CN108048531B

CN108048531B - Ultra-blocking fluorescent quantitative PCR method for detecting rare mutation with high sensitivity

Info

Publication number: CN108048531B
Application number: CN201711136473.4A
Authority: CN
Inventors: 饶品彬; 石立立; 张佩琢
Original assignee: Suzhou Genepharma Co ltd; Shanghai Genepharma Co Ltd
Current assignee: Shanghai Genepharma Co Ltd
Priority date: 2017-11-16
Filing date: 2017-11-16
Publication date: 2021-06-18
Anticipated expiration: 2037-11-16
Also published as: CN108048531A

Abstract

The invention discloses an ultra-blocking fluorescent quantitative PCR method for detecting rare mutation with high sensitivity. The invention adopts specific primer and probe technology, and can realize rapid detection of blood sample gene point mutation. The advantages of the invention include: (1) LNA modification is added on the basis of the conventional ARMS primer or mismatched bases are additionally introduced at the 3' end two-bit to the end three-bit, so that the mutation detection sensitivity is not influenced, and the specificity of detecting single base mutation by using the mutation specific primer is ensured; (2) introducing a modification blocking probe which is completely complementary with the wild type, and further reducing the background interference of the wild type on the basis of considering the amplification efficiency of the mutant type; (3) on the basis of introducing the high-specificity modification blocking probe, the ARMS primer can simultaneously detect different base mutation forms of the same locus, so that the multiple loci of the same gene can be subjected to degenerate detection only by using a plurality of systems; (4) the sensitivity is high; (5) the detection speed is high.

Description

Ultra-blocking fluorescent quantitative PCR method for detecting rare mutation with high sensitivity

Technical Field

The invention relates to the field of clinical diagnosis molecular biology, in particular to an ultra-retardation fluorescence quantitative PCR method for detecting rare mutation with high sensitivity.

Background

With the continuous deepening of people in the process of generating tumors, the cancer generation is considered to be related to gene mutation at present, so that the tumor cells are subjected to uncontrolled proliferation, so that the tumor screening can be carried out according to the condition of the gene mutation, then the tumor susceptibility gene detection is carried out, and a pointed prompting effect can be played if definite abnormality is found. Chemotherapy is one of the most important means for clinically treating malignant tumors at present, but with the wide clinical application, the drug resistance problem is highlighted, tumor cells often generate drug resistance to chemotherapeutic drugs to cause patients to be insensitive to the treatment, and finally chemotherapy failure and disease recurrence are caused, wherein the T790M mutation of the EGFR gene accounts for more than 60% of clinical drug-resistant NSCLC patients, the K-ras gene state is closely related to colorectal cancer (CRC) treatment, mRC patients carrying wild-type K-ras genes benefit more from the treatment of EGFR inhibitors, and mRC patients carrying K-ras gene mutations cannot benefit from the combination of EGFR inhibitors and chemotherapy.

The traditional tumor detection sample acquisition generally needs to be performed in situ through operation or puncture, so that the patient has the limitations of wound generation, incapability of dynamic monitoring and the like, and a novel liquid biopsy obtains free nucleic acid or cells from a body fluid sample represented by blood plasma, so that the novel liquid biopsy is applied to early diagnosis of tumor, selection of targeted drugs, dynamic curative effect monitoring and the like.

At present, there are many methods for detecting gene mutation, including direct DNA sequencing, Pyrosequencing (Pyrosequencing), denaturing high performance liquid chromatography (HDPLC), high resolution melting curve technique (HRM), restriction fragment length polymorphism analysis (RFLP), ARMS-PCR, etc. In which direct sequencing of DNA is the gold standard for mutation detection, but this method has the following disadvantages: the detection sensitivity is not high enough, and if the content of the mutant gene accounts for less than 10 percent of the total amount of the genome DNA, the existence of the mutant sample can not be detected by using a direct sequencing method; the operation process is complex, the detection time is long, and the requirement on operators is high; the non-closed tube operation relates to the operation after PCR amplification, so the operation is easy to be polluted, the false negative rate is high, and the result is not ideal; the interpretation subjectivity of the sequencing result is strong; the sample size detected in one experiment is limited, and only 8-24 samples can be detected at most. Therefore, the direct sequencing method is difficult to be widely developed in clinic. The ARMS-PCR method can reach 1% of sensitivity, can meet the detection requirement on a tumor tissue sample, but for a blood sample with convenient sampling, such as circulating tumor cells in blood plasma or blood, the tumor DNA content is often lower than 1%, and the ARMS-PCR method is not enough for detecting the blood, thereby limiting the judgment of a clinician on the curative effect of a targeted drug. In addition, the drug resistance mutation generated in the clinical medication process cannot be detected by the prior method because of insufficient sensitivity. Therefore, there is a need to find a mutation detection method with high sensitivity, strong specificity, easy operation and simple result judgment for detecting mutated genes from these low-content samples.

Disclosure of Invention

The invention aims to provide an ultra-block fluorescent quantitative PCR method for detecting rare mutation with high sensitivity.

The invention provides a primer probe combination for detecting specific sites in nucleic acid, which is (a1) or (a 2):

(a1) the kit consists of a universal upstream primer, a universal downstream primer, a general detection probe, a specific amplification upstream primer and a blocking probe;

the universal upstream primer is a universal upstream primer-I or a universal upstream primer-II; the universal downstream primer is a universal downstream primer-I or a universal downstream primer-II; the combination of the universal upstream primer-I and the universal downstream primer-I is used for amplifying a target sequence; the target sequence is a fragment of the nucleic acid and contains the specific site; the universal upstream primer-I and the universal downstream primer-I do not contain the specific sites; the universal upstream primer-II is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the universal upstream primer-I and has the same function as the universal upstream primer-I; the universal downstream primer-II is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the universal downstream primer-I and has the same function as the universal downstream primer-I;

the general detection probe is a reverse general detection probe or a forward general detection probe; the reverse general detection probe is a reverse general detection probe-I or a reverse general detection probe-II; the reverse general detection probe-I is reversely complementary with a segment except the specific site in the target sequence; the reverse general detection probe-II is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides into the reverse general detection probe-I and has the same function as the reverse general detection probe-I; the forward general detection probe is a forward general detection probe-I or a forward general detection probe-II; the forward general detection probe-I is the same as a segment of the target sequence except the specific site; the forward general detection probe-II is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to the forward general detection probe-I and has the same function as the forward general detection probe-I; two tail ends of the general detection probe are respectively modified by a fluorescent group and a quenching group;

the specific amplification upstream primer is a specific amplification upstream primer-I or a specific amplification upstream primer-II or a specific amplification upstream primer-III; the specific amplification upstream primer-I meets the following three conditions: the nucleotide at the 3 'terminal which is the same as a certain segment in the target sequence corresponds to the specific site, and the nucleotide at the 3' terminal is the same as the mutated nucleotide; the specific amplification upstream primer-II is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides except the specific site to the specific amplification primer-I and has the same function as the specific amplification upstream primer-I; the specific amplification upstream primer-III is a DNA molecule obtained by performing LNA modification on 1-5 nucleotides in the specific amplification upstream primer-I or the specific amplification upstream primer-II;

the blocking probe is a blocking probe-I, a blocking probe-II or a blocking probe-III; the blocking probe-I satisfies the following two conditions: the nucleotide is the same as a certain section in the target sequence, a certain nucleotide corresponds to the specific site, and the nucleotide is the same as the nucleotide before mutation; the blocking probe-II is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides except the specific site to the blocking probe-I and has the same function as the blocking probe-I; the blocking probe-III is a DNA molecule obtained by performing LNA modification on 1-5 nucleotides in the blocking probe-I or the blocking probe-II; blocking modification is carried out on the 3' end of the blocking probe;

