A primer set, application, kit and method for detecting EGFR gene T790M and C797S mutations

Abstract

This application relates to the field of biological detection technology, and in particular to a primer set, application, kit, and method for detecting EGFR gene T790M and C797S mutations. The primer set includes a forward primer SEQ ID No:1, a reverse primer SEQ ID No:2, and probes SEQ ID No:3 to SEQ ID No:6. The primer set of this application is used to detect EGFR gene T790M and C797S mutations.

Description

A primer set, application, kit, and method for detecting T790M and C797S mutations in the EGFR gene. Technical Field

[0001] This application relates to the field of biological detection technology, and in particular to a primer set, application, kit, and method for detecting EGFR gene T790M and C797S mutations. Background Technology

[0002] EGFR (epidermal growth factor receptor) is a transmembrane receptor activated by extracellular ligands of the EGF family. EGFR can homodimerize or heterodimerize with other ErbB family members to initiate signal transduction, activating downstream signaling pathways, including the typical MAPK and PI3K / AKT / mTOR signaling cascades, ultimately leading to cell proliferation, migration, and differentiation. In lung cancer, activating mutations in EGFR result in a constitutively activated form of the receptor sensitive to EGFR tyrosine kinase inhibition. Tyrosine kinase inhibitors targeting EGFR, including afatinib, erlotinib, and gefitinib, are known as first- / second-generation inhibitors and have been approved for first-line treatment of non-small cell lung cancer (NSCLC). After 10 months of treatment with first- / second-generation inhibitors, some lung cancer patients develop second-site resistance mutations, including Met amplification, EGFR T790M mutations, and KRAS / NRAS / PIK3CA / HER2 mutations, with the EGFR T790M mutation being the most frequent, accounting for over 50%. Osimertinib, an EGFR tyrosine kinase inhibitor and a third-generation inhibitor, has been approved by the FDA for patients with recurrent non-small cell lung cancer exhibiting the EGFR resistance mutation T790M. After osimertinib treatment, patients may develop secondary resistance mutations, primarily the C797S mutation. If the EGFR T790M and C797S mutations are cis-mutations, the recommended treatment is a combination of first- or second-generation inhibitors such as afatinib, erlotinib, or gefitinib with the third-generation inhibitor osimertinib. If the EGFR T790M and C797S mutations are trans-mutations, the recommended treatment is brigiation combined with cetuximab or panitumumab.

[0003] Sanger sequencing is an effective method for detecting EGFR T790M and C797S cis / trans variants. However, it has a high detection limit, only detecting mutations at frequencies above 20%; it is also complex and time-consuming, requiring over 24 hours, which does not meet the rapid requirements of current clinical diagnostics. Another commonly used method is NGS sequencing, which is expensive, costing tens or even hundreds of times more than Sanger sequencing, and is also complex and time-consuming. Summary of the Invention

[0004] This application provides a primer set, application, kit, and method for detecting EGFR gene T790M and C797S mutations. This method utilizes a kit to detect EGFR gene T790M and C797S mutations. It features high specificity, high sensitivity, and a short detection cycle.

[0005] To achieve the above objectives, the embodiments of this application adopt the following technical solutions:

[0006] In a first aspect, embodiments of this application provide a primer set for detecting EGFR gene T790M and C797S mutations, the primer set including amplification primer pairs and probes.

[0007] The amplification primer pair sequences include:

[0008] Forward primer: 5'-TGGGCATCTGCCTCACCTCCAC-3'SEQ ID No:1; and,

[0009] Reverse primer: 5'-GGTACTGGGAGCCAATATTG-3'SEQ ID No:2.

[0010] The probe sequence includes:

[0011] 5'-CAGCTCATCATGCAGCTCATGCC-3'SEQ ID No:3.

[0012] 5'-CGGCAGCCTCCTGGACTATGT-3'SEQ ID No:4.

[0013] 5'-CGGCTCCCTCCTGGACTATGT-3'SEQ ID No:5.

[0014] 5'-CGGAAGCCTCCTGGACTATGT-3'SEQ ID No:6.

