CN111662986B

CN111662986B - Method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S

Info

Publication number: CN111662986B
Application number: CN202010726064.5A
Authority: CN
Inventors: 唐东江; 赵计昌; 黄雅菁
Original assignee: Zhuhai Livzon Cynvenio Diagnostics Ltd
Current assignee: Zhuhai Livzon Cynvenio Diagnostics Ltd
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2022-07-12
Anticipated expiration: 2040-07-24
Also published as: CN111662986A

Abstract

The invention provides a method and a probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, and relates to the technical field of gene detection. The method comprises the steps of respectively using a probe system for selectively inhibiting T790 wild type and a probe system for selectively inhibiting C797 wild type to carry out PCR amplification on a sample, sequencing each amplification product, and judging the mutation configuration of T790 and C797 in the sample according to the sequencing result. The method can effectively distinguish cis-trans mutation configurations of T790M and C797S, has very high specificity, and can directly give out sample sequence information; meanwhile, the kit has the advantage of high sensitivity, and can detect mutation as low as 0.1 percent by adopting two-stage PCR amplification sequentially subjected to biased amplification and non-biased amplification and adopting Sanger sequencing with low cost; NGS sequencing can reduce cost by reducing sequencing depth while maintaining high sensitivity.

Description

Method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S

Technical Field

The invention relates to the technical field of gene detection, in particular to a method and a probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S.

Background

EGFR mutation mainly occurs on the first four exons (18-21) of an intracellular Tyrosine Kinase (TK) region, and more than 30 TK region mutations are discovered at present. Among these, the T790M and C797S mutations on exon 20 are the most troublesome types of gene mutations found in tumor patients. The EGFR gene T790M specifically refers to that the 790 th amino acid site of the No. 20 exon of an Epidermal Growth Factor Receptor (EGFR) is mutated from threonine (T) to methionine (G), and the gene level is expressed as c.2369C > T; the EGFR gene C797S specifically refers to EGFR No. 20 exon 797 amino acid position mutated from cysteine (C) to serine (S), and the gene level is expressed as c.2389T > A or c.2390G > C. When T790M and C797S are mutated simultaneously, T790M and C797S are located on the same chromosome, and this configuration is called cis (as the major type, accounting for 85%); T790M and C797S are located on different chromosomes in a configuration referred to as trans, and a schematic representation of cis and trans gene mutations for T790M and C797S is shown in fig. 1.

TKI is always an important drug in targeted therapy of EGFR mutant NSCLC, but most patients with sensitive EGFR mutation have drug resistance development in 9-13 months of first-generation EGFR TKI treatment. The EGFR T790M mutation accounted for 40% to 60% of all resistance mechanisms. The third generation EGFR-TKI Tagrusso/Tagrisso (also known as Oxitinib/Osimetinib, AZD9291) is irreversible selective TKI, can effectively cope with the new generation targeted drug of T790M mutation, brings hope for overcoming the secondary drug resistance of T790M, and finally can resist the drug. After about 11 months of using ocitinib, 30% to 40% will have secondary EGFR C797S mutation.

The results of the current research show that the T790M and C797S mutations and different configurations are extremely related to the drug resistance, curative effect prediction and prognosis of tumor patients, for example: when EGFR genes C797S and T790M are trans-mutated, it is clinically effective to use first and third generation EGFR-TKI combination therapies (e.g., Tarceva in combination with Oxititinib). Whereas when C797S and T790M were cis-mutated, tumors were resistant to all two or three generations of EGFR-TKI, see table 1.

TABLE 1

In the prior art, a second generation gene sequencing technology (NGS) is the only high-sensitivity and high-specificity detection method capable of directly providing sequence information to clearly distinguish cis-trans mutation configurations of C797S and T790M, and other PCR technologies such as ARMS PCR or digital PCR can only indirectly infer mutation configurations through fluorescence signals detected by single points, cannot provide sequence information and cannot directly judge the configurations accurately without disputes. However, the NGS technique has the characteristics of large throughput, high accuracy and abundant information, but is long in time, complex in experimental apparatus and procedures, high in cost and difficult to meet the detection requirements of all patients. Therefore, a method and a product which have high detection sensitivity, short detection period and low cost and can directly obtain T790M and C797S mutation sequence information so as to accurately judge cis-trans mutation configurations are still lacking at present.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The first purpose of the invention is to provide a method for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, wherein the method can at least distinguish the cis mutation or the trans mutation of EGFR-T790M and EGFR-C797S.

The second purpose of the invention is to provide a probe for blocking amplification of EGFR-T790 wild type and a probe for blocking amplification of EGFR-C797 wild type respectively.

The third purpose of the invention is to provide a reagent and/or a kit for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S.

In order to solve the technical problems, the invention adopts the following technical scheme:

according to one aspect of the invention, the invention provides a method for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, comprising:

amplifying a sample by using a PCR system for selectively inhibiting EGFR-T790 wild type amplification to obtain a first amplification product, wherein the PCR system for selectively inhibiting EGFR-T790 wild type amplification contains a probe for blocking amplification of EGFR-T790 wild type;

amplifying the sample by using a PCR system for selectively inhibiting EGFR-C797 wild type amplification to obtain a second amplification product; the PCR system for selectively inhibiting EGFR-C797 wild type amplification contains a probe for blocking amplification of EGFR-C797 wild type;

sequencing the first amplification product and the second amplification product respectively, and judging the mutation configuration of EGFR-T790M and EGFR-C797S in the sample according to the sequencing result.

According to another aspect of the invention, the invention also provides a probe for blocking amplification of EGFR-T790 wild type, wherein the nucleotide sequence of the probe is shown in SEQ ID NO. 1: 5' -CACCGTGCAGCTCATCACGCAGCTC-3'; or as shown in SEQ ID NO. 7: 5' -GCTCATCACGCAGCTCAT-3'; the underlined nucleotides in the probe shown in SEQ ID No.1 and the probe shown in SEQ ID No.7 are nucleotides that have been modified to enhance the thermal stability of the probe with the complementary strand; the 3' end of the probe is modifiedTo block extension of the probe during amplification.

