectrometry detection technology, and realizing the application of MassARRAY system to quickly and effectively detect p53 gene mutations in test samples. At the same time, the primer combination covers a total of 47 mutation types in 25 SNP sites of the p53 gene, and the detection sites are more comprehensive. The method for detecting p53 gene mutations using the primer combination for detecting p53 gene mutations provided by the present invention also has the advantages of strong accuracy, high sensitivity, good repeatability, low cost, short detection cycle, more comprehensive detection sites, and intuitive results without the need for bioinformatics analysis.

Description

Technical Field

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

[0002] The human p53 gene is located on the short arm of chromosome 17, at 17p13.1. It is 20 kb long and consists of 11 exons and 10 introns. The p53 gene transcribes a 2.5 kb mRNA encoding a 393-amino acid protein with a molecular weight of approximately 53 kDa. The p53 gene is the most frequently mutated gene in human cancers (>50%), and p53 mutations significantly increase the risk of cancer development. p53 plays a role in regulating or progressing the cell cycle, apoptosis, and genomic stability through multiple mechanisms. Wild-type p53 promotes the elimination of newly formed cancer cells by inducing various forms of cell death or senescence, responses that help limit tumor progression. p53 can also induce reversible cell cycle arrest in response to cellular damage or stress, allowing cells to resume proliferation once the damage or stress is resolved. However, when p53 mutations lead to loss of function, it loses its original tumor suppressor functions, including cell cycle arrest, induction of apoptosis, maintenance of genomic stability, and mismatch DNA repair. Moreover, p53 mutants may play an important role in tumorigenesis and metastasis, promoting tumorigenesis.

[0003] Types of p53 mutations include gene segment deletions, insertions, missense mutations caused by point mutations, and loss of heterozygosity. Approximately 80% of these mutations are missense mutations caused by point mutations, primarily in the region encoding the DBD domain of the p53 protein. Within this region, eight 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 referred to as hotspot mutations. Mutated p53 can disrupt the regulation of its encoded product, leading to impaired cell differentiation, uncontrolled growth, and even cancer.

[0004] Abnormal p53 expression is associated with tumor metastasis, recurrence, and poor prognosis. Therefore, detecting p53 mutations in cancer patients is of great significance. Currently, numerous methods are available for detecting p53 mutations, including Sanger sequencing, PCR, and high-throughput sequencing. However, Sanger sequencing has high sample requirements, a limited detection range, relatively low sensitivity, low throughput, and prolonged detection time. PCR-based detection methods, such as ARMS-PCR, fluorescent quantitative PCR, and digital PCR, offer ease of use, rapidity, and simplified data analysis. However, due to throughput limitations, they cannot meet the clinical needs of multi-site p53 detection. While high-throughput sequencing overcomes these throughput limitations and enables comprehensive detection of p53 mutations, it is cumbersome, time-consuming, and expensive, and requires a high level of technical expertise from both the experimentalist and the data analyst. Therefore, a cost-effective, rapid, and user-friendly detection method is urgently needed. 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 mutations based on nucleic acid flight mass spectrometry, the primer combination comprising an amplification primer set and an extension primer set for detecting p53 gene mutations, 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 ID NO.35-SEQ ID NO.39.

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

[0010] The present invention also provides a p53 gene mutation detection kit, which comprises the above 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 Primer MIX, 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 as follows:

[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 distribute them into 96-well plates; add the sample DNA to be tested to the W1, W2, and W3 reaction systems respectively; seal the 96-well plate with film, vortex and centrifuge, and 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 the iPLEX extension mix by adding W1 UEP MIX, W2 UEP MIX, and W3 UEPMIX to prepare the iPLEX extension mix for the W1, W2, and W3 reaction systems, respectively. Add the corresponding iPLEX extension mix to each reaction well and mix. Seal the 96-well plate with film, vortex, and centrifuge. Place the 96-well plate in a PCR instrument for thermal cycling and single-base extension.

[0019] S5. Time-of-flight mass spectrometry: Add 41 μl of ddH2O to each well of a 96-well plate containing a sample, followed by centrifugation. Desalt the sample using CPM, apply the sample to a chip, and perform time-of-flight mass spectrometry to obtain data. Determine the mutation status of the corresponding site based on the peak position corresponding to the product molecular weight.

[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 volume of the sample DNA to be tested was 2 μL;

[0023] 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 a 96-well plate, for a total volume of 7 μl after addition of the SAP mixture; seal the 96-well plate with film, vortex, and centrifuge at 4000 rpm for 5 seconds; place the 96-well plate in a PCR instrument for reaction, and the reaction conditions are as follows:

[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 mutations 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 mutations, wherein the amplification primer set includes the primer sequences shown in SEQ ID NO.1-SEQ ID NO.14, and the extension primer set includes the primer sequences shown in SEQ ID NO.15-SEQ ID NO.39. The primer combination for detecting p53 gene mutations disclosed in the present invention can accurately detect p53 gene mutations, while meeting the requirements of mass spectrometry detection technology, and realizing the rapid and effective detection of p53 gene mutations in the sample to be tested using the MassARRAY system. At the same time, the primer combination for detecting p53 gene mutations 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 mutations using a primer combination for detecting p53 gene mutations provided by the present invention has the advantages of high accuracy, high sensitivity, good repeatability, low cost, short detection cycle, more comprehensive detection sites, and intuitive results without the need for bioinformatics analysis, 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 diagram of LC520 in lung cancer tissue samples. DETAILED DESCRIPTION

[0040] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative of and explain 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 contents of the present invention are encompassed 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 examples of the present invention can all be obtained commercially, and the experimental methods for which specific conditions are not indicated in the examples of the present invention are generally carried out in accordance with conventional experimental methods or in accordance with the methods recommended by the experimental material manufacturers.

