EGFR L858R mutation site gene detection kit and application thereof

Abstract

The invention discloses an EGFR L858R mutation site gene detection kit and application thereof, and belongs to the technical field of molecular biology. The kit comprises an LAMP primer group and a strand displacement probe, wherein the LAMP primer group comprises a pair of outer primers F3 / B3, a pair of inner primers FIP / BIP and a loop primer LP; the strand displacement probe comprises an F strand and a Q strand, the 3'end of the nucleotide sequence of the F strand is modified by a fluorophore, and the 5 'end of the nucleotide sequence of the Q strand is modified by a quenching group. According to the invention, an improved loop-mediated isothermal amplification-nucleic acid molecule line technology is utilized, a novel primer design method is utilized to reduce non-specific amplification, a blank chain segment or mismatch repair is introduced to adjust multiple probe dynamics, and an easily programmed logic line is utilized to realize intelligent analysis. A novel sensitive, rapid and portable EGFR-TKIs series mutant gene multiple + heavy and intelligent detection principle is obtained; in combination with clinical sample analysis, the lung cancer EGFR liquid biopsy gene detection kit with low cost, high efficiency and high precision is developed.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular to an EGFR L858R mutant gene detection kit and application thereof. Background Art

[0002] Targeted therapy is the main method of precision diagnosis and treatment of lung cancer, among which targeted drugs, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), are the most widely used. Efficient diagnosis of sensitive and resistant gene mutation sites of targeted drugs and scientific use of this drug have become the first choice for targeted treatment of lung cancer patients. At present, the main methods for clinical detection of EGFR gene mutations are ARMS-PCR and second-generation sequencing technology, but these two technologies cannot meet the needs of early, accurate and low-cost detection.

[0003] The ARMS-PCR method has technical problems of non-specific amplification and false positives; although the second-generation sequencing technology has high sensitivity, the detection process is complex and the cycle is long. It has high requirements for equipment, personnel, and sites. At the same time, due to its high price, its application is greatly restricted. Therefore, the development of new detection technology to overcome the problems of non-specific amplification and false positives, while reducing the cost of detection and improving the operability and universality of the detection technology, is the hot spot and difficulty in the development of EGFR liquid biopsy gene detection kits. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide an EGFR L858R mutant gene detection kit and its application, which aims at the deficiencies of the prior art, uses constant temperature amplification technology and one-step chain displacement probe coupling, and realizes signal output by modifying the chain displacement probe with a fluorescent group, so as to effectively distinguish the wild-type gene sequence from the mutant gene sequence. The established new method is used for early screening of NSCLC patients and guiding the use of targeted drugs.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0006] A kit for detecting EGFR L858R mutant genes, comprising a LAMP primer set and a strand displacement probe, wherein:

[0007] The LAMP primer set includes: a pair of outer primers F3 / B3, a pair of inner primers FIP / BIP, and a loop primer LP, and its nucleotide sequence is as follows:

[0008] External primer F3: 5′-GCAGCCAGGAACGTACTG-3′ (SEQ ID No. 1);

[0009] External primer B3: 5′-ATCCTCCCCTGCATGTGT-3′ (SEQ ID No. 2);

[0010] Inner primer FIP: 5'-TTCCGCACCCAGCAGTTTGGGTGAAAACACCGCAGCATG-3' (SEQ IDNo.3);

[0011] Inner primer BIP: 5'-GGAGGCAAAGTAAGGAGGTGGCACAGCTAGTGGGAAGGCA-3' (SEQ IDNo. ​​4);

[0012] Loop primer LP: 5′-TTTAGGTCAGCCAGCATTTTCC-3′ (SEQ ID No. 5);

[0013] The strand displacement probe includes strand F and strand Q, and the nucleotide sequence is as follows:

[0014] F chain: 5'-GCCCGCCCAAAATCTGTGATCTTGACATGCCGA-3' (SEQ ID No. 6), the 3' end of the nucleotide sequence is modified with a fluorescent group;

[0015] Q strand: 5'-TCGGCATGTCAAGATCACAGATTTTG-3' (SEQ ID No. 7), the 5' end of the nucleotide sequence is modified with a quenching group and the 3' end is modified with a reverse dT nucleic acid sequence.