According to another aspect of the present invention, the present invention also provides a probe for blocking amplification of EGFR-C797 wild type, the nucleotide sequence of the probe is represented by SEQ ID No. 2: 5' -GCCCTTCGGCTGCCTCCT-3'; underlined nucleotides are nucleotides that are modified to enhance the thermal stability of the probe to the complementary strand; the 3' end of the probe is modified to block extension of the probe during amplification.

According to another aspect of the invention, the invention also provides a set of probes for detecting the cis-trans mutation configuration of EGFR-T790M and EGFR-C797S, which comprises a probe for blocking amplification of EGFR-T790 wild type and a probe for blocking amplification of EGFR-C797 wild type.

According to one aspect of the invention, the invention also provides a reagent and/or a kit for detecting the cis-trans mutation configuration of EGFR-T790M and EGFR-C797S, wherein the reagent and/or the kit comprises the probe set.

Compared with the prior art, the invention has the following beneficial effects:

according to the method for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, provided by the invention, a probe capable of blocking a wild-type template is added in the PCR amplification process, so that the templates in a sample containing a small amount of mutant templates are greatly enriched for subsequent sequencing analysis, and at least the following mutation configurations of the sample can be detected: wild type, T790M single point mutation, C797S single point mutation, T790M and C797S cis mutation, and T790M and C797S trans mutation. The detection method provided by the invention solves the problems of complex experimental design, large data analysis amount and high cost caused by directly sequencing a sample by adopting second-generation sequencing. Can effectively distinguish cis-trans mutation configurations of T790M and C797S, has very high specificity, and can directly provide sample sequence information; meanwhile, the method also has the advantage of high sensitivity, and low-cost Sanger sequencing can detect mutation as low as 0.1%; NGS sequencing can reduce cost by reducing sequencing depth while maintaining high sensitivity.

The probe for blocking amplification of the EGFR-T790 wild type and the probe for blocking amplification of the EGFR-C797 wild type, which are provided by the invention, can inhibit amplification of wild type genes and avoid influence on amplification of variant genes at the same time by optimizing the sequence of the probes, and play a role in high enrichment efficiency and good detection sensitivity of the variant genes, wherein the probe for blocking amplification of the EGFR-T790 wild type can block amplification of the wild type and single-point C797S mutant DNA, and does not block or hardly blocks amplification of the single-point T790M mutation and cis-mutation T790M C & T797S T > A or G > C DNA; the probe for blocking amplification of EGFR-C797 wild type can block amplification of DNA of wild type and single point T790M mutation, and does not block or slightly block amplification of DNA of single point C797S T > A or G > C and cis mutation T790M C > T & C797S T > A or G > C. The probe provided by the invention can detect 0.1% mutation frequency by adopting Sanger sequencing.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic representation of cis-and trans-gene mutations of T790M and C797S;

FIG. 2 is a schematic diagram of the detection of the method for detecting the mutant configurations of EGFR-T790M and EGFR-C797S;

FIG. 3 shows the amplification result of T790M C > T plasmid template amplified by the amplification system containing Blocker 1;

FIG. 4 shows the amplification result of the amplification system containing Blocker1 for the C797S T > A plasmid template;

FIG. 5 shows the amplification result of the amplification system containing Blocker1 for amplification of the C797S G > C plasmid template;

FIG. 6 shows the amplification result of the amplification system containing Blocker1 for the T790M C > T & C797S T > A plasmid template;

FIG. 7 shows the amplification result of the amplification system containing Blocker1 for the T790M C > T & C797S G > C plasmid template;

FIG. 8 shows the amplification result of the wild-type plasmid template amplified by the amplification system containing Blocker 1;

FIG. 9 shows the amplification result of T790M C > T plasmid template amplified by the amplification system containing Blocker 2;

FIG. 10 shows the amplification result of the amplification system containing Blocker2 for the C797S T > A plasmid template;

FIG. 11 shows the amplification result of the amplification system containing Blocker2 for amplification of the C797S G > C plasmid template;

FIG. 12 shows the result of amplification of T790M C > T & C797S T > A plasmid template by an amplification system containing Blocker 2;

FIG. 13 shows the amplification result of the amplification system containing Blocker2 for the T790M C > T & C797S G > C plasmid template;

FIG. 14 shows the amplification result of the amplification of wild-type plasmid template by the amplification system containing Blocker 2;

FIG. 15 shows the sequencing of the Blocker1 product from plasmid 001 (wild-type);

FIG. 16 shows the sequencing of the Blocker2 product from plasmid 001 (wild-type);

FIG. 17 shows the sequencing result of plasmid 002(T790M C > T single point mutant) Blocker1 product;

FIG. 18 shows the sequencing results of the plasmid 002(T790M C > T single point mutant) Blocker2 product;

FIG. 19 shows the sequencing of plasmid 003(C797S T > A single point mutant) Blocker 1;

FIG. 20 shows the sequencing of plasmid 003(C797S T > A single point mutant) Blocker 2;

FIG. 21 shows the sequencing results of the Blocker1 product from plasmid 004(C797S G > C single point mutant);

figure 22 is the result of sequencing of plasmid 004(C797S G > C single point mutant) Blocker2 product;

FIG. 23 is the result of sequencing the Blocker1 product of sample TC 01;

FIG. 24 shows the result of sequencing Blocker2 products of sample TC 01;

FIG. 25 is the result of sequencing the Blocker1 product of sample TC 02;

FIG. 26 is the result of sequencing the Blocker2 product of sample TC 02;

FIG. 27 is the result of sequencing the Blocker1 product of sample TC 03;

FIG. 28 is the result of sequencing the Blocker2 product of sample TC 03;

FIG. 29 is the result of sequencing the Blocker1 product of sample TC 04;

FIG. 30 is the result of sequencing the Blocker2 product of sample TC 04;

FIG. 31 is the result of sequencing the Blocker1 product of sample TC 05;

FIG. 32 is the result of sequencing the Blocker2 product of sample TC 05;

FIG. 33 is the result of sequencing the Blocker1 product of sample TC 06;

FIG. 34 is the result of sequencing the Blocker2 product of sample TC 06;