(a2) the kit consists of a universal upstream primer, a universal downstream primer, a general detection probe, a specific amplification downstream primer and a blocking probe;

the universal upstream primer and the universal downstream primer are the universal upstream primer described in (a 1);

the general detection probe is the general detection probe in (a 1);

the specific amplification downstream primer is a specific amplification downstream primer-I or a specific amplification downstream primer-II or a specific amplification downstream primer-III; the specific amplification downstream primer-I meets the following three conditions: the reverse complement of a certain segment in the target sequence, the 3 'terminal nucleotide corresponding to the specific site, and the 3' terminal nucleotide complementary to the mutated nucleotide; the specific amplification downstream primer-II is a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides except the specific site to the specific amplification downstream primer-I and has the same function as the specific amplification downstream primer-I; the specific amplification upstream primer-III is a DNA molecule obtained by performing LNA modification on 1-5 nucleotides in the specific amplification downstream primer-I or the specific amplification downstream primer-II;

the blocking probe is a blocking probe-IV, a blocking probe-V or a blocking probe-VI; the blocking probe-IV meets the following two conditions: a nucleotide which is complementary to a segment of the target sequence in the reverse direction, corresponds to the specific site and is complementary to the nucleotide before mutation; the blocking probe-V is a DNA molecule which enables the blocking probe-IV to be subjected to substitution and/or deletion and/or addition of one or more nucleotides except the specific site and has the same function as the blocking probe-IV; the blocking probe-VI is a DNA molecule obtained by performing LNA modification on 1-5 nucleotides in the blocking probe-IV or the blocking probe-V; blocking modification is carried out on the 3' end of the blocking probe;

the specific site is a site where a target point mutation occurs in nucleic acid; at the specific site, the nucleotide in which the target point mutation does not occur is named as a pre-mutation nucleotide, and the nucleotide in which the target point mutation occurs is named as a post-mutation nucleotide.

The blocking modification may specifically be an amination, dideoxy or phosphorylation modification.

The general detection probe can select 1-5 nucleotides for LNA modification.

The general detection probe can be modified by MGB. When MGB modification is adopted, one end of the general detection probe is modified by a fluorescent group, and the other end of the general detection probe is modified by a quenching group coupled with MGB or modified by an MGB group with a quenching function.

The size of the target sequence is 100-350 bp.

The size of the universal upstream primer and the universal downstream primer is 13-25 nt.

The size of the general detection probe is 12-30 nt.

The size of the specific amplification upstream primer or the specific amplification downstream primer is 12-25 nt.

The size of the blocking probe is 12-30 nt.

The specific amplification upstream primer can introduce mismatched bases which are different from the target sequence from the 2 nd to the 3 rd position of the 3' end.

The specific amplification downstream primer can introduce mismatched bases which are not complementary with the target sequence from the 2 nd to the 3 rd position of the 3' end.

One end of the general detection probe can be specifically marked by FAM fluorescent group, and the other end of the general detection probe can be specifically marked by BHQ1 quenching group. In the embodiment of the invention, the FAM fluorophore label is positioned at the 5 'end of the general detection probe, and the BHQ1 quencher label is positioned at the 3' end of the general detection probe.

The blocking probe may specifically be modified with an amino group. In embodiments of the invention, the amino modification is located at the 3' end of the blocking probe.

The TM values of the universal upstream primer and the universal downstream primer are 56-66 ℃.

The TM value of the general detection probe is 66-72 ℃.

The TM value of the blocking probe was 66-72 ℃.

When the primer probe combination described in (a1) is used to detect a specific site, the detection method is as follows: (a) taking nucleic acid to be detected as a template, and performing fluorescent quantitative PCR by adopting the universal upstream primer, the universal downstream primer and the general detection probe; (b) taking nucleic acid to be detected as a template, and carrying out fluorescent quantitative PCR by adopting the specific amplification upstream primer, the general downstream primer, the general detection probe and the blocking probe; (c) if the fluorescence quantitative PCR of the step (a) can obtain positive amplification and the fluorescence quantitative PCR of the step (b) can not obtain positive amplification or the ct value is more than or equal to 37, the specific site in the nucleic acid to be detected is the nucleic acid before mutation, if the fluorescence quantitative PCR of the step (a) can obtain positive amplification and the fluorescence quantitative PCR of the step (b) can obtain positive amplification and the ct value is less than 37, and the specific site in the nucleic acid to be detected is the mutant nucleic acid.

When the primer probe described in (a2) is used for detecting a specific site, the detection method is as follows: (a) taking nucleic acid to be detected as a template, and performing fluorescent quantitative PCR by adopting the universal upstream primer, the universal downstream primer and the general detection probe; (b) taking nucleic acid to be detected as a template, and carrying out fluorescent quantitative PCR by adopting the specific amplification downstream primer, the universal upstream primer, the general detection probe and the blocking probe; (c) if the fluorescence quantitative PCR of the step (a) can obtain positive amplification and the fluorescence quantitative PCR of the step (b) can not obtain positive amplification or the ct value is more than or equal to 37, the specific site in the nucleic acid to be detected is the nucleic acid before mutation, if the fluorescence quantitative PCR of the step (a) can obtain positive amplification and the fluorescence quantitative PCR of the step (b) can obtain positive amplification and the ct value is less than 37, and the specific site in the nucleic acid to be detected is the mutant nucleic acid.

When one of the pre-mutation nucleic acids is present at the specific site corresponding to a plurality of mutant nucleic acids, detection of a plurality of mutations can be achieved using one specific amplification primer.

The invention also provides a method for detecting the specific site in the nucleic acid, which is the method A or the method B.

The method A comprises the following steps: (a) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and adopting the universal upstream primer, the universal downstream primer and the universal detection probe in the primer probe combination of (a 1); (b) taking nucleic acid to be detected as a template, and carrying out fluorescent quantitative PCR by using the specific amplification upstream primer, the universal downstream primer, the universal detection probe and the blocking probe in the primer probe combination of (a 1); (c) if the fluorescence quantitative PCR of the step (a) can obtain positive amplification and the fluorescence quantitative PCR of the step (b) can not obtain positive amplification or the ct value is more than or equal to 37, the specific site in the nucleic acid to be detected is the nucleic acid before mutation, if the fluorescence quantitative PCR of the step (a) can obtain positive amplification and the fluorescence quantitative PCR of the step (b) can obtain positive amplification and the ct value is less than 37, and the specific site in the nucleic acid to be detected is the mutant nucleic acid.

The method B comprises the following steps: (a) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and adopting the universal upstream primer, the universal downstream primer and the universal detection probe in the primer probe combination of (a 2); (b) taking nucleic acid to be detected as a template, and carrying out fluorescent quantitative PCR by using the specific amplification downstream primer, the universal upstream primer, the general detection probe and the blocking probe in the primer probe combination of (a 2); (c) if the fluorescence quantitative PCR of the step (a) can obtain positive amplification and the fluorescence quantitative PCR of the step (b) can not obtain positive amplification or the ct value is more than or equal to 37, the specific site in the nucleic acid to be detected is the nucleic acid before mutation, if the fluorescence quantitative PCR of the step (a) can obtain positive amplification and the fluorescence quantitative PCR of the step (b) can obtain positive amplification and the ct value is less than 37, and the specific site in the nucleic acid to be detected is the mutant nucleic acid.

In the method A or the method B, the specific site is a site where a target point mutation occurs in the nucleic acid; at the specific site, the nucleotide in which the target point mutation does not occur is named as a pre-mutation nucleotide, and the nucleotide in which the target point mutation occurs is named as a post-mutation nucleotide.

In the method a, the PCR reaction system in the step (a) may specifically be: 10 XPCR Buffer (Vazyme, cat # P122-d2) 2. mu.L, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl₂1.5mM, universal upstream primer 0.2. mu.M-0.5. mu.M, universal downstream primer 0.2. mu.M-0.5. mu.M, universal probe 0.2. mu.M-0.5. mu.M, 50 XROX (Invitrogen, cat. No.: 12223-012) 0.4. mu.L, template 2. mu.L, sterile water to 20. mu.L.

In the method a, the PCR reaction procedure of the step (a) may specifically be: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles.

In the method a, the PCR reaction system in the step (b) may specifically be: 10 XPCR Buffer (Vazyme, cat # P122-d2) 2. mu.L, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl₂1.5mM, 0.2-0.5. mu.M universal downstream primer, 0.2-0.5. mu.M specific amplification upstream primer, 0.2-0.5. mu.M universal probe, 0.1-0.5. mu.M blocking probe, 0.4. mu.L 50 XROX (Invitrogen, cat. No.: 12223-012), 2. mu.L template, and sterile water to make up to 20. mu.L.

In the method a, the PCR reaction procedure in the step (b) may specifically be: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles. The fluorescent signal is detected during the reaction.