[0015] The primer set provided in this application is used to detect EGFR T790M and C797S cis or trans mutations. The detection method includes EGFR T790M and C797S amplification primers F and R, labeled with specific probes FAM-Probe1-MGB and VIC-Probe2-MGB, VIC-Probe3-MGB, and VIC-Probe4-MGB, respectively, and uses TaqMan-qPCR to detect EGFR T790M and C797S mutations. The optimized peptide nucleic acid PNA-Primer1, which specifically targets the wild-type EGFR T790M sequence, is selected. Utilizing the stable binding of peptide nucleic acid to DNA and its resistance to Taq polymerase degradation, the EGFR T790M wild-type site is blocked, thus preventing effective amplification and inhibiting the amplification of both wild-type EGFR T790M and C797S mutant (trans) DNA fragments. Simultaneous mutations of GFR T790M and C797S (cis) can be effectively amplified, allowing for the detection of EGFR T790M and C797S cis or trans mutations. This detection method is highly sensitive (0.1%), highly specific, and rapid.

[0016] Optionally, a primer set for detecting T790M and C797S mutations in the EGFR gene also includes PNA. The PNA sequence includes: 5'-CAGCTCATCACGCAGCTCATGCC-3'SEQ ID No:7.

[0017] Optionally, a fluorescent group is attached to the 5' end of probe SEQ ID No:3, and a quenching group is attached to the 3' end of probe SEQ ID No:3; and / or a fluorescent group is attached to the 5' end of probe SEQ ID No:4, and a quenching group is attached to the 3' end of probe SEQ ID No:4; and / or a fluorescent group is attached to the 5' end of probe SEQ ID No:5, and a quenching group is attached to the 3' end of probe SEQ ID No:5; and / or a fluorescent group is attached to the 5' end of probe SEQ ID No:6, and a quenching group is attached to the 3' end of probe SEQ ID No:6.

[0018] Optionally, probe SEQ ID No:3 has a fluorescent group FAM attached to its 5' end and a quencher group MGB attached to its 3' end. Probe SEQ ID No:4 has a fluorescent group VIC attached to its 5' end and a quencher group MGB attached to its 3' end. Probe SEQ ID No:5 has a fluorescent group VIC attached to its 5' end and a quencher group MGB attached to its 3' end. Probe SEQ ID No:6 has a fluorescent group VIC attached to its 5' end and a quencher group MGB attached to its 3' end.

[0019] Secondly, embodiments of this application provide a kit for detecting EGFR gene T790M and C797S mutations, including PCR amplification reagents and the aforementioned primer set.

[0020] Optionally, the concentration of the forward primer is 10 μM, the concentration of the reverse primer is 10 μM, the concentration of probe SEQ ID No:3 is 10 μM, the concentration of probe SEQ ID No:4 is 10 μM, the concentration of probe SEQ ID No:5 is 10 μM, the concentration of probe SEQ ID No:6 is 10 μM, and the concentration of PNA is 10 μM.

[0021] Thirdly, embodiments of this application provide a method for detecting EGFR gene T790M and C797S mutations, comprising: taking DNA extracted from the sample to be tested; performing PCR amplification on the DNA using the kit of claim 5 or 6; and detecting the PCR amplification product.

[0022] Optionally, DNA was extracted from genomic DNA of paraffin-embedded lung cancer samples.

[0023] Optionally, the PCR amplification reaction conditions are as follows: pre-denaturation at 92–98℃ for 2–5 min; denaturation at 92–98℃ for 30 s–1 min, first annealing at 65–70℃ for 30 s–1 min, second annealing at 58–63℃ for 30 s–1 min, extension at 65–75℃ for 30 s–1 min, for 45 cycles; amplification followed by extension at 65–75℃ for 2–5 min, and storage at 4–12℃.

[0024] Optionally, the PCR amplification reaction conditions are as follows: 98℃ pre-denaturation for 5 min; 98℃ denaturation for 30 s, 67℃ first annealing for 30 s, 60℃ second annealing for 30 s, 72℃ extension for 30 s, for 45 cycles; 72℃ amplification followed by extension for 5 min, and storage at 10℃.

[0025] Fourthly, embodiments of this application provide an application of the primer set described above in detecting EGFR gene T790M and C797S mutations. This application is for non-diagnostic purposes. Attached Figure Description

[0026] Figure 1 is a schematic diagram of T790M and C797S cis-mutation and trans-mutation provided in an embodiment of this application;

[0027] Figure 2 is a schematic diagram of the principle of detecting T790M and C797S mutations provided in an embodiment of this application;

[0028] Figure 3 is a schematic diagram of the principle of detecting T790M and C797S cis-mutations and trans-mutations provided in an embodiment of this application. Detailed Implementation

[0029] The embodiments of this application will now be described in detail with reference to the accompanying drawings.