FIG. 35 is the result of sequencing the Blocker1 product of sample TC 07;

FIG. 36 is the result of sequencing the Blocker2 product of sample TC 07;

FIG. 37 is the result of sequencing the Blocker1 product of sample TC 08;

FIG. 38 is the result of sequencing the Blocker2 product of sample TC 08;

FIG. 39 is the result of sequencing the Blocker1 product of sample TC 09;

FIG. 40 is the result of sequencing the Blocker2 product of sample TC 09;

FIG. 41 is the result of sequencing the Blocker1 product of sample TC 10;

FIG. 42 is the result of sequencing the Blocker2 product of sample TC 10;

FIG. 43 is the result of sequencing the Blocker1 product of sample TC 11;

FIG. 44 is the result of sequencing the Blocker2 product of sample TC 11;

FIG. 45 is the result of sequencing the Blocker1 product of sample TC 12;

FIG. 46 is the result of sequencing the Blocker2 product of sample TC 12;

FIG. 47 is the result of sequencing the Blocker1 product of sample TC 13;

FIG. 48 is the result of sequencing the Blocker2 product of sample TC 13;

FIG. 49 shows the result of sequencing the Blocker1 product of sample TC 14;

FIG. 50 is the result of sequencing the Blocker2 product of sample TC 14;

FIG. 51 is the result of sequencing the Blocker1 product of sample TC 15;

FIG. 52 is the result of sequencing the Blocker2 product of sample TC 15;

FIG. 53 is the result of sequencing the Blocker1 product of sample TC 16;

FIG. 54 is the result of sequencing the Blocker2 product of sample TC 16;

FIG. 55 shows the sequencing of the Blocker3 product from plasmid 001 (wild-type);

FIG. 56 shows the sequencing of the Blocker4 product from plasmid 001 (wild-type);

FIG. 57 shows the sequencing results of the plasmid 002(T790M C > T single point mutant) Blocker3 product;

FIG. 58 shows the sequencing results of the plasmid 002(T790M C > T single point mutant) Blocker4 product;

FIG. 59 shows the sequencing of plasmid 003(C797S T > A single point mutant) Blocker 3;

FIG. 60 shows the sequencing of plasmid 003(C797S T > A single point mutant) Blocker 4;

FIG. 61 shows the sequencing of plasmid 004(C797S G > C single point mutant) Blocker3 products;

FIG. 62 shows the sequencing of plasmid 004(C797S G > C single point mutant) Blocker4 products;

FIG. 63 shows the amplification results of the amplification system containing Blocker5 for wild-type plasmid template;

FIG. 64 shows the amplification result of T790M C > T plasmid template amplified by the amplification system containing Blocker 5;

FIG. 65 shows the amplification result of the amplification system containing Blocker5 for the C797S T > A plasmid template;

FIG. 66 shows the amplification result of the amplification system containing Blocker5 for the C797S G > C plasmid template;

FIG. 67 is the result of amplification of T790M C > T & C797S T > A plasmid template by an amplification system containing Blocker 5;

FIG. 68 shows the result of amplification of T790M C > T & C797S G > C plasmid template by an amplification system containing Blocker 5;

FIG. 69 shows the amplification result of the wild-type plasmid template amplified by the amplification system containing Blocker 6;

FIG. 70 shows the amplification result of T790M C > T plasmid template amplified by the amplification system containing Blocker 6;

FIG. 71 shows the amplification result of the amplification system containing Blocker6 for the C797S T > A plasmid template;

FIG. 72 is an amplification result of the amplification of C797S G > C plasmid template by the amplification system containing Blocker 6;

FIG. 73 shows the amplification result of the amplification system containing Blocker6 for the T790M C > T & C797S T > A plasmid template;

FIG. 74 shows the amplification results of the amplification system containing Blocker6 for the T790M C > T & C797S G > C plasmid template.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

According to another aspect of the invention, the invention provides a method for detecting the cis-trans mutation configuration of EGFR-T790M and EGFR-C797S, the method comprising: amplifying a sample by using a PCR system for selectively inhibiting EGFR-T790 wild type amplification to obtain a first amplification product; amplifying the sample by using a PCR system for selectively inhibiting EGFR-C797 wild type amplification to obtain a second amplification product; and sequencing the first amplification product and the second amplification product respectively, and judging the mutation configurations of EGFR-T790M and EGFR-C797S in the sample according to the sequencing result. The invention enables a PCR system to selectively inhibit the amplification of a corresponding wild type sample during amplification by respectively using a probe for blocking amplification of EGFR-T790 wild type and a probe for blocking amplification of EGFR-C797 wild type.

In the invention, the EGFR-T790 wild type refers to that the EGFR is wild type at the T790 locus, and other sites of the EGFR can be either wild type or non-wild type, for example, a sample is wild type at the T790 locus, and the C797 locus is mutated, and can also be understood as EGFR-T790 wild type; the EGFR-C797 wild type refers to the EGFR which is wild type at the C797 site, and other sites of the EGFR can be wild type or non-wild type, for example, the sample is wild type at the C797 site, and the T790 site is mutated, and the EGFR-C797 wild type can also be understood.

In the invention, the first amplification product refers to a product obtained after PCR amplification of a system containing a probe for blocking amplification of EGFR-T790 wild type, and is also called a Blocker1 product, a Blocker3 product or a Blocker5 product in some embodiments; the second amplification product refers to a product obtained after PCR amplification of a system containing a probe for blocking amplification of the wild type of EGFR-C797, and is also referred to as a Blocker2 product, a Blocker4 product or a Blocker6 product in some embodiments; it is understood that the first amplification product and the second amplification product are only used for distinguishing the two groups of amplification products, and are not to be construed as limiting the importance of the amplification products and the order of amplification reactions in PCR; similarly, the numbering of the probes, for example, Blocker 1-6, is not intended to limit the order of importance of the probes. The skilled person can arbitrarily name the probes with different sequences and the amplification products of different amplification systems according to the actual needs, and the invention is not limited to this.