[0041] Based on nucleic acid flight mass spectrometry, the present invention designs primer combinations for detecting 47 mutation types in 25 SNP sites of the p53 gene (Table 1). The primer combinations include 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 sequences shown in SEQ ID NO: 1 to SEQ ID NO: 14, and the extension primer set includes the primer sequences shown in SEQ ID NO: 15 to SEQ ID NO: 39.

[0042] Table 1: 47 mutation types at 25 SNP sites in the 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 Primer MIX, 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 as follows:

[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 distribute them into 96-well plates; add the sample DNA to be tested to the W1, W2, and W3 reaction systems respectively; seal the 96-well plate with film, vortex and centrifuge, and 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 the iPLEX extension mix by adding W1 UEP MIX, W2 UEP MIX, and W3 UEPMIX to prepare the iPLEX extension mix for the W1, W2, and W3 reaction systems, respectively. Add the corresponding iPLEX extension mix to each reaction well and mix. Seal the 96-well plate with film, vortex, and centrifuge. Place the 96-well plate in a PCR instrument for thermal cycling and single-base extension.

[0051] S5. Time-of-flight mass spectrometry: Add 41 μl of ddH2O to each well of a 96-well plate containing a sample, followed by centrifugation. Desalt the sample using CPM, apply the sample to a chip, and perform time-of-flight mass spectrometry to obtain data. Determine the mutation status of the corresponding site based on the peak position corresponding to the product molecular weight.

[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 volume of the sample DNA to be tested was 2 μL;

[0055] 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 a 96-well plate, for a total volume of 7 μl after addition of the SAP mixture; seal the 96-well plate with film, vortex, and centrifuge at 4000 rpm for 5 seconds; place the 96-well plate in a PCR instrument for reaction, and the reaction conditions are as follows:

[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] DNA from human cell lines was extracted using the QIAamp kit. The specific extraction method is as follows:

[0070] (1.1) Wash cells with 1 mL of PBS and digest with 1 mL of trypsin for 5 minutes.

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

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

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

[0074] (1.6) Add 200 μL of 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) Aspirate the liquid into the spin column sleeve and centrifuge at 8000 rpm for 1 min;

[0077] (1.9) Discard the liquid in the collection tube, add 500 μL of 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 12,000 rpm for 3 minutes; (1.11) Discard the liquid in the collection tube, and centrifuge at 12,000 rpm for 1 minute;

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

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

[0081] (2) Primer configuration

[0082] Configure an amplification primer set and an extension primer set, 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 a PCR mixture (single test volume) according to the number of test copies. Add W1 Primer MIX, W2 Primer MIX, and W3 Primer MIX to prepare three tubes of W1, W2, and W3 reaction systems respectively. Aliquot 3 μL into each well of 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 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 the SAP mixture to the reaction wells of a 96-well plate, bringing the total volume to 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 cycle: 37°C, 40 min; 85°C, 5 min; 4°C, hold.

[0098] (5) Extension reaction

[0099] (5.1) Prepare the iPLEX extension mix by adding W1 UEP MIX, W2 UEP MIX, and W3 UEP MIX to prepare the iPLEX extension mixes of the W1, W2, and W3 reaction systems, respectively. The iPLEX extension mix is ​​prepared as follows:

[0100]

[0101] (5.2) Add the corresponding iPLEX extension mix 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 for thermal cycling reaction and 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) Desalt the sample using CPM.

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

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

[0110]

[0111]

[0112] Note: “ / ” represents no mutation 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 mutations 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] Configure an amplification primer set and an extension primer set, 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 into each well of 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 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 the SAP mixture to the reaction wells of a 96-well plate, bringing the total volume to 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 cycle: 37°C, 40 min; 85°C, 5 min; 4°C, hold.

[0133] (5) Extension reaction

[0134] (5.1) Prepare the iPLEX extension mix by adding W1 UEP MIX, W2 UEP MIX, and W3 UEP MIX to prepare the iPLEX extension mixes of the W1, W2, and W3 reaction systems, respectively. The iPLEX extension mix is ​​prepared as follows:

[0135]

[0136] (5.2) Add the corresponding iPLEX extension mix 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 for thermal cycling reaction and 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) Desalt the sample using CPM.

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

[0144] 27 lung cancer tissues were tested for p53 mutations, and the results were compared with the NGS results. The experimental results are shown in Table 2 and Table 3. The corresponding p53 gene mutation detection patterns are shown in Figure 1-5 The experimental results showed that the test results were consistent with those of NGS, accurately detecting mutations at 25 SNP sites within the p53 gene in 27 lung cancer tissues. Compared with NGS, this method is less expensive and faster, completing the test within 8 hours. Data analysis is also simplified, eliminating the need for bioinformatics analysis.

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

[0146]

[0147] Note: “ / ” represents no mutation detected

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

[0149]

[0150]

[0151] Note: “ / ” represents no mutation 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 scope of protection 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 to SEQ ID NO.14, and the extension primer set includes primer sequences shown in SEQ ID NO.15 to 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 sequences 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 W3 Primer MIX includes the sequences 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 UEP MIX includes the primer sequences shown in SEQ ID NO.15-SEQ ID NO.25, W2 UEP MIX includes the primer sequences shown in SEQ ID NO.26-SEQ ID NO.34, and W3 UEP MIX includes the sequences 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.

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