In the method B, the PCR reaction system in the step (a) may specifically be: 10 XPCR Buffer (Vazyme, cat # P122-d2) 2. mu.L, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl₂1.5mM, universal upstream primer 0.2. mu.M-0.5. mu.M, universal downstream primer 0.2. mu.M-0.5. mu.M, universal probe 0.2. mu.M-0.5. mu.M, 50 XROX (Invitrogen, cat. No.: 12223-012) 0.4. mu.L, template 2. mu.L, sterile water to 20. mu.L.

In the method B, the PCR reaction procedure of the step (a) may specifically be: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles.

In the method B, the PCR reaction system in the step (B) may specifically be: 10 XPCR Buffer (Vazyme, cat # P122-d2) 2. mu.L, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl₂1.5mM, 0.2-0.5. mu.M universal upstream primer, 0.2-0.5. mu.M specific amplification downstream primer, 0.2-0.5. mu.M universal probe, 0.1-0.5. mu.M blocking probe, 0.4. mu.L 50 XROX (Invitrogen, cat # 12223-012), 2. mu.L template, and sterile water to make up to 20. mu.L.

In the method B, the PCR reaction procedure in the step (B) may specifically be: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles. The fluorescent signal is detected during the reaction.

The invention also discloses a primer probe combination (primer probe combination A) for detecting whether the EGFR gene T790M mutation exists in nucleic acid, which consists of a universal upstream primer, a universal downstream primer, a general detection probe, a specific amplification downstream primer and a retardation probe; the universal upstream primer is a single-stranded DNA molecule shown in a sequence 1 of a sequence table; the universal downstream primer is a single-stranded DNA molecule shown in a sequence 2 of a sequence table; the specific amplification downstream primer is a single-stranded DNA molecule shown in a sequence 3 of a sequence table, wherein LNA modification is carried out on 5 th nucleotide, 6 th nucleotide and 7 th nucleotide at the 5' end; the universal detection probe is a single-stranded DNA molecule shown in a sequence 4 of a sequence table, the 5 'end is marked by an FAM fluorescent group, and the 3' end is marked by a BHQ1 quenching group; the blocking probe is a single-stranded DNA molecule shown in a sequence 5 of a sequence table, wherein LNA modification is carried out on a 5 th nucleotide, a 6 th nucleotide and a 7 th nucleotide from a 5 'end, and amino modification is carried out on a 3' end.

The invention also discloses a primer probe combination (primer probe combination B) for detecting whether K-ras gene mutation exists in nucleic acid, which consists of a universal upstream primer, a universal downstream primer, a general detection probe, a specific amplification upstream primer and a blocking probe;

the universal upstream primer is shown as a sequence 8 in a sequence table;

the universal downstream primer is shown as a sequence 9 in a sequence table;

the specific amplification upstream primer is (b1) and/or (b2) and/or (b3) as follows:

(b1) a single-stranded DNA molecule shown in sequence 10 of the sequence table;

(b2) a single-stranded DNA molecule shown in sequence 11 of the sequence table;

(b3) a single-stranded DNA molecule shown in sequence 12 of the sequence table;

the general detection probe is shown as a sequence 13 in a sequence table, wherein LNA modification is carried out from the 7 th nucleotide, the 8 th nucleotide, the 11 th nucleotide, the 12 th nucleotide and the 14 th nucleotide of a 5' end; the blocking probe is shown as a sequence 14 in a sequence table, wherein LNA modification is carried out on the 7 th nucleotide, the 9 th nucleotide and the 10 th nucleotide from the 5 'end, and amino modification is carried out on the 3' end.

The invention also provides a method for detecting whether the EGFR gene T790M mutation exists in nucleic acid, which comprises the following steps: (1) taking nucleic acid to be detected as a template, and performing fluorescent quantitative PCR by adopting the universal upstream primer, the universal downstream primer and the universal detection probe in the primer probe combination A; (2) taking nucleic acid to be detected as a template, and carrying out fluorescent quantitative PCR by adopting the specific amplification downstream primer, the universal upstream primer, the general detection probe and the blocking probe in the primer probe combination A; (3) if the fluorescence quantitative PCR of the step (1) can obtain positive amplification and the fluorescence quantitative PCR of the step (2) can not obtain positive amplification or the ct value is more than or equal to 37, and the EGFR gene T790M mutation does not exist in the nucleic acid to be detected, if the fluorescence quantitative PCR of the step (1) can obtain positive amplification and the fluorescence quantitative PCR of the step (2) can obtain positive amplification and the ct value is less than 37, and the EGFR gene T790M mutation exists in the nucleic acid to be detected.

The invention also protects a method for detecting whether the mutation of the exon 2 of the K-ras gene exists in nucleic acid, which is the method A or the method B or the method C.

The method A comprises the following steps: (1) taking nucleic acid to be detected as a template, and performing fluorescent quantitative PCR by adopting the universal upstream primer, the universal downstream primer and the universal detection probe in the primer probe combination B; (2) taking nucleic acid to be detected as a template, and carrying out fluorescent quantitative PCR by adopting the specific amplification upstream primer (b1), the universal downstream primer, the universal detection probe and the blocking probe in the primer probe combination B; (3) if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can not obtain positive amplification or the ct value is more than or equal to 37, no K-ras gene exon 2G 12A mutation exists in the nucleic acid to be detected, no K-ras gene exon 2G 12V mutation exists, and no K-ras gene exon 2G 12D mutation exists, if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can obtain positive amplification and the ct value is less than 37, and the K-ras gene exon 2G 12A mutation or the K-ras gene exon 2G 12V mutation or the K-ras gene exon 2G 12D mutation exists in the nucleic acid to be detected;

the method B comprises the following steps: (1) taking nucleic acid to be detected as a template, and performing fluorescent quantitative PCR by adopting the universal upstream primer, the universal downstream primer and the universal detection probe in the primer probe combination B; (2) taking nucleic acid to be detected as a template, and carrying out fluorescent quantitative PCR by adopting the specific amplification upstream primer (b2), the universal downstream primer, the universal detection probe and the blocking probe in the primer probe combination B; (3) if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can not obtain positive amplification or the ct value is more than or equal to 37, no K-ras gene exon 2G 12S mutation exists in the nucleic acid to be detected, no K-ras gene exon 2G 12C mutation exists, and no K-ras gene exon 2G 12R mutation exists, if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can obtain positive amplification and the ct value is less than 37, and the K-ras gene exon 2G 12S mutation or the K-ras gene exon 2G 12C mutation or the K-ras gene exon 2G 12R mutation exists in the nucleic acid to be detected;

the method C comprises the following steps: (1) taking nucleic acid to be detected as a template, and performing fluorescent quantitative PCR by adopting the universal upstream primer, the universal downstream primer and the universal detection probe in the primer probe combination B; (2) taking nucleic acid to be detected as a template, and carrying out fluorescent quantitative PCR by adopting the specific amplification upstream primer (b3), the universal downstream primer, the universal detection probe and the blocking probe in the primer probe combination B; (3) if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can not obtain positive amplification or the ct value is more than or equal to 37, and the K-ras gene exon 2G 13D mutation does not exist in the nucleic acid to be detected, if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can obtain positive amplification and the ct value is less than 37, and the K-ras gene exon 2G 13D mutation exists in the nucleic acid to be detected.

The invention adopts specific primer and probe technology, and can realize rapid detection of blood sample gene point mutation. The advantages of the invention include: (1) in the design of a primer probe, LNA modification is added on the basis of a conventional ARMS primer or mismatched bases are additionally introduced into the 3' last two bits to the last three bits, compared with the conventional ARMS, the method has the advantages that the mutation detection sensitivity is not influenced, and the specificity of detecting single base mutation by using a mutation specific primer is ensured; (2) in the design of a primer probe, a modification blocking probe which is completely complementary with a wild type is introduced, and the background interference of the wild type is further reduced on the basis of considering the amplification efficiency of a mutant type; (3) on the basis of introducing the high-specificity modification blocking probe, the ARMS primer can simultaneously detect different base mutation forms of the same locus, and the sensitivity is not influenced by mutual interference, so that the simultaneous detection of a plurality of loci of the same gene is realized only by using a plurality of systems; (4) the sensitivity is high, the detection sensitivity can reach 0.01 percent, and the kit is more suitable for detecting circulating tumor cells in blood; (5) the detection speed is high, the detection process can be completed in 120 minutes, and the time consumption is only one half of that of digital PCR.