[0030] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this application, unless otherwise stated, "a plurality of" means two or more.

[0031] In this application, DNA (deoxyribonucleic acid) is a long-chain polymer composed of four deoxynucleotides: adenine deoxynucleotide (dAMP), thymine deoxynucleotide (dTMP), cytosine deoxynucleotide (dCMP), and guanine deoxynucleotide (dGMP).

[0032] A reagent kit is a box used to hold chemical reagents for detecting chemical components, drug residues, virus types, etc. Of course, those skilled in the art will understand that the box can also be a tube or other container for holding chemical reagents.

[0033] EGFR: Epidermal growth factor receptor, is a member of the epidermal growth factor receptor (HER). Mutations in the tyrosine kinase domain of EGFR lead to overactivation and are closely related to the development of lung cancer. Relevant mutations include EGFR T790M, L858R, and 19Exon Deletion.

[0034] TaqMan probes: TaqMan fluorescent probes are oligonucleotide probes with a fluorescent group at the 5' end, such as 5-carboxyfluorescein-5-succimidyl ester (5-FAM), tetrachlorofluorescein (6-TET), VIC, and hexachlorofluorescein (HEX), etc., and a quencher group at the 3' end, such as 5-carboxytetramethylrhodamine (5-TAMRA) and BHQ carboxylic acid (BHQ). During PCR amplification, fluorescence is released by cleaving the fluorescent group at the 5' end, thereby detecting the target mutation.

[0035] Peptide nucleic acids (PNAs) are a class of DNA analogs in which the sugar-phosphate backbone is replaced by a polypeptide backbone. Specifically, the neutral peptide chain amide 2-aminoethylglycine bond replaces the pentose phosphate diester bond backbone in DNA, while the rest is the same as DNA. PNAs can recognize and bind to DNA or RNA sequences through Watson-Crick base pairing to form a stable double helix structure.

[0036] EGFR (Epidermal growth factor receptor, abbreviated as EGFR, ErbB-1, or HER1) is a member of the epidermal growth factor receptor family. Activating mutations in the tyrosine kinase domain of the EGFR receptor are one of the main driving factors in lung cancer development. The EGFR T790M mutation is the target of osimertinib treatment, but the inevitable development of a resistance mutation, EGFR C797S, is a consequence. As shown in Figure 1, T790M and C797S mutations located on the same chromosome are cis mutations, while those located on separate alleles are trans mutations. Patients with EGFR T790M and C797S trans mutations are sensitive to combination therapy with first- and third-generation EGFR-TKI drugs; therefore, detecting EGFR T790M and C797S trans or cis mutations is extremely important.

[0037] Currently, the main methods for detecting EGFR T790M and C797S trans or cis mutations are Sanger sequencing or next-generation sequencing technologies. Sanger sequencing suffers from low sensitivity, while next-generation sequencing is time-consuming and expensive. The TaqMan fluorescent probe method offers high sensitivity, ease of operation, short detection cycle, and low cost. As shown in Figure 2, the TaqMan fluorescent probe method can simultaneously detect EGFR T790M and C797S mutations, but it cannot determine whether they are cis or trans mutations. Based on existing detection methods, this application designs a peptide nucleic acid (Blocker) targeting the wild-type sequence corresponding to the T790M mutation site. For cis-mutations, the Blocker binding to the wild-type site has no effect on EGFR T790M and C797S mutations, and can still release FAM and VIC fluorescence. For trans-mutations, the Blocker blocks the wild-type site. During PCR amplification, the Blocker blocks the detection of the C797S mutation located at its 3' end. As shown in Figure 3, only FAM fluorescence can be detected. Therefore, EGFR T790M and C797S can be determined as cis-mutations or trans-mutations by using FAM and VIC signals.

[0038] This application provides a primer set for detecting EGFR gene T790M and C797S mutations. The primer set includes a forward primer (F), a reverse primer (R), a probe, and a peptide nucleic acid (PNA). The sequences of the forward primer (F) and the reverse primer (R) are shown in Table 1 below, the sequence of the probe is shown in Table 2 below, and the sequence of the peptide nucleic acid (PNA) is shown in Table 3 below. Examples 1 and 2 below use the forward primer (F), the reverse primer (R), the probe, and the peptide nucleic acid to detect mutations in the EGFR gene T790M and C797S and perform sensitivity experiments.