In the method provided by the invention, sequencing can be performed by any sequencing method acceptable in the field, the sequencing is used for obtaining the base type of the mutation site, so any sequencing method capable of obtaining the nucleotide sequence of the amplification product can be used, the method is not limited to the method, and the method comprises but is not limited to Sanger sequencing or NGS sequencing, and experiments show that mutation as low as 0.1% can be detected by low-cost Sanger sequencing; the use of NGS sequencing also reduces cost by reducing sequencing depth while maintaining high sensitivity.

In some preferred embodiments, the probe for blocking amplification of EGFR-T790 wild type and the probe for blocking amplification of EGFR-C797 wild type are independently in accordance with (a) - (C):

(a) the nucleotide sequence is completely matched with the wild target gene segment, and is mismatched with the variant target gene segment at the variant position.

(b) The length is 24-50 bp, such as but not limited to 24bp, 25bp, 26bp, 27bp, 28bp, 29bp, 30bp, 31bp, 32bp, 33bp, 34bp, 35bp, 36bp, 37bp, 38bp, 39bp, 40bp, 41bp, 42bp, 43bp, 44bp, 45bp, 46bp, 47bp, 48bp, 49bp or 50bp, wherein within the range of 1-5 bp at the variation position and two sides thereof, such as but not limited to 1bp, 2bp, 3bp, 4bp or 5 bp; at least 1 to 6 nucleotides are modified to enhance the thermal stability of the probe to the complementary strand, such as but not limited to 1, 2, 3, 4, 5 or 6 nucleotides, preferably including locked nucleic acid or peptide nucleic acid modifications.

(c) When annealed, can bind to the wild-type target gene fragment and the variant target gene fragment, and when extended, only binds to the wild-type target gene fragment.

The probe meeting the above conditions can be combined with the wild type template with high binding force in an annealing state and combined with the variant template with low binding force, and can be combined with the wild type template only in an extension state, so that the balance is achieved on the inhibition of the amplification of the wild type gene fragment and the inhibition of the amplification of the variant gene fragment is achieved, the inhibition of the amplification of the wild type gene fragment is limited to the maximum extent, and the influence on the amplification of the variant gene fragment is avoided. Meanwhile, the probe meeting the conditions is adopted, and the probe and the primer of the amplified target segment are in a non-competitive relationship, namely the design of the primer is not limited to be close to a mutation point, so that the selection range of the primer is wider, and the requirements of various technical platforms on the primer can be met.

In some preferred embodiments, the probe for blocking amplification of the EGFR-T790 wild type and the probe for blocking amplification of the EGFR-C797 wild type are respectively and independently a non-quenched probe, the non-quenched probe does not need labeling modification of a fluorescent group and a quenching group, the probe cost is reduced, the application range of the non-quenched probe is wide, the non-quenched probe is suitable for various platforms, and the amplified and enriched PCR product can be directly used for sequencing.

In some preferred embodiments, the 3 'end of the probe for blocking amplification of EGFR-T790 wild type and the 3' end of the probe for blocking amplification of EGFR-C797 wild type are independently modified to block extension of the probes during amplification, preventing the probes from increasing in Tm due to increased length, which results in inhibition of amplification of the variant gene. The modification of the 3' end of the probe preferably comprises a dideoxy modification, an amino modification or a phosphorylation modification.

In some preferred embodiments, the nucleotide sequence of the probe for blocking amplification of the wild-type EGFR-T790 is set forth in SEQ ID No. 1: 5' -CACCGTGCAGCTCATCACGCAGCTC-3'; or as shown in SEQ ID NO. 7: 5' -GCTCATCACGCAGCTCAT-3'; the underlined nucleotides in the probe shown in SEQ ID NO.1 and the probe shown in SEQ ID NO.7 are locked nucleic acid modified nucleotides. The probe can block amplification of wild type and single-point C797S mutant DNA, and does not block or slightly blocks single-point T790M mutation and cis-mutation T790M C>T&C797S T>A or G>C, DNA amplification.

In some preferred embodiments, the probe for blocking amplification of EGFR-C797 wild type has the nucleotide sequence set forth in SEQ ID No. 2: 5' -GCCCTTCGGCTGCCTCCT-3'; underlined nucleotides are locked nucleic acid modified nucleotides. The probe can block amplification of wild type and single-point T790M mutant DNA, and does not block or slightly blocks single-point C797S T>A or G>C and cis mutation T790M C>T&C797S T>A or G>C, amplifying the DNA.

In some preferred embodiments, the samples are PCR amplified using the following primer pairs:

SEQ ID NO.3：5’-TGGACAACCCCCACGTGT-3’；

SEQ ID NO.4：5’-AGCCAATATTGTCTTTGTGTTCC-3’。

in some preferred embodiments, PCR amplification comprises biased amplification with an annealing extension temperature of 53 ℃ to 63 ℃, such as, but not limited to, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, or 63 ℃, preferably 53 ℃ to 59 ℃, and more preferably 56 ℃.

Biased amplification means that a Blocker (probe) strictly blocks amplification wild-type PCR reaction conditions, namely Blocker-PCR reaction conditions, such as annealing extension temperature of 56 ℃ in some embodiments, so that the Blocker and a wild-type template are tightly combined to efficiently block wild-type amplification; and in the biased amplification stage, Blocker is not combined with the mutant template, and the mutant template can be normally amplified or slightly blocked.

In some preferred embodiments, the PCR amplification is a two-stage amplification comprising a biased amplification followed by a non-biased amplification; the non-biased amplification means that Blocker does not bind with a wild type or mutant template sequence at a higher extension temperature, and loses the blocking function, and both the wild type and the mutant template can be normally amplified. The objective is to increase the amount of PCR product and to enable the sequencing method to meet the requirement for the amount of sample. When the Sanger sequencing method is adopted for sequencing, a two-section amplification PCR amplification method is preferably adopted, if the whole process is biased amplification, the quantity of the wild sample PCR end products is too small, the requirement of Sanger sequencing sample loading quantity cannot be met, and the wild sample cannot give out sequence information. The two-stage PCR design has the advantages that the mutant template in the sample can be enriched in the biased amplification stage of the mutant template; and in the non-biased amplification stage, the sample size is synchronously amplified, and both wild type samples and mutant type samples can meet the requirement of Sanger sequencing sample loading.