Drawings

FIG. 1 is a schematic diagram of primer probe design for a super-block detection system.

FIG. 2 is a graph of PCR amplification of EGFR wild type plasmid and T790M mutant plasmid using the EGFR gene T790M fluorescent PCR reaction control detection system with universal primers.

FIG. 3 is a PCR amplification plot of EGFR wild type plasmid and T790M mutant plasmid using a specific primer EGFR gene T790M fluorescent PCR reaction mutation detection system.

FIG. 4 is a graph of PCR amplification of EGFR wild type plasmid and T790M mutant type plasmid using EGFR gene T790M fluorescent PCR reaction super-block detection system with specific primers combined with locked nucleic acid block probes.

FIG. 5 is a sensitivity curve diagram of the EGFR gene T790M fluorescence PCR reaction super-block detection system.

FIG. 6 is a graph showing PCR amplification of K-ras wild-type plasmid and G12A, G12V, G12D, G12S, G12C, G12R, G13D mutant plasmids using a universal primer control assay system.

FIG. 7 is a graph showing PCR amplification of K-ras wild-type plasmid and mutant plasmids G12A, G12V, G12D, G12S, G12C, G12R and G13D using a mutation detection system using G12AVD specific primers.

FIG. 8 is a graph showing PCR amplification of K-ras wild-type plasmid and mutant plasmids G12A, G12V, G12D, G12S, G12C, G12R and G13D using a mutation detection system using G12SCR specific primers.

FIG. 9 is a graph showing PCR amplification of K-ras wild-type plasmid and mutant plasmids G12A, G12V, G12D, G12S, G12C, G12R and G13D using a mutation detection system using G13D specific primers.

FIG. 10 is a graph of PCR amplification of K-ras wild-type plasmid and G12A, G12V, G12D mutant-type plasmids using the super-block detection system of G12AVD specific primers combined with locked nucleic acid blocking probes.

FIG. 11 is a graph of PCR amplification of K-ras wild-type plasmid and G12R, G12S, G12R mutant-type plasmids using the G12SCR specific primer in combination with a nucleic acid blocking probe in a super-block detection system.

FIG. 12 is a graph of PCR amplification of K-ras wild-type plasmid and G13D mutant plasmid using the superblock assay system with G13D specific primers combined with locked nucleic acid blocking probes.

FIG. 13 is a graph of gradient dilution sensitivity detection amplification for G12A mutant plasmid using the G12AVD super-block system.

FIG. 14 is a graph of gradient dilution sensitivity detection amplification for G12V mutant plasmid using the G12AVD super-block system.

FIG. 15 is a graph of gradient dilution sensitivity detection amplification for G12D mutant plasmid using the G12AVD super-block system.

FIG. 16 is a graph of gradient dilution sensitivity detection amplification for G12S mutant plasmid using the G12SCR superblock system.

FIG. 17 is a graph of gradient dilution sensitivity detection amplification for G12C mutant plasmid using the G12SCR superblock system.

FIG. 18 is a graph of gradient dilution sensitivity detection amplification for G12R mutant plasmid detection using the G12SCR superblock system.

FIG. 19 is a graph of gradient dilution sensitivity detection amplification for G13D mutant plasmid detection using the G13D super-block system.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 design of ultra-block fluorescent quantitative PCR primer Probe

The primer probe combination for detecting the gene rare mutation site consists of an upstream universal primer F, a downstream universal primer R, a general detection type probe P, a specific amplification primer AP and a locked nucleic acid blocking probe BL.

The sequences of the upstream universal primer F, the downstream universal primer R and the general detection type probe P are all from wild type and mutant homologous regions of a detection gene; the length of the upstream universal primer F and the downstream universal primer R is 13-25nt, and the TM value is 56-66 ℃; the length of the general detection type probe P is 12-30nt, and the TM value is 66-72 ℃; the general detection type probe P is modified by LNA or MGB; the number of modified LNA is 1-5, ATCG 4 base monomers can be modified by LNA, and the 5 'end and the 3' end are respectively marked with a fluorescent group and a quenching group; the MGB modifies a 5 'end marker fluorescent group, a 3' end marker and MGB coupled quenching group or MGB group with quenching function (MGB is a minor groove combination, which can be coupled with a quenching group during synthesis and integrally connected to the 3 'end of the probe, or the quenching group can be not coupled, and the quenching group is independently connected to the 3' end of the probe and then connected to the MGB).

The specific amplification primer AP can be an upstream primer (the same as the mutant sequence of the gene to be detected) or a downstream primer (complementary to the mutant sequence of the gene to be detected); the length of the specific amplification primer AP is 12-25nt, and the shorter the length is, the stronger the specificity is. The 3' end base of the specific amplification primer AP is designed to be a site where mutation exists; the specific amplification primer AP can contain LNA (low noise amplifier) modifications, the number of the LNA modifications is 1-5, ATCG 4 base monomers can be modified by LNA, and the LNA modifications are positioned near the middle and 5' ends of the primer; the specific amplification primer AP can additionally add 1 mismatched base at the 2 nd to 3 rd position from the penultimate end of the 3' end so as to increase the specificity; when different base mutations occur in the same site, the specific amplification primer AP can be designed to be specifically combined with different mutation templates with different base mutations in the same site.

The locked nucleic acid blocking probe BL sequence comprises a wild type and a mutant type differential site of a gene to be detected and is completely matched with (same as or reverse complementary with) the wild type sequence of the gene to be detected; the 3' end of the locked nucleic acid blocking probe BL can be modified by amination, dideoxy, phosphorylation and the like; the length of the locked nucleic acid blocking probe BL is 12-30nt, and the TM value is 66-72 ℃; the locked nucleic acid blocking probe BL and the forward primer are combined with the same template strand; the TM value of the locked nucleic acid blocking probe BL is 5-10 ℃ higher than that of the forward primer TM; the locked nucleic acid blocking probe BL can contain LNA modification, the number of LNA modification is 1-5, and all ATCG 4 base monomers can be modified by LNA; the base of the different site of the locked nucleic acid blocking probe BL contains LNA modification.

The optimal length of the target sequence of the primer probe combination is 100-350 bp.

The design schematic diagram of the primer probe combination is shown in FIG. 1.

Example 2 establishment of the ultra-blocking fluorescent quantitative PCR detection method

1. Extracting total RNA of a sample to be detected, and performing reverse transcription to obtain cDNA.

2. Carrying out contrast PCR reaction and super-block PCR reaction by taking the cDNA obtained in the step 1 as a template;

control PCR reaction (20. mu.L system as an example): 10 XPCR Buffer (Vazyme, cat # P122-d2) 2. mu.L, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl₂1.5mM, 0.2. mu.M-0.5. mu.M universal upstream primer F, 0.2. mu.M-0.5. mu.M universal downstream primer R, 0.2. mu.M-0.5. mu.M universal probe P, 0.4. mu.L 50 XROX (Invitrogen, cat. No.: 12223-012), 2. mu.L template, sterile water to 20. mu.L.

Control PCR reaction program: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles.

The fluorescent signal is detected during the reaction.

When the specific amplification primer is a downstream primer, the super-block PCR reaction system (taking a 20 μ L system as an example): 10 XPCRBUFFER (Vazyme, cat # P122-d2) 2. mu.L, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl₂1.5mM, universal upstream primer F0.2-0.5. mu.M, specific amplification primer AP 0.2-0.5. mu.M, universal probe P0.2-0.5. mu.M, locked nucleic acid blocking probe BL 0.1-0.5. mu.M, 50 × ROX (Invitrogen, cat # 12223-012) 0.4. mu.L, template 2. mu.L, and sterile water to 20. mu.L.

When the specific amplification primer is an upstream primer, the super-block PCR reaction system (taking a 20 μ L system as an example): 10 XPCRBUFFER (Vazyme, cat # P122-d2) 2. mu.L, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl₂1.5mM, universal downstream primer R0.2. mu.M-0.5. mu.M, specific amplification primer AP 0.2. mu.M-0.5. mu.M, universal test probe P0.2. mu.M-0.5. mu.M, locked nucleic acid blocking probe BL 0.1. mu.M-0.5. mu.M, 50 XROX (Invitrogen, cat # 12223-012) 0.4. mu.L, template 2. mu.L, sterile water to 20. mu.L.