[0039] Table 1. Forward primer (F) and reverse primer (R) sequences

[0040] Primer Name Sequence (5'→3') SEQ Number Forward Primer (F) TGGGCATCTGCCTCACCTCCACSEQ ID NO: 1 Reverse Primer (R) GGTACTGGGAGCCAATATTGSEQ ID NO: 2 surface

[0041] Table 2 Probe Sequences

[0042] Probe Name Sequence (5'→3') SEQ Sequence Number FAM-Probe1-MGBCAGCTCATCATGCAGCTCATGCC SEQ ID NO: 3 VIC-Probe1-MGBCGGCAGCCTCCTGGACTATGT SEQ ID NO: 4 VIC-Probe2-MGBCGGCTCCCTCCTGGACTATGT SEQ ID NO: 5 VIC-Probe3-MGBCGGAAGCCTCCTGGACTATGT SEQ ID NO: 6 surface

[0043] Table 3 Peptide Nucleic Acid Sequences

[0044] Peptide Nucleic Acid Name Sequence (5'→3') SEQ Sequence Number PNA-Primer1CAGCTCATCACGCAGCTCATGCCSEQ ID NO: 7 surface

[0045] Example 1:

[0046] This embodiment is used to detect EGFR T790M and C797S cis or trans mutations, including PCR amplification reagents, forward and reverse primers, specific probes FAM-Probe1-MGB, VIC-Probe1-MGB, VIC-Probe2-MGB and VIC-Probe3-MGB, and specific peptide nucleic acid PNA-Primer1.

[0047] 1. Material preparation

[0048] 1) The PCR amplification reagents were selected from Thermo's Platinum series. TM Hot Start PCR Master Mix(2X);

[0049] 2) Prepare the forward and reverse primers, probes FAM-Probe1-MGB, VIC-Probe1-MGB, VIC-Probe2-MGB, VIC-Probe3-MGB, and peptide nucleic acid PNA-Primer1 to 10 μM using DNase-Free & RNase-Free Water.

[0050] 3) Genomic DNA samples were extracted into paraffin-embedded lung cancer tissue (FFPE) DNA and prepared into concentrations of 32.5 ng / μL, 21.4 ng / μL, 25.6 ng / μL and 30.2 ng / μL, respectively.

[0051] 2. Testing Steps

[0052] 1) Place Platinum TM Hot StartPCRMasterMix (2X), forward and reverse primers, probes FAM-Probe1-MGB, VIC-Probe1-MGB, VIC-Probe2-MGB and VIC-Probe3-MGB, peptide nucleic acid PNA-Primer1 and genomic DNA samples were taken out of the refrigerator and placed on ice to thaw naturally.

[0053] 2) Divide 8 PCR tubes into 4 groups, numbered 1-2 in each group. Add the components to all reaction tubes numbered 1 according to Table 4, and add the components to all reaction tubes numbered 2 according to Table 5. Specifically, add 23.5 ng / μL (0.31 μL) of Sample 1 DNA to reaction tubes 1-2 in group 1, add 21.4 ng / μL (0.47 μL) of Sample 2 DNA to reaction tubes 1-2 in group 2, add 25.6 ng / μL (0.39 μL) of Sample 3 DNA to reaction tubes 1-2 in group 3, and add 23.5 ng / μL (0.33 μL) of Sample 4 DNA to reaction tubes 1-2 in group 4. The concentrations of the forward primer, reverse primer, probe, and peptide nucleic acid in Tables 4 and 5 can also be selected as 20 μM, 30 μM, or 50 μM, and the specific experimental procedure is the same.

[0054] Table 4. Concentration and volume of each component

[0055]

[0056]

[0057] Table 5. Concentration and volume of each component

[0058]

[0059]

[0060] 3) Turn on the real-time PCR instrument (Roche 480 or ABI 7500), set the PCR reaction program according to Table 6, place the PCR reaction tubes side by side into the PCR instrument, and run the reaction program;

[0061] Table 6 Reaction Temperature and Time

[0062]

[0063] 4) After the reaction is complete, analyze the detection results.