The EGFR-T790M and EGFR-C797S mutant configurations provided by the invention comprise wild type, T790M single-point mutation, C797S single-point mutation, T790M and C797S cis-mutation, and T790M and C797S trans-mutation. The principle is shown in fig. 2.

If the sample is wild type, the sites of T790 and C797 on both chromosomes can be blocked by both probes. Optionally, reference is made to the following manner: if the T790 site and the C797 site in the first amplification product and the second amplification product are both C and TG; the sample is judged to be wild type.

If the sample is a T790M single point mutation, the sample is a C797 wild type, the probe for blocking amplification of the EGFR-C797 wild type can also block amplification of the target fragment, and a T790M mutation site cannot be detected in the second amplification product, and the probe for blocking amplification of the EGFR-T790 wild type can enrich a T790M single point mutation fragment, so that a T790M single point mutation is detected in the first amplification product; optionally, reference is made to the following manner: if the T790 site in the first amplification product is T, the C797 site is TG; the T790 site in the second amplification product is C, and the C797 site is TG; the sample is judged to be a T790M single point mutation.

If the sample is a C797S single point mutation, the sample is a T790 wild type, and a probe for blocking amplification of the EGFR-T790 wild type can block amplification of a target fragment and cannot detect a C797S mutation site in a second amplification product; the probe used to block amplification of EGFR-C797 wild type was able to enrich for the C797S single point mutation fragment, thereby detecting a C797S single point mutation in the second amplification product; optionally, reference is made to the following manner: if the T790 site in the first amplification product is C, the C797 site is TG; and if the T790 site in the second amplification product is C and the C797 site is AG or TC, the sample is judged to be the C797S single point mutation. More specifically, if the C797 site is AG, the sample is a C797S T > a type mutation; if the C797 site is TC, the sample is C797S G > C type mutation.

If the sample is T790M and C797S cis-form mutation, one chromosome in the sample contains two kinds of mutation at the same time, and the other chromosome does not contain mutation, then the two probes can be enriched to obtain fragments containing mutation, the amplification of the fragments which are expressed as wild type is blocked, and thus the amplification products of the two probes can detect two kinds of mutation sites. Optionally, reference is made to the following manner: if the T790 site in both the first amplification product and the second amplification product is T; if the C797 site in the first amplification product and the second amplification product is AG or TC at the same time, the samples are T790M and C797S cis-mutations; more specifically, if the C797 site in the first amplification product and the second amplification product is AG at the same time, the mutation configuration is T790M C > T & C797S T > A cis-mutation; if the C797 site in the first amplification product and the second amplification product is TC at the same time, the mutation configuration is T790M C > T & C797S G > C cis-mutation.

If the sample is T790M and C797S trans mutation, the mutation sites contained in the two chromosomes in the sample are different; the fragment containing only the C797S mutation is also wild-type for an amplification system containing a probe for blocking amplification of the wild-type EGFR-T790, so that the amplification system can only amplify the fragment containing the T790M, and only the T790M mutation site can be detected in an amplification product; the fragment containing only the T790M mutation is also wild-type for an amplification system containing a probe for blocking amplification of EGFR-C797 wild-type, so that the amplification system can only amplify the fragment containing C797S, and only the C797S mutation site can be detected in an amplification product; i.e., the mutation sites in the amplification products of the two probes are different. Optionally, reference is made to the following manner: if the T790 site in the first amplification product is T, the C797 site is TG; if the T790 site in the second amplification product is C and the C797 site is AG or TC, the sample is judged to be T790M and C797S trans mutation; more specifically, if the C797 site in the second amplification product is AG, the mutation configuration is T790M C > T & C797S T > a trans mutation; if the C797 site in the second amplification product is TC, the mutation configuration is T790M C > T & C797S G > C trans mutation.

The T790 site is C or T, which refers to the gene level expression of the amplified product corresponding to the 790 th amino acid site of the EGFR No. 20 exon, i.e., the base type of the 2369 th nucleotide; the above-mentioned AG or TC at the C797 site means that the expression level of the gene corresponding to the 797 amino acid site of the EGFR exon 20 in the amplified product, i.e., the base types of nucleotides 2389 and 2390, is expressed.

It is noted that the method for detecting the cis-trans mutation configurations of EGFR-T790M and EGFR-C797S provided by the present invention is a method for non-diagnostic and therapeutic purposes, and can be used for studies including, but not limited to, the relationship between tumor resistance and the pre-cis mutation configurations of EGFR-T790M and EGFR-C797S; for the study of the relationship between tumor incidence or tumor type and EGFR-T790M and EGFR-C797S mutation configuration; the method is used for developing new drugs for treating tumors or evaluating the drug effect of known drugs for treating tumors. It is understood that the knowledge of the EGFR-T790M and EGFR-C797S mutation patterns does not provide a direct diagnosis of whether a tumor is present or the type of tumor, nor is the method provided herein directly used to guide the treatment of tumors.

Based on the method for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, the invention also provides a probe for blocking amplification of EGFR-T790 wild type and a probe for blocking amplification of EGFR-C797 wild type respectively.

The nucleotide sequence of the probe for blocking amplification of the EGFR-T790 wild type is shown as SEQ ID NO. 1: 5' -CACCGTGCAGCTCATCACGCAGCTC-3'; or as shown in SEQ ID NO. 7: 5' -GCTCATCACGCAGCTCAT-3'; the underlined nucleotides in the probe shown in SEQ ID No.1 and the probe shown in SEQ ID No.7 are nucleotides that have been modified to enhance the thermal stability of the probe with the complementary strand; preferably, the underlined nucleotides are locked nucleic acid modified nucleotides or peptide nucleic acid modified nucleotides; the 3' end of the probe is modified to block extension of the probe during amplification; preferably, the 3' end of the probe is dideoxy modified, amino modified or phosphorylated. The probe for blocking amplification of the wild type EGFR-T790 can block amplification of wild type and single-point C797S mutant DNA, and does not block or slightly block single-point T790M mutation and cis-mutation T790M C>T&C797S T>A or G>C, DNA amplification.