Ultra-block PCR reaction procedure: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles.

The fluorescent signal is detected during the reaction.

3. And (4) judging according to the reaction result of the step (2), wherein the judging method comprises the following steps:

the amplification curve of the contrast PCR reaction system has an obvious S curve, the ultra-block PCR reaction system has no obvious S curve or ct is more than or equal to 37, and the sample result is judged to be wild type; and (3) comparing an amplification curve of the PCR reaction system with an obvious S curve, and the ultra-block PCR reaction system also has an obvious S curve, and the ct value is less than 37, and judging the sample result as the mutant type.

Example 3 ultra-block fluorescent quantitative PCR of rare mutation of EGFR Gene T790M

Detection method

1. A primer probe is designed according to the method of example 1 aiming at the T790M mutation of the EGFR gene (the wild-type target sequence of the EGFR gene is shown as the sequence 6 in the sequence table; the T790M mutant target sequence of the EGFR gene is shown as the sequence 7 in the sequence table), and the following steps are carried out:

the general upstream primer EGFR-T790M-F: 5'-CCTCCAGGAAGCCTACGTGATGG-3' (SEQ ID NO: 1);

the general downstream primer EGFR-T790M-R: 5'-CAGTTGAGCAGGTACTGGGAG-3' (SEQ ID NO: 2);

specifically amplifying a downstream primer EGFR-T790M-AP: 5 '-AGGG + C + A + TGAGCTGCA-3' (SEQ ID NO: 3); wherein "+" is an LNA modification and "+" indicates a subsequent base modification;

general detection type probe EGFR-T790M-P: 5'-TGAGCTGCACGGTGGAGGTGA-3' (SEQ ID NO: 4); wherein the 5 'end of the probe is marked by FAM, and the 3' end is marked by BHQ 1;

a blocking probe EGFR-T790M-BL 5 '-AGGG + C + A + TGAGCTGCG-3' (sequence 5); wherein "+" is an LNA modification and "+" indicates a subsequent base modification; the 3' end of the probe is modified by amino.

2. A double-stranded DNA molecule (EGFR gene wild-type target sequence) shown in a sequence 6 and a double-stranded DNA molecule (EGFR gene mutant target sequence) shown in a sequence 7 are artificially synthesized.

3. Inserting the double-stranded DNA molecule shown in the sequence 6 into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain a wild type plasmid (the wild type plasmid is verified by sequencing), and diluting the wild type plasmid to 1 × 10 by adopting 1 × TE buffer solution⁷copy/mL concentration, to obtain wild type standard. Inserting the double-stranded DNA molecule shown in the sequence 7 into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain a mutant plasmid (the sequence is verified), and adopting the mutant plasmid with the concentration of 1 × 10⁷Dilution of copy/mL wild-type standard to give mutant plasmid concentration of 1X 10³copy/mL, 1X 10⁴copy/mL, 1X 10⁵copy/mL, 1X 10⁶copy/mL, 1X 10⁷The copy/mL mutant standard is understood to mean that the background content of the mutant standard in the wild-type standard is 0.01%, 0.1%, 1%, 10%, 100%, respectively.

4. Respectively adopting the wild type standard prepared in the step 3 and the concentration of the wild type standard is 1 multiplied by 10⁷copy/mL mutant standard was used as template for control PCR and ultrablock PCR reactions, followed by a set of mutant PCR reactions as in example 2.

Mutation PCR reaction System: 10 XPCR Buffer (Vazyme, cat # P122-d2) 2. mu.L, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl₂1.5mM, 0.2-0.5. mu.M universal upstream primer F, 0.2-0.5. mu.M specific amplification primer AP, 0.2-0.5. mu.M universal probe P, 0.4. mu.L 50 XROX (Invitrogen, cat. No.: 12223-012), 2. mu.L template, and sterile water to 20. mu.L.

Mutation PCR reaction procedure: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles. FAM fluorescence signal was detected during the reaction.

The results of the control PCR reaction are shown in FIG. 2. The results of the mutation PCR reaction are shown in FIG. 3. The detection result of the super-block PCR reaction is shown in FIG. 4. As a result, the Ct values of both the wild type and the mutant type were about 22 in the control PCR reaction results. In the mutant PCR reaction results, the Ct value of the mutant was the same as that in the control PCR reaction and was also 22; and because the specific amplification primer is used in the reaction system, the Ct value of the wild type is delayed, which shows that the length of the primer is shortened and the specificity is obviously enhanced after the specific amplification primer is introduced into the LNA locked nucleic acid for modification. In the results of the ultrablocking PCR reaction, the Ct value of the mutant type is the same as the Ct value in the control PCR reaction and is also 22; and because the blocking probe is added into the system, the wild type does not obtain an amplification curve, which indicates that the locked nucleic acid blocking probe is added, further enhances the specificity of the reaction system, and is suitable for detecting rare mutant genes in the high-background wild type environment.

Second, sensitivity

And (3) respectively adopting the wild type standard substance prepared in the step one and the mutant type standard substance with different concentrations as templates, and carrying out the ultra-block PCR reaction according to the method in the embodiment 2 to detect the sensitivity.

The results are shown in FIG. 5. The result shows that when the background content of the mutant standard substance in the wild standard substance in the ultra-blocking detection system is 100%, the Ct value is about 21.95; when the background content of the mutant standard substance in the wild standard substance is 10%, the Ct value is about 25.23; when the background content of the mutant standard substance in the wild standard substance is 1%, the Ct value is about 28.61; when the background content of the mutant standard substance in the wild standard substance is 0.1%, the Ct value is about 32.42; when the background content of the mutant standard substance in the wild type standard substance is 0.01%, the Ct value is about 35.88; the method shows that the ultra-block PCR has high sensitivity and can detect the mutation as low as 0.01 percent.

Example 4 Superblock fluorescent quantitative PCR of exon 2 codon 12 and codon 13 mutation of K-ras Gene

Method and device

1. A primer is designed according to the method of example 1 aiming at the 12 th codon mutation and the 13 th codon mutation of exon 2 of K-ras gene (the wild-type target sequence of exon 2 of K-ras gene is shown in a sequence 15 in a sequence table, the mutant target sequence of exon 2G 12A of K-ras gene is shown in a sequence 16 in the sequence table, the mutant target sequence of exon 2G 12V of K-ras gene is shown in a sequence 17 in the sequence table, the mutant target sequence of exon 2G 12D of K-ras gene is shown in a sequence 18 in the sequence table, the mutant target sequence of exon 2G 12S of K-ras gene is shown in a sequence 19 in the sequence table, the mutant target sequence of exon 2G 12C of K-ras gene is shown in a sequence 20 in the sequence table, the mutant target sequence of exon 2G 12R of K-ras gene is shown in a sequence 21 in the sequence table, and the mutant target sequence of exon 2G 13D of K-ras gene is shown in a sequence 22 in the sequence table), as follows:

the general upstream primer K-ras-F: 5'-GCCTGCTGAAAATGACTGAA-3' (SEQ ID NO: 8);

the general downstream primer K-ras-R: 5'-CCTGACATACTCCCAAGGAAAG-3' (SEQ ID NO: 9);

the specific amplification upstream primers K-ras-G12AVD-AP aiming at three sites of G12A, G12V and G12D are as follows:

5'-AACTTGTGGTAGTTGGAGCTGC-3' (SEQ ID NO: 10);

specific amplification upstream primers K-ras-G12SCR-AP aiming at three sites of G12S, G12C and G12R:

5'-ATAAACTTGTGGTAGTTGGAGCTC-3' (SEQ ID NO: 11);

specific amplification upstream primer K-ras-G13D-AP aiming at G13D site:

5'-GTGGTAGTTGGAGCTGGAGA-3' (SEQ ID NO: 12);

general detection type probe K-ras-P: 5 '-ACTTGA + A + ACC + C + AA + GGTAC-3' (SEQ ID NO: 13); wherein "+" is an LNA modification and "+" indicates a subsequent base modification; the 5 'end of the probe is marked by FAM, and the 3' end of the probe is marked by BHQ 1;

blocking probe K-ras-BL 5 '-TGGAGC + TG + G + TGGC-3' (SEQ ID NO: 14); wherein "+" is an LNA modification and "+" indicates a subsequent base modification; the 3' end of the probe is modified by amino.