[0016] Preferably, in the above technical solution, the fluorescent group at the 3' end of the F chain is 6-FAM; and the quenching group at the 5' end of the Q chain is BHQ1.

[0017] Preferably, in the above technical solution, the mass ratio of F3, B3, FIP, BIP and LP of the LAMP primer set is 1:1:4:4:2.

[0018] Preferably, in the above technical solution, the molar ratio of F chain to Q chain is 1:3.

[0019] Preferably, in the above technical solution, the kit specifically comprises: each 25 μL reaction system comprises: 2.0 μL of primer premix consisting of outer primers and inner primers, 2.0 μL of LP primer, 2.0 μL of dNTPs, 0.5 μL of Mg 2+ , 1.125 μL strand displacement probe, 5.0 μL betaine, 2.5 μL isothermal amplification buffer, 1.0 μL Bst 2.0 DNA polymerase, 1 μL EGFR gene template, 7.875 μL deionized water.

[0020] Preferably, in the above technical solution, the concentration of the strand F of the strand displacement probe is 1 μM, the concentration of dNTPs is 10 mM, and the concentration of Mg is 10 mM. 2+ The concentration of isothermal amplification buffer was 10×, the concentration of Bst 2.0 DNA polymerase was 8 U, and the concentration of EGFR gene template was 2×10 6 Copies / μL. dNTPs include dATP, dCTP, dGTP, and dTTP, and the mass ratio of the four is 1:1:1:1. The primer premix includes 5 μM outer primer F3, 5 μM outer primer B3, 20 μM inner primer FIP, and 20 μM inner primer BIP. The isothermal amplification buffer consists of 0.2 M Tris, 0.1 M (NH4)2SO4, 0.5 M KCl, 0.02 M MgSO4, and 0.4 g Tween-20.

[0021] An EGFR L858R mutation gene detection kit is used in the early screening of non-small cell lung cancer for non-disease purposes and / or the preparation of EGFR-TKIs targeted drugs.

[0022] A method for detecting EGFR L858R mutant genes for non-disease purposes, the method using isothermal amplification technology, specifically comprising the following steps:

[0023] (1) Prepare the DNA template sample to be tested;

[0024] (2) Prepare the reaction system, which includes primer premix, strand displacement probe, dNTPs, betaine, isothermal amplification buffer, Mg 2+ and Bst 2.0 DNA polymerase;

[0025] (3) Add the sample to be tested into the reaction system and perform a constant temperature amplification reaction at 62°C for 60 minutes;

[0026] (4) Determine the presence of the EGFR L858R mutant gene by monitoring changes in the fluorescence signal.

[0027] Preferably, in the above technical solution, in step (2), in the amplification system, the concentration of the F strand of the strand displacement probe is 1 μM, the concentration of dNTPs is 10 mM, and the concentration of Mg is 2+ The concentration of is 100 mM, the concentration of betaine is 5 M, the concentration of isothermal amplification buffer is 10×, the concentration of Bst 2.0 DNA polymerase is 8 U, and the concentration of the DNA sample to be tested is 5 ng / μL.

[0028] The above technical solution of the present invention has the following beneficial effects:

[0029] This application is based on the independently developed precision molecular diagnostic technology. It intends to use the improved "loop-mediated isothermal amplification-nucleic acid molecular circuit" technology, use new primer design methods to reduce non-specific amplification, introduce blank segments or mismatch repair to adjust the dynamics of multiple probes, and easily programmable logic circuits to achieve intelligent analysis, so as to obtain a new principle of multiple and intelligent detection of EGFR-TKIs series mutant genes that is sensitive, rapid, and portable; combined with clinical sample analysis, develop a low-cost, high-efficiency, and high-precision lung cancer EGFR liquid biopsy gene detection kit. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0031] Figure 1 To design and optimize LAMP amplification primers and reaction systems, where: A is the screening of LAMP amplification primers; B is the optimization of LAMP amplification primers; C is the optimization of LAMP amplification system; D is the screening of loop primers LP.

[0032] Figure 2 To design and optimize the sequence and reaction system of one-step strand displacement probe, where: A is non-denaturing polypropylene gel electrophoresis to verify the feasibility of the strand displacement probe; B is the optimization of the F:Q ratio of the strand displacement probe; C is the optimization of the strand displacement probe concentration; D is the detection of target mimics of different concentrations by the strand displacement probe.