The nucleotide sequence of the probe for blocking amplification of the wild type EGFR-C797 is shown as SEQ ID NO. 2: 5' -GCCCTTCGGCTGCCTCCT-3'; underlined nucleotides are nucleotides that are modified to enhance the thermal stability of the probe to the complementary strand; preferably, the underlined nucleotides are locked nucleic acid modified nucleotides or peptide nucleic acid modified nucleotides; the 3' end of the probe is modified to block extension of the probe during amplification; preferably, the 3' end of the probe is dideoxy modified, amino modified or phosphorylated. The probe for blocking amplification of the EGFR-C797S wild type can block amplification of wild type and single-point T790M mutant DNA, and does not block or slightly blocks single-point C797S T>A or G>C and cis mutation T790M C>T&C797S T>A or G>C ofAnd (3) DNA amplification.

Based on the method for detecting the cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, the invention also provides a set of probes for detecting the cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, wherein the set of probes comprises a probe for blocking amplification of the EGFR-T790 wild type and a probe for blocking amplification of the EGFR-C797 wild type. Preferably, the probe for blocking amplification of the wild type EGFR-T790 is the probe with the nucleotide sequence shown in SEQ ID NO.1 or SEQ ID NO.7, and/or the probe for blocking amplification of the wild type EGFR-C797 is the probe with the nucleotide sequence shown in SEQ ID NO. 2.

Based on the method for detecting the cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, the invention also provides a reagent and/or a kit for detecting the cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, and the kit comprises the probe set. The method for using the reagent and/or the kit can refer to the method for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S, and preferably the reagent and/or the kit provided by the invention can be used by adopting the method for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S.

In some alternative embodiments, the kit further comprises other amplification reagents and/or amplification consumables. Alternatively, the probe for blocking amplification of EGFR-T790 wild type, the probe for blocking amplification of EGFR-C797 wild type are provided in a separate package with the other amplification reagents, or in a single reagent mixed with the other amplification reagents. Optionally, the additional amplification reagents comprise one or more of primer pairs, DNA polymerase, buffers, dNTPs, enzyme-free water, and double-stranded DNA dyes; further optionally, the other amplification reagents are provided in a single package when they are plural, or at least two of the other amplification reagents are provided in the form of a mixed single reagent.

In some preferred embodiments, the nucleotide sequences of the primer pairs are as follows:

SEQ ID NO.3：5’-TGGACAACCCCCACGTGT-3’；

SEQ ID NO.4：5’-AGCCAATATTGTCTTTGTGTTCC-3’。

the technical solution and the advantages of the present invention will be further explained with reference to the preferred embodiments.

Example 1

This example provides a kit for detecting the cis-trans mutation types of EGFR-T790M and EGFR-C797S, having the composition shown in the following table:

TABLE 2

(2) The experimental procedure of the kit is as follows

TABLE 3

First, comparison of blocking performance of Blocker 1:

plasmid DNA with different mutation information was used as template, and the plasmid DNA information is shown in the following table:

TABLE 4

Name (R)	Type of mutation
		Plasmid 001	Wild Type (WT)
Plasmid 002	T790M C>T
		Plasmid 003	C797S T>A
Plasmid 004	C797S G>C
		Plasmid 005	T790M C>T&C797S T>A
Plasmid 006	T790M C>T&C797S G>C

The system was formulated according to the following formulation and the PCR procedure is shown in Table 3.

TABLE 5

The results are shown in FIGS. 3-8, where Blocker1 blocks amplification of wild-type and single-site C797S mutant DNA, and does not block or hardly block amplification of DNA of single-site T790M mutation and cis-mutation T790M C > T & C797S T > A or G > C.

(II) comparison of blocking Performance of Blocker2

TABLE 6

Name(s)	Type of mutation
		Plasmid 001	Wild Type (WT)
Plasmid 002	T790M C>T
		Plasmid 003	C797S T>A
Plasmid 004	C797S G>C
		Plasmid 005	T790M C>T&C797S T>A
Plasmid 006	T790M C>T&C797S G>C

TABLE 7

The results are shown in FIGS. 9-14, where Blocker2 blocked amplification of wild-type and single-site T790M mutant DNAs, and did not block or little block amplification of single-site C797S T > A or G > C and cis-mutation T790M C > T & C797S T > A or G > C.

(III) detection of samples with different mutation ratios

Plasmid DNA with different mutation information is used as a template to prepare samples to be tested with different mutation forms and different mutation frequencies, and the preparation method is shown in the following table:

TABLE 8

TABLE 9

The judgment method is as follows:

watch 10

And (3) checking the product, and performing primary sequencing: the results are as follows:

the sequencing results of the plasmid 001 (wild type) Blocker1 product are shown in fig. 15; the result of sequencing the Blocker2 product is shown in FIG. 16: the T790 site base C and the C797 site base TG are on the same DNA strand, so the sample is wild type.

The sequencing result of the plasmid 002(T790M C > T single point mutant) Blocker1 product is shown in FIG. 17; the result of sequencing the Blocker2 product is shown in FIG. 18: the T790M C > T mutation and the C797 site base TG are on the same DNA strand, so the sample is T790M C > T single point mutation.

The sequencing results of the plasmid 003(C797S T > A single point mutant) Blocker1 product are shown in FIG. 19; the result of sequencing the Blocker2 product is shown in FIG. 20: the base C of T790 site and the mutation of C797S T > A are on the same DNA chain, so the sample is the single point mutation of C797S T > A.

The sequencing results of the plasmid 004(C797S G > C single point mutant) Blocker1 product are shown in fig. 21; the sequencing results of the Blocker2 product are shown in fig. 22: the base C of the T790 site and the mutation of the C797S G > C are on the same DNA chain, so the sample is the single point mutation of the C797S G > C.

The sequencing results of sample TC01(T790M C > T & C797S T > A transmutation frequency 1%) Blocker1 products are shown in FIG. 23; the sequencing results of Blocker2 products are shown in fig. 24: the T790M C > T mutation and the C797S T > A mutation are not on the same DNA strand, so the sample is a trans mutation.