2. Artificially synthesizing a double-stranded DNA molecule shown in a sequence 15 (K-ras gene wild-type target sequence), a double-stranded DNA molecule shown in a sequence 16 (K-ras gene G12A mutant-type target sequence), a double-stranded DNA molecule shown in a sequence 17 (K-ras gene G12V mutant-type target sequence), a double-stranded DNA molecule shown in a sequence 18 (K-ras gene G12D mutant-type target sequence), a double-stranded DNA molecule shown in a sequence 19 (K-ras gene G12S mutant-type target sequence), a double-stranded DNA molecule shown in a sequence 20 (K-ras gene G12C mutant-type target sequence), a double-stranded DNA molecule shown in a sequence 21 (K-ras gene G12R mutant-type target sequence) and a double-stranded DNA molecule shown in a sequence 22 (K-ras gene G13D mutant-type target sequence).

3. Inserting the double-stranded DNA molecule shown in the sequence 15 into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain a wild type plasmid (the sequence is verified). The double-stranded DNA molecule shown in the sequence 16 is inserted into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain the G12A mutant plasmid (the sequencing of which is verified). The double-stranded DNA molecule shown in the sequence 17 is inserted into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain the G12V mutant plasmid (the sequencing of which is verified). The double-stranded DNA molecule shown in the sequence 18 is inserted into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain the G12D mutant plasmid (the sequencing of which is verified). The double-stranded DNA molecule shown in the sequence 19 is inserted into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain the G12S mutant plasmid (the sequencing of which is verified). The double-stranded DNA molecule shown in the sequence 20 is inserted into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain the G12C mutant plasmid (the sequencing of which is verified). The double-stranded DNA molecule shown in the sequence 21 is inserted into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain the G12R mutant plasmid (the sequencing of which is verified). The double-stranded DNA molecule shown in the sequence 22 is inserted into the EcoRV enzyme cutting site of the PUC57 plasmid to obtain the G13D mutant plasmid (the sequencing of which is verified).

Wild type plasmid was diluted to 1 × 10 with 1 × TE buffer⁷copy/mL concentration, to obtain wild type standard. The G12A mutant plasmid, G12V mutant plasmid, G12D mutant plasmid, G12C mutant plasmid, G12S mutant plasmid, G12R mutant plasmid and G13D mutant plasmid were used at a concentration of 1X 10⁷Dilution of copy/mL wild-type standard to give mutant plasmid concentration of 1X 10³copy/mL, 1X 10⁴copy/mL, 1X 10⁵copy/mL, 1X 10⁶copy/mL, 1X 10⁷copy/mL mutant standard, which is understood to be a mutantThe background content of the variant standard substance in the wild-type standard substance is 0.01%, 0.1%, 1%, 10% and 100%, respectively.

4. Respectively taking the wild type standard substance obtained in the step 3 and the concentration of the wild type standard substance as 1 multiplied by 10⁷A control PCR reaction was performed in the same manner as in example 2 using each mutant standard in copies/mL as a template. The results are shown in FIG. 6.

5. Respectively taking the wild type standard substance obtained in the step 3 and the concentration of the wild type standard substance as 1 multiplied by 10⁷Each mutant standard was copied/mL as a template, and subjected to a mutation PCR reaction.

And (3) detecting mutation PCR reaction systems of three sites of G12A, G12V and G12D: 10 XPCR Buffer (Vazyme, cat # P122-d2)2 uL, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl21.5 mM, universal downstream primer K-ras-R0.2 uM-0.5 uM, specific amplification upstream primer K-ras-G12AVD-AP 0.2 uM-0.5 uM, universal probe K-ras-P0.2 uM-0.5 uM, 50 XPROX (Invitrogen, cat # 12223 and 012)0.4 uL, template 2 uL, and sterile water to make up to 20 uL. Mutation PCR reaction procedure: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles. FAM fluorescence signal was detected during the reaction. The results are shown in FIG. 7.

And (3) detecting mutation PCR reaction systems of three sites of G12S, G12C and G12R: 10 XPCR Buffer (Vazyme, cat # P122-d2)2 uL, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl21.5 mM, universal downstream primer K-ras-R0.2 uM-0.5 uM, specific amplification upstream primer K-ras-G12SCR-AP 0.2 uM-0.5 uM, universal detection probe K-ras-P0.2 uM-0.5 uM, 50 XPROX (Invitrogen, cat # 12223 and 012)0.4 uL, template 2 uL, and sterile water to make up to 20 uL. Mutation PCR reaction procedure: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles. FAM fluorescence signal was detected during the reaction. The results are shown in FIG. 8.

Detection of mutation at site G13D PCR reaction System: 10 XPCR Buffer (Vazyme, cat # P122-d2)2 uL, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl21.5 mM, universal downstream primer K-ras-F0.2 uM-0.5 uM, specific amplification upstream primer K-ras-G13D-AP 0.2 uM-0.5 uM, universal probe K-ras-P0.2 uM-0.5 uM, 50 XPROX (Invitrogen, cat # 12223 and 012)0.4 uL, template 2 uL, and sterile water to make up to 20 uL. Mutation PCR reaction procedure: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles. FAM fluorescence signal was detected during the reaction. The results are shown in FIG. 9.

6. Respectively taking the wild type standard substance obtained in the step 3 and the concentration of the wild type standard substance as 1 multiplied by 10⁷copy/mL of G12A mutant, G12V mutant and G12 variant standard was used as template to perform a super-block PCR reaction. The results are shown in FIG. 10.

And (3) detecting an ultrablock PCR reaction system of three sites of G12A, G12V and G12D: 10 XPCR Buffer (Vazyme, cat # P122-d2)2 uL, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl21.5 mM, universal downstream primer K-ras-R0.2 uM-0.5 uM, specific amplification upstream primer K-ras-G12AVD-AP 0.2 uM-0.5 uM, universal test probe K-ras-P0.2 uM-0.5 uM, blocking probe K-ras-BL0.1 uM-0.5 uM, 50 XPROX (Invitrogen, cat # 12223-012)0.4 uL, template 2 uL, sterile water make up to 20 uL. Mutation PCR reaction procedure: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles. FAM fluorescence signal was detected during the reaction.

7. Respectively taking the wild type standard substance obtained in the step 3 and the concentration of the wild type standard substance as 1 multiplied by 10⁷copy/mL of G12C mutant, G12S mutant and G12R mutant as templates, and performing a super-block PCR reaction. The results are shown in FIG. 11.

And (3) detecting an ultrablock PCR reaction system of three sites of G12S, G12C and G12R: 10 XPCR Buffer (Vazyme, cat # P122-d2)2 uL, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl21.5 mM, universal downstream primer K-ras-R0.2 uM-0.5 uM, specific amplification upstream primer K-ras-G12SCR-AP 0.2 uM-0.5 uM, universal detection probe K-ras-P0.2 uM-0.5 uM, blocking probe K-ras-BL0.1 uM-0.5 uM, 50 XPROX (Invitrogen, cat # 12223-012)0.4 uL, template 2 uL, and sterile water to make up to 20 uL. Mutation PCR reaction procedure: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles. FAM fluorescence signal was detected during the reaction.

8. Respectively taking the wild type standard substance obtained in the step 3 and the concentration of the wild type standard substance as 1 multiplied by 10⁷A G13D mutant standard in a copy/mL manner is used as a template to perform a super-block PCR reaction. The results are shown in FIG. 12.

And (3) detecting an ultra-block PCR reaction system of the G13D site: 10 XPCR Buffer (Vazyme, cat # P122-d2)2 uL, dNTP (Vazyme, cat # P031-01)0.2mM, MgCl21.5 mM, universal downstream primer K-ras-R0.2 uM-0.5 uM, specific amplification upstream primer K-ras-G13D-AP 0.2 uM-0.5 uM, universal test probe K-ras-P0.2 uM-0.5 uM, blocking probe K-ras-BL0.1 uM-0.5 uM, 50 XPROX (Invitrogen, cat # 12223-012)0.4 uL, template 2 uL, sterile water make up to 20 uL. Mutation PCR reaction procedure: 3 minutes at 95 ℃; 95 ℃ for 15s, 62 ℃ for 30s, 72 ℃ for 30s, 40 cycles. FAM fluorescence signal was detected during the reaction.