[0033] Figure 3 To design and optimize the isothermal amplification-strand displacement probe coupling reaction system, where: A is the fluorescence signal generated by the reaction system at 58°C for 30 min; B is the fluorescence signal generated by the reaction system at 60°C for 30 min; C is the fluorescence signal generated by the reaction system at 62°C for 30 min; D is the detection of different concentrations of EGFR L858R mutant templates under the optimal system. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0035] Unless otherwise specified, the reagents used in this application are all commercially available or obtained through commercial channels, or can be prepared by referring to existing chemical methods.

[0036] Genomic sequence of EGFRL858R, a site associated with pathogenic mutations in non-small cell lung cancer and drug sensitivity to gefitinib and osimertinib:

[0037] LAMP Primers:

[0038] External primer F3: 5′-GCAGCCAGGAACGTACTG-3′ (SEQ ID No. 1);

[0039] External primer B3: 5′-ATCCTCCCTGCATGTGT-3′ (SEQ ID No. 2);

[0040] Inner primer FIP: 5'-TTCCGCACCCAGCAGTTTGGGTGAAAACACCGCAGCATG-3' (SEQ IDNo.3);

[0041] Inner primer BIP: 5'- GGAGGCAAAGTAAGGAGGTGGCACAGCTAGTGGGAAGGCA-3' (SEQ IDNo. ​​4);

[0042] Loop primer LP: 5'-TTTAGGTCAGCCAGCATTTTCC-3' (SEQ ID No. 5), react at 55-65°C for 2h.

[0043] A specific fluorescent quenching strand displacement probe was designed for a single-stranded DNA circle generated by LAMP amplification:

[0044] F chain: 5'-GCCCGCCCAAAATCTGTGATCTTGACATGCCGA-3' (SEQ ID No. 6), the 3' end of the nucleotide sequence is modified with a fluorescent group, and the modified sequence is 5'-GCCCGCCCAAAATCTGTGATCTTGACATGCCGA-FAM-3';

[0045] Q chain: 5'-TCGGCATGTCAAGATCACAGATTTTG-3' (SEQ ID No.7), the 5' end of the nucleotide sequence is modified with a quenching group, and the 3' end is modified with Inverted dT (reverse dT). The modified sequence is 5'-BHQ1-TCGGCATGTCAAGATCACAGATTTTG-Inverted dT. The amplified product containing the mutation site displaces the fluorophore-modified chain and restores the fluorescence signal. The reaction is carried out at a constant temperature and does not require special instruments to achieve real-time detection of the EGFR L858R site. It can not only eliminate false positive signals, but also distinguish between wild-type and mutant genes.

[0046] The following is a detailed description of the embodiments:

[0047] Example 1 Design and optimization of LAMP amplification primers and reaction system

[0048] (1) Design and screening of LAMP reaction primers:

[0049] The primers for LAMP amplification of the EGFR gene were screened using LAMP primer design software (http: / / primerexplorer.jp / e / ).

[0050] Prepare 25 μL reaction system, including inner primers, outer primers, dNTPs, Mg 2+ , betaine, and the buffer was 10× isothermal amplification buffer. 1 μL of 0, 2×10 6 copies / μL of EGFR wild-type template, 2×10 6 The EGFR mutant template was 100 copies / μL; after annealing, Evagreen dye and Bst 2.0 DNA polymerase were added respectively; finally, it was placed in a real-time fluorescence quantitative PCR instrument and reacted at 65°C for 80 min.

[0051] (2) Optimization of primer and dNTPs dosage:

[0052] Take 1.0 μL, 2.0 μL, and 3.0 μL of primer premix (including inner and outer primers), dNTPs, and Mg 2+ , betaine, the buffer was 10× isothermal amplification buffer, and 1 μL of 0, 2×10 6 copies / μL of EGFR wild-type template, 2×10 6 The EGFR mutant template was 100 copies / μL; after annealing, Evagreen dye and Bst 2.0 DNA polymerase were added respectively; finally, the real-time fluorescence quantitative PCR instrument was placed and reacted at 65°C for 60 min.