Sample TC02(T790M C > T & C797S T > a transmutation frequency 0.5%) the sequencing results of the Blocker1 product are shown in fig. 25; the result of sequencing the Blocker2 product is shown in FIG. 26: the T790M C > T mutation and the C797S T > A mutation are not on the same DNA strand, so the sample is a trans mutation.

Sample TC03(T790M C > T & C797S T > A transmutation frequency 0.2%) Blocker1 product sequencing results are shown in FIG. 27; the sequencing results of the Blocker2 product are shown in fig. 28: the T790M C > T mutation and the C797S T > A mutation are not on the same DNA strand, so the sample is a trans mutation.

Sample TC04(T790M C > T & C797ST > 0.1% frequency of a transmutation) the results of the sequencing of the Blocker1 product are shown in fig. 29; the sequencing results of the Blocker2 product are shown in fig. 30: the T790M C > T mutation and the C797S T > A mutation are not on the same DNA strand, so the sample is a trans mutation.

Sample TC05(T790M C > T & C797S G > 1% C transmutation frequency) the results of the sequencing of the Blocker1 product are shown in fig. 31; the sequencing results of the Blocker2 product are shown in fig. 32: the T790M C > T mutation and the C797SG > C mutation are not on the same DNA strand, so the sample is a trans mutation.

Sample TC06(T790M C > T & C797S G > 0.5% frequency of C transmutation) the sequencing results of the Blocker1 product are shown in FIG. 33; the result of sequencing the Blocker2 product is shown in fig. 34: the T790M C > T mutation and the C797S G > C mutation are not on the same DNA strand, so the sample is a trans mutation.

Sample TC07(T790M C > T & C797S G > 0.2% C transmutation frequency) the results of the sequencing of the Blocker1 product are shown in fig. 35; the result of sequencing the Blocker2 product is shown in FIG. 36: the T790M C > T mutation and the C797S G > C mutation are not on the same DNA strand, so the sample is a trans mutation.

Sample TC08(T790M C > T & C797S G > 0.1% C transmutation frequency) the results of the sequencing of the Blocker1 product are shown in fig. 37; the sequencing results of the Blocker2 product are shown in fig. 38: the T790M C > T mutation and the C797S G > C mutation are not on the same DNA strand, so the sample is a trans mutation.

The sequencing results of sample TC09(T790M C > T & C797S T > 1% of the frequency of cis mutation of a) Blocker1 products are shown in fig. 39; the result of sequencing the Blocker2 product is shown in FIG. 40: the T790M C > T mutation and the C797S T > A mutation are on the same DNA strand, so the sample is a cis-mutation.

The sequencing results of the sample TC10(T790M C > T & C797S T > A cis mutation frequency 0.5%) Blocker1 product are shown in FIG. 41; the result of sequencing the Blocker2 product is shown in FIG. 42: the T790M C > T mutation and the C797S T > A mutation are on the same DNA strand, so the sample is a cis-mutation.

Sample TC11(T790M C > T & C797S T > 0.2% frequency of cis mutation of a) the results of sequencing the Blocker1 product are shown in fig. 43; the sequencing results of the Blocker2 product are shown in fig. 44: the T790M C > T mutation and the C797S T > A mutation are on the same DNA strand, so the sample is a cis-mutation.

The sequencing results of the sample TC12(T790M C > T & C797S T > A cis mutation frequency 0.1%) Blocker1 products are shown in FIG. 45; the result of sequencing the Blocker2 product is shown in FIG. 46: the T790M C > T mutation and the C797S T > A mutation are on the same DNA strand, so the sample is a cis-mutation.

The sequencing results of sample TC13(T790M C > T & C797S G > 1% C cis mutation frequency) Blocker1 products are shown in fig. 47; the result of sequencing the Blocker2 product is shown in FIG. 48: the T790M C > T mutation and the C797S G > C mutation are on the same DNA strand, so the sample is a cis-mutation.

The sequencing results of the sample TC14(T790M C > T & C797S G > C cis mutation frequency 0.5%) Blocker1 product are shown in fig. 49; the sequencing results of the Blocker2 product are shown in fig. 50: the T790M C > T mutation and the C797S G > C mutation are on the same DNA strand, so the sample is a cis-mutation.

The sequencing results of the sample TC15(T790M C > T & C797S G > C cis mutation frequency 0.2%) Blocker1 product are shown in FIG. 51; the sequencing results of the Blocker2 product are shown in fig. 52: the T790M C > T mutation and the C797S G > C mutation are on the same DNA strand, so the sample is a cis-mutation.

The sequencing results of the sample TC16(T790M C > T & C797S G > C cis-mutation frequency 0.1%) Blocker1 product are shown in FIG. 53; the result of sequencing the Blocker2 product is shown in FIG. 54: the T790M C > T mutation and the C797S G > C mutation are on the same DNA strand, so the sample is a cis-mutation.

The results show that: the method adopts Blocker1 and Blocker2 to carry out PCR amplification on a sample to be detected respectively, can detect T790M C > T mutation and C797S T > A or G > C mutation in the sample, and can distinguish whether the mutation type is cis-trans, and the detected mutation frequency is as low as 0.1%.

Example 2

This example provides a kit for detecting the cis-trans mutation types of EGFR-T790M and EGFR-C797S, differing from the examples in that the following probes are used:

TABLE 11

TABLE 12

Name (R)	Type of mutation
		Plasmid 001	Wild Type (WT)
Plasmid 002	T790M C>T
		Plasmid 003	C797S T>A
Plasmid 004	C797S G>C

Watch 13

And (3) checking the product, and performing primary sequencing: the results are as follows

The sequencing results of the plasmid 001 (wild type) Blocker3 product are shown in fig. 55; the sequencing results of the Blocker4 products are shown in fig. 56: the Blocker3 product has T790M C > T mutation, does not conform to the plasmid 001, and is false positive; the Blocker4 product was wild-type, consistent with plasmid 001.

The sequencing result of the plasmid 002(T790M C > T single point mutant) Blocker3 product is shown in fig. 57; the sequencing results of the Blocker4 product are shown in fig. 58: both the Blocker3 and Blocker4 products exhibited a T790M C > T single point mutation, consistent with plasmid 002.