[0064] 3. Results

[0065] 3.1 TaqMan PCR detection data are shown in Table 7 below: CT values ​​of the samples.

[0066] Table 7. CT Values ​​of Samples

[0067]

[0068] 3.2 Analysis of Test Results

[0069] In sample 1, tube 1 showed FAM and VIC signal CT values ​​both <42, indicating the presence of both T790M and C797S mutations. Tube 2 showed FAM and VIC signal CT values ​​both <42, suggesting that the T790M and C797S mutations are located on the same chromosome, i.e., a cis mutation. In sample 2, tube 1 showed FAM signal CT values ​​<42, indicating a positive T790M mutation, while the VIC signal CT value was >42, indicating a negative C797S mutation. In sample 3, tube 1 showed FAM and VIC signal CT values ​​both <42, indicating the presence of both T790M and C797S mutations. Tube 2 showed FAM signal CT values ​​<42, suggesting that the T790M and C797S mutations are located on different chromosomes, i.e., a trans mutation. Sample 4 was the same as sample 3. Conclusion: Sample 1 is a cis mutation, while samples 3 and 4 are trans mutations.

[0070] Example 2:

[0071] This embodiment is used to test the sensitivity of detecting cis or trans mutations of EGFR T790M and C797S, including PCR amplification reagents, forward and reverse primers, specific probes FAM-Probe1-MGB, VIC-Probe1-MGB, VIC-Probe2-MGB and VIC-Probe3-MGB, and specific peptide nucleic acid PNA-Primer1.

[0072] 1. Material preparation

[0073] 1) The PCR amplification reagents were selected from Thermo's Platinum series. TM Hot Start PCR Master Mix(2X);

[0074] 2) Prepare the forward and reverse primers, probes FAM-Probe1-MGB, VIC-Probe1-MGB, VIC-Probe2-MGB, VIC-Probe3-MGB, and peptide nucleic acid PNA-Primer1 to 10 μM using DNase-Free & RNase-Free Water.

[0075] 3) One cis-mutant and one trans-mutant standard sample of EGFR T790M and C797S were selected, with a mutation frequency of 5%, as standard genomic DNA samples. The samples were diluted to 1%, 0.5%, 0.2%, and 0.1% using wild-type genomic DNA. The nucleic acid concentration was 20 ng / μL.

[0076] 2. Testing Steps

[0077] 1) Place Platinum TM Hot Start PCR MasterMix (2X), primers F and R, probes FAM-Probe1-MGB, VIC-Probe1-MGB, VIC-Probe2-MGB and VIC-Probe3-MGB, peptide nucleic acid PNA-Primer1, and standard genomic DNA samples were removed from the refrigerator and placed on ice to thaw naturally.

[0078] 2) Divide 16 PCR tubes into 8 groups, numbered 1-2 in each group. Add the components to all reaction tubes numbered 1 according to Table 4, and add the components to all reaction tubes numbered 2 according to Table 5. For reaction tubes in groups 1-4, add EGFR T790M and C797S cis-mutant DNA at a serial dilution of 1%-0.1%, with a concentration of 20 ng / μL and a volume of 1 μL. For reaction tubes in groups 5-8, add EGFR T790M and C797S trans-mutant DNA at a serial dilution of 1%-0.1%, with a concentration of 20 ng / μL and a volume of 1 μL.

[0079] 3) Turn on the real-time PCR instrument (Roche 480 or ABI 7500), set the PCR reaction program according to Table 6, place the PCR reaction tubes side by side into the PCR instrument, and run the reaction program.

[0080] 4) After the test is completed, the test results are analyzed.

[0081] 3. Results

[0082] 3.1 TaqMan PCR detection data are shown in Table 8 below (CT value table for sample detection).

[0083] Table 8. CT Values ​​of Samples

[0084]

[0085] 3.2 Analysis of Test Results

[0086] As shown in Table 8, when the cis-mutant and trans-mutant standard samples with a mutation frequency of 5% were serially diluted to 1%, 0.5%, 0.2%, and 0.1%, the samples with a mutation frequency of 0.1% were all effectively detected, indicating that the sensitivity of the detection method reached 0.1%.