[0053] Take 1.0 μL, 2.0 μL, and 3.0 μL of dNTPs, inner primers, outer primers, and Mg 2+ , betaine, and the buffer was 10× isothermal amplification buffer. 1 μL of 0, 2×10 6 copies / μL of EGFR wild-type template, 2×10 6 The EGFR mutant template was 100 copies / μL; after annealing, Evagreen dye and Bst 2.0 DNA polymerase were added respectively; finally, the real-time fluorescence quantitative PCR instrument was placed and reacted at 65°C for 60 min.

[0054] (3) Design and screening of loop primers:

[0055] LAMP primer design software (http: / / primerexplorer.jp / e / ) was used to screen loop primers for LAMP amplification of the EGFR gene.

[0056] Prepare a 25 μL reaction system, including 2.0 μL of internal primer and external primer premix, 2.0 μL of dNTPs, Mg 2 +, betaine, the buffer was 10× isothermal amplification buffer, 2.0 μL loop primer. 1 μL of 0 and 2×10 6 copies / μL of EGFR wild-type template, 2×10 6 The EGFR mutant template was 100 copies / μL; after annealing, Evagreen dye and Bst 2.0 DNA polymerase were added respectively; finally, the real-time fluorescence quantitative PCR instrument was placed and reacted at 65°C for 60 min.

[0057] in conclusion:

[0058] The present invention designs and optimizes LAMP amplification primers and reaction systems to screen out amplification primers with high amplification efficiency and significant genotype discrimination. The fluorescent signal of the LAMP reaction is collected to prove that the designed LAMP primers have high amplification efficiency (refer to Figure 1 Figure A).

[0059] By optimizing the reaction system, 2.0 μL of primer premix and 2.0 μL of dNTPs were used as the optimal amount, and 2.0 μL of LP primer was used to ensure efficient amplification of the LAMP reaction system (refer to Figure 1 BD diagram).

[0060] Example 2 Design and optimization of the sequence and reaction system of the one-step strand displacement probe

[0061] (1) Probe design:

[0062] The NUPACL website (https: / / www.nupack.org / partition / new) was used to simulate the strand displacement efficiency of the wild-type EGFR gene template and the mutant EGFR gene template with the sequence modified with the FAM fluorescent group (F chain) and the sequence modified with the BHQ quenching group (Q chain), respectively.

[0063] (2) Non-denaturing polypropylene gel electrophoresis:

[0064] 40% polyacrylamide 4.5 mL, 5×TBE solution 750 μL, glycerol 750 μL, 10% ammonium persulfate (AP) 50 μL, tetramethylethylenediamine (TEMED) 16 μL, ultrapure water to 15 mL, prepare 12% non-denaturing polyacrylamide gel. Each 15 μL sample was mixed with 3 μL 6×DNA Loading, and the mixture was added to the electrophoresis tank. In a 4°C refrigerator, the electrophoresis system was used to perform 120 V constant voltage electrophoresis in 1×TBE for 120 min. The non-denaturing polyacrylamide gel after electrophoresis was placed in 50 mL 1×TBE containing 13 μL Safe Red nucleic acid dye for staining for 15 min, and imaged using iBright FL1000 gel imager, and the grayscale of the running band was analyzed using Image J software to determine the extent of the reaction.

[0065] (3) Optimization of strand displacement probe ratio and concentration:

[0066] Take 1 μL, 2 μL, 3 μL, and 4 μL of 10 μM Q chain, 1 μL of 10 μM F chain, 1 μL of 10× isothermal amplification buffer, and deionized water to prepare a 10 μL chain displacement probe system. After annealing, incubate at 95°C for 5 minutes and cool to 25°C at a rate of 0.1°C per second to prepare the chain displacement probe buffer. Take 3.0 μL of chain displacement probe buffer and add 12 μL of 10 mM Mg 2+ and 6.0 μL of 10× isothermal amplification buffer, mixed with 6 μL of 0 and 1 μM EGFR L858R wild-type target mimics and 1 μM EGFR L858R mutant target mimics, respectively, and added deionized water to the volume of 60 μL. After mixing, react at 65 ℃ for 30 minutes. Take 13 μL and put it into a 384-well plate. Set up four parallel samples in each group, detect the fluorescence intensity of the product with an enzyme reader, and screen out the optimal ratio of probe F:Q.