The sequencing results of the plasmid 003(C797S T > A single point mutant) Blocker3 product are shown in FIG. 59; the sequencing results of the Blocker4 product are shown in fig. 60: the Blocker3 product has T790M C > T mutation and C797S T > A mutation, and does not conform to plasmid 003; the Blocker4 product only showed the C797S T > A mutation, consistent with plasmid 003.

The sequencing results of the plasmid 004(C797S G > C single point mutant) Blocker3 product are shown in fig. 61; the sequencing results of the Blocker4 product are shown in fig. 62: the Blocker3 product is wild type, does not conform to plasmid 004, and is false negative; the Blocker4 product exhibited a C797S G > C mutation, consistent with plasmid 004.

From the above it can be seen that: plasmid 001 and plasmid 003 are both EGFR-T790 wild type, but under the action of Blocker3, a T790M C > T mutation appears, which is false positive; plasmid 004 is EGFR-C797S G > C mutant, but does not have C797S G > C mutation under the action of Blocker3, and is false negative, which indicates that the probe provided by example 1 has better effect.

Example 3

TABLE 14

The system was formulated according to the following table, with DNA samples in Table 4 and PCR procedure in Table 3.

Watch 15

The amplification results of Blocker5 are shown in FIGS. 63-68: blocker5 blocks amplification of wild-type and single-point C797S mutant DNA, and does not block or rarely block amplification of single-point T790M mutant and cis-mutant T790M C > T & C797S T > A or G > C DNA.

The amplification result of the Blocker6 is shown in FIGS. 69-74, and Blocker6 blocks the amplification of wild-type and single-point T790M mutant DNAs, and does not block or hardly block the amplification of single-point C797S T > A or G > C and cis-mutation T790M C > T & C797S T > A or G > C.

Comparison of blocking effect Δ Ct for each Blocker in examples 1 to 3:

TABLE 16

Note: delta. DELTA. Ct ═ DELTA Ct_(WT)(Ct_(-BL)–Ct_(+BL))-△Ct_(MT)(Ct_(-BL)-Ct_(+BL)) WT means wild type, MT means mutant, -BL means no Blocker is added in PCR reaction, and + BL means Blocker is added in PCR reaction.

By comparing the Ct differences of ± Blocker, the following conclusions can be drawn: blocker1 and Blocker2 performed better than Blocker5 and Blocker6 in EGFR T790M and C797S cis-trans mutant samples.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Zhuhaishengmei bio-diagnostic technology Co., Ltd

<120> method and probe for detecting cis-trans mutation configurations of EGFR-T790M and EGFR-C797S

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> DNA

<213> Artificial sequence

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caccgtgcag ctcatcacgc agctc 25

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gcccttcggc tgcctcct 18

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tggacaaccc ccacgtgt 18

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agccaatatt gtctttgtgt tcc 23

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ctcatcacgc agctcatgcc cttcggctgc ctc 33

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catgcccttc ggctgcctcc tggactatg 29

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gctcatcacg cagctcat 18

Claims

1. A method for detecting the cis-trans mutation configuration of EGFR-T790M and EGFR-C797S for non-diagnostic and therapeutic purposes, comprising:

amplifying a sample by using a PCR system for selectively inhibiting EGFR-T790 wild type amplification to obtain a first amplification product, wherein the PCR system for selectively inhibiting EGFR-T790 wild type amplification contains a probe for blocking amplification of EGFR-T790 wild type; the nucleotide sequence of the probe for blocking amplification of EGFR-T790 wild type is shown as SEQID NO. 1: 5' -CACCGTGCAGCTCATCACGCAGCTC-3’；

Amplifying the sample by using a PCR system for selectively inhibiting EGFR-C797 wild type amplification to obtain a second amplification product; the PCR system for selectively inhibiting EGFR-C797 wild type amplification contains a probe for blocking amplification of EGFR-C797 wild type; the nucleotide sequence of the probe for blocking amplification of the wild type EGFR-C797 is shown as SEQ ID NO. 2: 5' -GCCCTTCGGCTGCCTCCT-3’；

Sequencing the first amplification product and the second amplification product respectively, and judging the mutation configurations of EGFR-T790M and EGFR-C797S in the sample according to the sequencing result;

underlined nucleotides in the probes shown are locked nucleic acid modified nucleotides;

the 3' end of the probe is subjected to dideoxy modification;

the samples were PCR amplified using the following primer pairs:

SEQ ID NO.3：5’-TGGACAACCCCCACGTGT-3’；

SEQ ID NO.4：5’-AGCCAATATTGTCTTTGTGTTCC-3’；

the PCR amplification is two-stage amplification, wherein the two-stage amplification comprises firstly carrying out biased amplification and then carrying out non-biased amplification;

the annealing extension temperature of the biased amplification is 56 ℃, the annealing temperature of the non-biased amplification is 56 ℃, and the extension temperature is 72 ℃.

2. The method of claim 1, wherein the sequencing comprises sequencing with NGS or Sanger.

3. The method of claim 1, wherein the mutant configuration of the sample is determined according to the following method:

if the T790 site and the C797 site in the first amplification product and the second amplification product are both C and TG; judging the sample to be wild type;

if the T790 site in the first amplification product is T, the C797 site is TG; the T790 site in the second amplification product is C, and the C797 site is TG; judging the sample to be T790M single point mutation;

if the T790 site in the first amplification product is C, the C797 site is TG; if the T790 site in the second amplification product is C and the C797 site is AG or TC, the sample is judged to be C797S single-point mutation;

if the T790 site in both the first amplification product and the second amplification product is T; if the C797 site in the first amplification product and the second amplification product is AG or TC at the same time, the samples are T790M and C797S cis-mutations;

if the T790 site in the first amplification product is T, the C797 site is TG; and if the T790 site in the second amplification product is C and the C797 site is AG or TC, the samples are judged to be T790M and C797S trans-mutation.

4. The method of claim 1, wherein the two-stage amplified amplification products are sequenced using Sanger sequencing.