[0087] In the description of this specification, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

[0088] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims. (Sequence List) <110> BOE Technology Group Co., Ltd. <120> A primer set, application, kit, and method for detecting T790M and C797S mutations in the EGFR gene. <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> twenty two <212> DNA <213> Artificial Sequence <400> 1tgggcatctg cctcacctcc ac 22 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2ggtactggga gccaatattg 20 <210> 3 <211> twenty three <212> DNA <213> Artificial Sequence <400> 3cagctcatca tgcagctcat gcc 23 <210> 4 <211> twenty one <212> DNA <213> Artificial Sequence <400> 4cggcagcctc ctggactatg t 21 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <400> 5cggctccctc ctggactatg t 21 <210> 6 <211> twenty one <212> DNA <213> Artificial Sequence <400> 6cggaagcctc ctggactatg t 21 <210> 7 <211> twenty three <212> DNA <213> Artificial Sequence <400> 7cagctcatca cgcagctcat gcc 23

Claims

1. A primer set for detecting T790M and C797S mutations in the EGFR gene, characterized in that, The primer set includes amplification primer pairs and probes; the amplification primer pairs include: forward primer: 5'-TGGGCATCTGCCTCACCTCCAC-3'SEQ ID No:1; and reverse primer: 5'-GGTACTGGGAGCCAATATTG-3'SEQ ID No:2; the probe sequences include: 5'-CAGCTCATCATGCAGCTCATGCC-3'SEQ ID No:3; 5'-CGGCAGCCTCCTGGACTATGT-3'SEQ ID No:4; 5'-CGGCTCCCTCCTGGACTATGT-3'SEQ ID No:5; 5'-CGGAAGCCTCCTGGACTATGT-3'SEQ ID No:6; the primer set also includes: PNA, the PNA sequence including 5'-CAGCTCATCACGCAGCTCATGCC-3'SEQ ID No:7; wherein the 5' end of the probe SEQ ID No:3 is connected to the fluorescent group FAM, the probe SEQ ID... The 3' end of probe No. 3 is connected to a quenching group MGB; the 5' end of probe SEQ ID No. 4 is connected to a fluorescent group VIC, and the 3' end of probe SEQ ID No. 4 is connected to a quenching group MGB; the 5' end of probe SEQ ID No. 5 is connected to a fluorescent group VIC, and the 3' end of probe SEQ ID No. 5 is connected to a quenching group MGB; the 5' end of probe SEQ ID No. 6 is connected to a fluorescent group VIC, and the 3' end of probe SEQ ID No. 6 is connected to a quenching group MGB.

2. A kit for detecting EGFR gene T790M and C797S mutations, characterized in that, Includes PCR amplification reagents and the primer set as described in claim 1.

3. The kit for detecting EGFR gene T790M and C797S mutations according to claim 2, characterized in that, The concentration of the forward primer is 10 μM, the concentration of the reverse primer is 10 μM, the concentration of the probe SEQ ID No:3 is 10 μM, the concentration of the probe SEQ ID No:4 is 10 μM, the concentration of the probe SEQ ID No:5 is 10 μM, the concentration of the probe SEQ ID No:6 is 10 μM, and the concentration of PNA is 10 μM.

4. The use of the primer set in the preparation of a kit for detecting EGFR gene T790M and C797S mutations by the following method, characterized in that, The method includes: taking DNA extracted from the sample to be tested; performing PCR amplification on the DNA using the kit described in claim 2 or 3; and detecting the PCR amplification product.

5. The use according to claim 4, characterized in that, The DNA was extracted from genomic DNA of a paraffin-embedded lung cancer sample.

6. The use according to claim 4, characterized in that, The PCR amplification reaction was performed as follows: pre-denaturation at 92-98℃ for 2-5 minutes; denaturation at 92-98℃ for 30 seconds to 1 minute, first annealing at 65-70℃ for 30 seconds to 1 minute, second annealing at 58-63℃ for 30 seconds to 1 minute, extension at 65-75℃ for 30 seconds to 1 minute, for 45 cycles; amplification followed by extension at 65-75℃ for 2-5 minutes, and storage at 4-12℃.

7. The use according to claim 6, characterized in that, The PCR amplification reaction was as follows: pre-denaturation at 98℃ for 5 min; denaturation at 98℃ for 30 s, first annealing at 67℃ for 30 s, second annealing at 60℃ for 30 s, extension at 72℃ for 30 s, for 45 cycles; amplification at 72℃ followed by extension for 5 min, and storage at 10℃.

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