[0067] At the optimal ratio, prepare a strand displacement probe buffer with a F chain concentration of 1 μM. Take 2.1 μL, 2.4 μL, 2.7 μL, and 3.0 μL of strand displacement probe buffer, respectively, and add 12 μL of 10 mM Mg 2+ and 6.0 μL of 10× isothermal amplification buffer, mixed with 6 μL of 0 and 1 μM EGFR L858R wild-type target mimics and 1 μM EGFR L858R mutant target mimics, respectively, and added deionized water to the volume of 60 μL. After mixing, react at 65 ℃ for 30 minutes. Take 13 μL and put it into a 384-well plate. Set up four parallel samples in each group, detect the fluorescence intensity of the product with an enzyme reader, and screen out the optimal concentration of the probe.

[0068] Finally, under the optimal probe ratio and concentration conditions, prepare a strand displacement probe buffer with an F chain concentration of 1 μM. Take 2.7 μL of strand displacement probe buffer and add 12 μL of 10 mM Mg 2+ and 6.0 μL of 10× isothermal amplification buffer, mixed with 6 μL of EGFR L858R mutant target mimics with concentrations of 0, 20 nM, 50 nM, 100 nM, 200 nM, 400 nM, 600 nM, 800 nM, 1000 nM, 1500 nM, 2000 nM, and 2500 nM, respectively, and the volume was supplemented to 60 μL with deionized water. After mixing, react at 65 ℃ for 30 min, take 13 μL and put it into a 384-well plate, set up four parallel samples in each group, and detect the fluorescence intensity of the product with an enzyme reader to detect the sensitivity of the probe.

[0069] in conclusion:

[0070] The present invention designs and optimizes the sequence and reaction system of a one-step strand displacement probe to screen out a strand displacement probe with high sensitivity and significant genotype discrimination.

[0071] The results of non-denaturing polyacrylamide gel electrophoresis showed that the synthesized strand displacement probe could effectively undergo strand displacement after reacting with the mutant EGFR gene sequence (ref. Figure 2 Figure A).

[0072] The fluorescence intensity of the system was read by an ELISA instrument, and the optimal usage ratio of 1:3 F chain:Q chain was optimized. The 45nM strand displacement probe produced the strongest fluorescence signal, the highest signal-to-noise ratio, and good sensitivity (reference Figure 2 BC diagram).

[0073] The probe has a strong linear correlation with the EGFR L858R mutant target mimic in the range of 0-100 nM, and the minimum detection limit can reach 0.531 nM (reference Figure 2 Figure D).

[0074] Example 3 Design and optimization of isothermal amplification-strand displacement probe coupling reaction system

[0075] The strand displacement probe optimized in Example 2 was added to the LAMP reaction system of Example 1, and the fluorescence signals generated by the EGFR L858R wild-type and mutant gene templates at 58°C, 62°C, and 65°C for 30 min were recorded respectively (see Figure 3 AC diagram).

[0076] The results showed that the fluorescence signal generated by the mutant gene sequence was significantly higher than that of the wild-type gene sequence. The strand displacement probe effectively distinguished the EGFR L858R genotype and could detect a minimum of 20 copies / μL of the EGFR L858R wild-type template within 60 min (see Figure 3 Figure D).

[0077] in conclusion:

[0078] The present invention designs and optimizes an isothermal amplification-strand displacement probe coupling reaction system to screen out reaction conditions with high amplification efficiency and significant genotype differentiation.

[0079] The fluorescence signal was collected to prove that the designed isothermal amplification-strand displacement probe coupling reaction system had good amplification efficiency, strong specificity and high sensitivity. By optimizing the reaction system and reacting at 62 °C for 60 min, the coupling reaction system could ensure efficient and specific amplification.