The above experimental results are as follows:

in the control PCR reaction system, the ct values of the wild type and each mutant are 24.00 +/-0.10.

In a mutation PCR reaction system for detecting three sites of G12A, G12V and G12D, the Ct values of G12A, G12V and G12D are all about 24.00 +/-0.10, the Ct values of G12S, G12C, G12R and G13D plasmids are about 34.00 +/-0.10, and the wild type Ct value is about 34.00. In a mutation PCR reaction system for detecting three sites of G12S, G12C and G12R, Ct values of G12S, G12C and G12R are about 24.00 +/-0.10, and Ct values of G12A, G12V, G12D and G13D plasmids and wild type Ct values are about 36.00 +/-0.10. In a mutation PCR reaction system for detecting the G13D locus, the Ct value of G13D is about 24.06, the plasmids G12A, G12V, G12D, G12S, G12C and G12R are not amplified, and the wild type plasmids are weakly amplified, which indicates that the specificity of the specific amplification primer is enhanced.

In a super-block PCR reaction system for detecting three sites of G12A, G12V and G12D, Ct values of mutant plasmids of G12A, G12D and G12V are 22.01, 22.12 and 23.08 respectively, and a wild type amplification curve does not exist. In a super-block PCR reaction system for detecting three sites of G12S, G12C and G12R, Ct values of mutant plasmids of G12R, G12S and G12C are 24.25, 27.14 and 24.88 respectively, and a wild type amplification curve is not generated. In the super-block PCR reaction system for detecting the G13D locus, the Ct value of the G13D mutant plasmid is about 24.11, and the wild type has no amplification curve. The addition of the locked nucleic acid blocking probe is demonstrated, the specificity of the reaction system is further enhanced, and the method is suitable for detecting rare mutant genes in high-background wild-type environment.

Second, sensitivity

1. And (3) respectively adopting the wild type standard prepared in the step one (3) and G12A mutant standard with different concentrations as templates, and carrying out the ultra-block PCR reaction according to the method in the step one, thereby detecting the sensitivity.

The results are shown in FIG. 13. The result shows that when the background content of the mutant standard substance in the wild standard substance in the ultra-blocking detection system is 100%, the Ct value is about 21.89; when the background content of the mutant standard substance in the wild standard substance is 10%, the Ct value is about 25.24; when the background content of the mutant standard substance in the wild standard substance is 1%, the Ct value is about 28.76; when the content of the mutant standard substance in the background of the wild standard substance is 0.1%, the Ct value is about 32.06; when the background content of the mutant standard substance in the wild type standard substance is 0.01%, the Ct value is about 35.94; the method shows that the ultra-block PCR has high sensitivity and can detect the mutation as low as 0.01 percent.

2. And (3) respectively adopting the wild type standard prepared in the step one (3) and G12V mutant standard with different concentrations as templates, and carrying out the ultra-block PCR reaction according to the method in the step one, thereby detecting the sensitivity.

The results are shown in FIG. 14. The result shows that when the background content of the mutant standard substance in the wild standard substance in the ultra-blocking detection system is 100%, the Ct value is about 22.72; when the background content of the mutant standard substance in the wild standard substance is 10%, the Ct value is about 24.91; when the background content of the mutant standard substance in the wild standard substance is 1%, the Ct value is about 28.31; when the background content of the mutant standard substance in the wild standard substance is 0.1%, the Ct value is about 31.67; when the background content of the mutant standard substance in the wild standard substance is 0.01%, the Ct value is about 35.82; the method shows that the ultra-block PCR has high sensitivity and can detect the mutation as low as 0.01 percent.

3. And (3) respectively adopting the wild type standard prepared in the step one (3) and G12D mutant standard with different concentrations as templates, and carrying out the ultra-block PCR reaction according to the method in the step one, thereby detecting the sensitivity.

The results are shown in FIG. 15. The result shows that when the background content of the mutant standard substance in the wild standard substance in the ultra-blocking detection system is 100%, the Ct value is about 21.66; when the background content of the mutant standard substance in the wild standard substance is 10%, the Ct value is about 24.93; when the background content of the mutant standard substance in the wild standard substance is 1%, the Ct value is about 28.34; when the background content of the mutant standard substance in the wild type standard substance is 0.1%, the Ct value is about 31.72; when the background content of the mutant standard substance in the wild standard substance is 0.01%, the Ct value is about 35.72; the method shows that the ultra-block PCR has high sensitivity and can detect the mutation as low as 0.01 percent.

4. And (3) respectively adopting the wild type standard prepared in the step one (3) and G12S mutant standard with different concentrations as templates, and carrying out the ultra-block PCR reaction according to the method in the step one, thereby detecting the sensitivity.

The results are shown in FIG. 16. The result shows that when the background content of the mutant standard substance in the wild standard substance in the super-block detection system is 100%, the Ct value is about 21.49; when the background content of the mutant standard substance in the wild standard substance is 10%, the Ct value is about 25.49; when the background content of the mutant standard substance in the wild standard substance is 1%, the Ct value is about 29.06; when the background content of the mutant standard substance in the wild standard substance is 0.1%, the Ct value is about 32.68; when the background content of the mutant standard substance in the wild standard substance is 0.01%, the Ct value is about 35.92; the method shows that the ultra-block PCR has high sensitivity and can detect the mutation as low as 0.01 percent.

5. And (3) respectively adopting the wild type standard prepared in the step one (3) and G12C mutant standard with different concentrations as templates, and carrying out the ultra-block PCR reaction according to the method in the step one, thereby detecting the sensitivity.

The results are shown in FIG. 17. The result shows that when the background content of the mutant standard substance in the wild standard substance in the ultra-blocking detection system is 100%, the Ct value is about 22.36; when the background content of the mutant standard substance in the wild standard substance is 10%, the Ct value is about 25.98; when the background content of the mutant standard substance in the wild standard substance is 1%, the Ct value is about 28.89; when the background content of the mutant standard substance in the wild type standard substance is 0.1%, the Ct value is about 33.21; when the background content of the mutant standard substance in the wild type standard substance is 0.01%, the Ct value is about 35.97 (the sensitivity of the ultra-block PCR is high, and the mutation can be detected as low as 0.01%).

6. And (3) respectively adopting the wild type standard prepared in the step one (3) and G12R mutant standard with different concentrations as templates, and carrying out the ultra-block PCR reaction according to the method in the step one, thereby detecting the sensitivity.

The results are shown in FIG. 18. The result shows that when the background content of the mutant standard substance in the wild standard substance in the ultra-blocking detection system is 100%, the Ct value is about 22.02; when the background content of the mutant standard substance in the wild standard substance is 10%, the Ct value is about 25.29; when the background content of the mutant standard substance in the wild standard substance is 1%, the Ct value is about 29.16; when the background content of the mutant standard substance in the wild standard substance is 0.1%, the Ct value is about 32.65; when the background content of the mutant standard substance in the wild standard substance is 0.01%, the Ct value is about 35.78; the method shows that the ultra-block PCR has high sensitivity and can detect the mutation as low as 0.01 percent.

7. And (3) respectively adopting the wild type standard prepared in the step one (3) and G12R mutant standard with different concentrations as templates, and carrying out the ultra-block PCR reaction according to the method in the step one, thereby detecting the sensitivity.

The results are shown in FIG. 19. The result shows that when the background content of the mutant standard substance in the wild standard substance in the ultra-blocking detection system is 100%, the Ct value is about 21.98; when the background content of the mutant standard substance in the wild standard substance is 10%, the Ct value is about 25.48; when the background content of the mutant standard substance in the wild standard substance is 1%, the Ct value is about 29; when the background content of the mutant standard substance in the wild standard substance is 0.1%, the Ct value is about 32.81; when the background content of the mutant standard substance in the wild standard substance is 0.01%, the Ct value is about 35.86; the method shows that the ultra-block PCR has high sensitivity and can detect the mutation as low as 0.01 percent.