[0080] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A kit for detecting EGFR L858R mutation gene, It is characterized in that The kit comprises a LAMP primer set and a strand displacement probe, wherein: The LAMP primer set includes: a pair of outer primers F3 / B3, a pair of inner primers FIP / BIP, and a loop primer LP, and its nucleotide sequence is as follows: External primer F3: 5′-GCAGCCAGGAACGTACTG-3′ (SEQ ID No. 1); External primer B3: 5′-ATCCTCCCCTGCATGTGT-3′ (SEQ ID No. 2); Internal primer FIP: 5′-TTCCGCACCCAGCAGTTTGGGTGAAAACACCGCAGCATG-3′ (SEQ ID No. 3); Internal primer BIP: 5′-GGAGGCAAAGTAAGGAGGTGGCACAGCTAGTGGGAAGGCA-3′ (SEQ ID No. 4); Loop primer LP: 5′-TTTAGGTCAGCCAGCATTTTCC-3′ (SEQ ID No. 5); The strand displacement probe includes strand F and strand Q, and the nucleotide sequence is as follows: F chain: 5'-GCCCGCCCAAAATCTGTGATCTTGACATGCCGA-3' (SEQ ID No. 6), the 3' end of the nucleotide sequence is modified with a fluorescent group; Q strand: 5'-TCGGCATGTCAAGATCACAGATTTTG-3' (SEQ ID No. 7), the 5' end of the nucleotide sequence is modified with a quenching group and the 3' end is modified with a reverse dT nucleic acid sequence.

2. The EGFR L858R mutant gene detection kit according to claim 1, It is characterized in that The fluorescent group at the 3' end of the F chain is 6-FAM; the quenching group at the 5' end of the Q chain is BHQ1.

3. The EGFR L858R mutant gene detection kit according to claim 1, It is characterized in that The mass ratio of F3, B3, FIP, BIP, and LP of the LAMP primer set is 1:1:4:4:

2.

4. The EGFR L858R mutant gene detection kit according to claim 1, It is characterized in that The molar ratio of F chain to Q chain was 1:

3.

5. The EGFR L858R mutant gene detection kit according to claim 1, It is characterized in that The kit specifically includes: each 25 μL reaction system contains: 2.0 μL of primer premix consisting of outer primers and inner primers, 2.0 μL of LP primer, 2.0 μL of dNTPs, 0.5 μL of Mg 2+ , 1.125 μL strand displacement probe, 5.0 μL betaine, 2.5 μL isothermal amplification buffer, 1.0 μL Bst 2.0 DNA polymerase, 1 μL EGFR gene template, and deionized water.

6. The EGFR L858R mutant gene detection kit according to claim 5, It is characterized in that The concentration of strand F of the strand displacement probe was 1 μM, the concentration of dNTPs was 10 mM, and the concentration of Mg was 10 mM. 2+ The concentration of isothermal amplification buffer was 10×, the concentration of Bst 2.0 DNA polymerase was 8 U, and the concentration of EGFR gene template was 2×10 6 copies / μL; dNTPs include dATP, dCTP, dGTP, and dTTP, and the mass ratio of the four is 1:1:1:1; the primer premix includes 5 μM outer primer F3, 5 μM outer primer B3, 20 μM inner primer FIP, and 20 μM inner primer BIP; The isothermal amplification buffer consisted of 0.2 M Tris, 0.1 M (NH 4 ) 2 SO 4 , 0.5 M KCl, 0.02 M MgSO 4 and 0.4 g Tween-20.

7. Use of the EGFR L858R mutant gene detection kit according to any one of claims 1 to 6 in the early screening of non-small cell lung cancer for non-disease purposes and / or the preparation of EGFR-TKIs targeted drugs.

8. A method for detecting EGFR L858R mutant gene for non-disease purposes, It is characterized in that The method adopts isothermal amplification technology and specifically comprises the following steps: (1) Prepare the DNA template sample to be tested; (2) preparing a reaction system, wherein the reaction system comprises the primer premix as described in claim 5 or 6, a strand displacement probe, dNTPs, betaine, an isothermal amplification buffer, Mg 2+ and Bst 2.0 DNA polymerase; (3) Add the sample to be tested into the reaction system and perform a constant temperature amplification reaction at 62°C for 60 minutes; (4) Determine the presence of the EGFR L858R mutant gene by monitoring changes in the fluorescence signal.

9. The method according to claim 8, It is characterized in that In step (2), in the amplification system, the primer premix includes 5 μM outer primer F3, 5 μM outer primer B3, 20 μM inner primer FIP and 20 μM inner primer BIP, the concentration of the strand displacement probe F chain is 1 μM, the concentration of dNTPs is 10 mM, and the concentration of Mg is 10 mM. 2+ The concentration of is 100 mM, the concentration of betaine is 5 M, the concentration of isothermal amplification buffer is 10×, the concentration of Bst 2.0 DNA polymerase is 8 U, and the concentration of the DNA sample to be tested is 5 ng / μL.

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