<110> Suzhou Jima Gene GmbH

<120> super-blocking fluorescent quantitative PCR method for detecting rare mutation with high sensitivity

<160> 22

<210> 1

<211> 23

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 1

cctccaggaa gcctacgtga tgg 23

<210> 2

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 2

cagttgagca ggtactggga g 21

<210> 3

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 3

agggcatgag ctgca 15

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 4

tgagctgcac ggtggaggtg a 21

<210> 5

<211> 15

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 5

agggcatgag ctgcg 15

<210> 6

<211> 174

<212> DNA

<213> human (Homo)

<400> 6

cctccaggaa gcctacgtga tggccagcgt ggacaacccc cacgtgtgcc gcctgctggg 60

catctgcctc acctccaccg tgcagctcat cacgcagctc atgcccttcg gctgcctcct 120

ggactatgtc cgggaacaca aagacaatat tggctcccag tacctgctca actg 174

<210> 7

<211> 174

<212> DNA

<213> human (Homo)

<400> 7

cctccaggaa gcctacgtga tggccagcgt ggacaacccc cacgtgtgcc gcctgctggg 60

catctgcctc acctccaccg tgcagctcat catgcagctc atgcccttcg gctgcctcct 120

ggactatgtc cgggaacaca aagacaatat tggctcccag tacctgctca actg 174

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 8

gcctgctgaa aatgactgaa 20

<210> 9

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 9

cctgacatac tcccaaggaa ag 22

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 10

aacttgtggt agttggagct gc 22

<210> 11

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 11

ataaacttgt ggtagttgga gctc 24

<210> 12

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 12

gtggtagttg gagctggaga 20

<210> 13

<211> 18

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 13

acttgaaacc caaggtac 18

<210> 14

<211> 13

<212> DNA

<213> Artificial sequence

<220>

<223>

<400> 14

tggagctggt ggc 13

<210> 15

<211> 319

<212> DNA

<213> human (Homo)

<400> 15

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctggtggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 16

<211> 319

<212> DNA

<213> human (Homo)

<400> 16

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctgctggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 17

<211> 319

<212> DNA

<213> human (Homo)

<400> 17

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctgttggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 18

<211> 319

<212> DNA

<213> human (Homo)

<400> 18

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctgatggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 19

<211> 319

<212> DNA

<213> human (Homo)

<400> 19

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctagtggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 20

<211> 319

<212> DNA

<213> human (Homo)

<400> 20

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agcttgtggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 21

<211> 319

<212> DNA

<213> human (Homo)

<400> 21

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctcgtggc gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

<210> 22

<211> 319

<212> DNA

<213> human (Homo)

<400> 22

gcctgctgaa aatgactgaa tataaacttg tggtagttgg agctggtgac gtaggcaaga 60

gtgccttgac gatacagcta attcagaatc attttgtgga cgaatatgat ccaacaatag 120

aggtaaatct tgttttaata tgcatattac tggtgcagga ccattctttg atacagataa 180

aggtttctct gaccattttc atgagtactt attacaagat aattatgctg aaagttaagt 240

tatctgaaat gtaccttggg tttcaagtta tatgtaacca ttaatatggg aactttactt 300

tccttgggag tatgtcagg 319

Claims

1. A primer probe combination for detecting whether EGFR gene T790M mutation exists in nucleic acid comprises a universal upstream primer, a universal downstream primer, a general detection probe, a specific amplification downstream primer and a blocking probe; the universal upstream primer is a single-stranded DNA molecule shown in a sequence 1 of a sequence table; the universal downstream primer is a single-stranded DNA molecule shown in a sequence 2 of a sequence table; the specific amplification downstream primer is a single-stranded DNA molecule shown in a sequence 3 of a sequence table, wherein LNA modification is carried out on 5 th nucleotide, 6 th nucleotide and 7 th nucleotide at the 5' end; the universal detection probe is a single-stranded DNA molecule shown in a sequence 4 of a sequence table, the 5 'end is marked by an FAM fluorescent group, and the 3' end is marked by a BHQ1 quenching group; the blocking probe is a single-stranded DNA molecule shown in a sequence 5 of a sequence table, wherein LNA modification is carried out on a 5 th nucleotide, a 6 th nucleotide and a 7 th nucleotide from a 5 'end, and amino modification is carried out on a 3' end.

2. A primer probe combination for detecting whether K-ras gene mutation exists in nucleic acid comprises a universal upstream primer, a universal downstream primer, a general detection probe, a specific amplification upstream primer and a blocking probe;

the universal upstream primer is shown as a sequence 8 in a sequence table;

the universal downstream primer is shown as a sequence 9 in a sequence table;

(b1) a single-stranded DNA molecule shown in sequence 10 of the sequence table;

(b2) a single-stranded DNA molecule shown in sequence 11 of the sequence table;

(b3) a single-stranded DNA molecule shown in sequence 12 of the sequence table;

the general detection probe is shown as a sequence 13 in a sequence table, wherein LNA modification is carried out from the 7 th nucleotide, the 8 th nucleotide, the 11 th nucleotide, the 12 th nucleotide and the 14 th nucleotide of a 5' end;

the blocking probe is shown as a sequence 14 in a sequence table, wherein LNA modification is carried out on the 7 th nucleotide, the 9 th nucleotide and the 10 th nucleotide from the 5 'end, and amino modification is carried out on the 3' end.

3. A method of detecting the presence or absence of a mutation in the EGFR gene T790M in a nucleic acid comprising the steps of: (1) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and adopting the universal upstream primer, the universal downstream primer and the universal detection probe in claim 1; (2) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and the specific amplification downstream primer, the general upstream primer, the general detection probe and the blocking probe in the claim 1; (3) if the fluorescence quantitative PCR of the step (1) can obtain positive amplification and the fluorescence quantitative PCR of the step (2) can not obtain positive amplification or the ct value is more than or equal to 37, and the EGFR gene T790M mutation does not exist in the nucleic acid to be detected, if the fluorescence quantitative PCR of the step (1) can obtain positive amplification and the fluorescence quantitative PCR of the step (2) can obtain positive amplification and the ct value is less than 37, and the EGFR gene T790M mutation exists in the nucleic acid to be detected;

the methods are non-disease diagnostic and therapeutic methods.

4. A method for detecting whether the mutation of the exon 2 of the K-ras gene exists in nucleic acid is a method A or a method B or a method C;

the method A comprises the following steps: (1) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and adopting the universal upstream primer, the universal downstream primer and the universal detection probe in claim 2; (2) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and the specific amplification upstream primer (b1), the universal downstream primer, the universal detection probe and the blocking probe in claim 2; (3) if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can not obtain positive amplification or the ct value is more than or equal to 37, no K-ras gene exon 2G 12A mutation exists in the nucleic acid to be detected, no K-ras gene exon 2G 12V mutation exists, and no K-ras gene exon 2G 12D mutation exists, if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can obtain positive amplification and the ct value is less than 37, and the K-ras gene exon 2G 12A mutation or the K-ras gene exon 2G 12V mutation or the K-ras gene exon 2G 12D mutation exists in the nucleic acid to be detected;

the method B comprises the following steps: (1) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and adopting the universal upstream primer, the universal downstream primer and the universal detection probe in claim 2; (2) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and the specific amplification upstream primer (b2), the universal downstream primer, the universal detection probe and the blocking probe in claim 2; (3) if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can not obtain positive amplification or the ct value is more than or equal to 37, no K-ras gene exon 2G 12S mutation exists in the nucleic acid to be detected, no K-ras gene exon 2G 12C mutation exists, and no K-ras gene exon 2G 12R mutation exists, if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can obtain positive amplification and the ct value is less than 37, and the K-ras gene exon 2G 12S mutation or the K-ras gene exon 2G 12C mutation or the K-ras gene exon 2G 12R mutation exists in the nucleic acid to be detected;

the method C comprises the following steps: (1) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and adopting the universal upstream primer, the universal downstream primer and the universal detection probe in claim 2; (2) performing fluorescent quantitative PCR by using nucleic acid to be detected as a template and the specific amplification upstream primer (b3), the universal downstream primer, the universal detection probe and the blocking probe in claim 2; (3) if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can not obtain positive amplification or the ct value is more than or equal to 37, and the exon 2G 13D mutation of the K-ras gene does not exist in the nucleic acid to be detected, if the fluorescent quantitative PCR in the step (1) can obtain positive amplification and the fluorescent quantitative PCR in the step (2) can obtain positive amplification and the ct value is less than 37, and the exon 2G 13D mutation of the K-ras gene exists in the nucleic acid to be detected;

the methods are non-disease diagnostic and therapeutic methods.