CN110938693A

CN110938693A - Primer group, kit and method for detecting BRAF gene mutation

Info

Publication number: CN110938693A
Application number: CN201911230036.8A
Authority: CN
Inventors: 徐雪; 宋萍; 汪进平; 陈苗苗; 杨玉霞; 罗俊峰
Original assignee: Review Genetic Technology (suzhou) Co Ltd
Current assignee: Review Genetic Technology (suzhou) Co Ltd
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2020-03-31
Anticipated expiration: 2039-12-04
Also published as: CN110938693B

Abstract

The invention discloses a primer group, a kit and a method for detecting BRAF gene mutation, and belongs to the technical field of gene detection. The primer group provided by the invention comprises a BRAF upstream primer and a BRAF downstream primer; the nucleotide sequence of the BRAF upstream primer is shown as a sequence table SEQ ID NO 1; the nucleotide sequence of the BRAF downstream primer is shown in a sequence table SEQ ID NO. 2. In addition, the kit provided by the invention comprises a PCR amplification reaction reagent, a positive quality control product, a negative quality control product, a primer mixed solution, a primer probe mixed solution and a standard curve equation. The method for detecting the BRAF gene mutation has the characteristics of high efficiency, simplicity, convenience, intuition, high sensitivity and the like, and can be used for carrying out quantitative analysis on the mutation frequency of the BRAF gene.

Description

Primer group, kit and method for detecting BRAF gene mutation

Technical Field

The invention relates to the technical field of gene detection, in particular to a primer group, a kit and a method for detecting BRAF gene mutation.

Background

The BRAF (v-RAF murine sarkoma viral oncogene homolog B) gene encodes B-RAF protein and is involved in cell signal regulation, growth and survival. Mutation of V600E in the BRAF gene can lead to constitutive activation of B-RAF protein monomers and subsequent activation of MEK1 and MEK2 proteins, thereby promoting cell proliferation and inhibiting apoptosis. BRAF mutations account for approximately 8% of human tumors, with a predominant occurrence in melanoma, colorectal, thyroid, and non-small cell lung cancers, with the vast majority of mutations being the BRAF V600E mutation. The research finds that the tumor with BRAF V600E mutation has poor prognosis, is not sensitive to chemotherapy and has short survival time. Currently, the FDA has approved different targeted therapeutic drugs against BRAF V600E mutations of different tumors, respectively.

The combination therapy of Dabrafenib (Dabraafinib) and Trametinib (Trametinib) can be used for adjuvant therapy of melanoma which is completely resected after the transfer of combined lymph nodes of BRAF V600E or BRAF V600K mutation and non-resectable or metastatic melanoma, and can also be used for adjuvant therapy of locally advanced or metastatic thyroid undifferentiated carcinoma which is mutated in BRAF V600E and has no suitable local treatment scheme and has metastatic NSCLC and BRAF V600E mutation. The combination of Vemurafenib (Vemurafenib) and Cobimetinib (Cobimetinib) can be used for adjuvant treatment of BRAF V600E or BRAF V600K mutant unresectable or metastatic melanoma.

The liquid biopsy technology is a breakthrough technology which can realize non-invasive sampling, early tumor progress detection and auxiliary treatment, particularly Circulating free DNA (cfDNA) detection from plasma, is rapidly becoming an important minimally invasive auxiliary means for standard tumor biopsy, and can dynamically reflect the genetic profile of tumors. Research also shows that free DNA in blood is extracellular DNA in a cell-free state, which is mainly double-stranded DNA with small fragments, and the characteristic change of tumor can be reflected by qualitative and quantitative detection of cfDNA (circulating tumor DNA) derived from tumor cells in plasma cfDNA. However, since plasma ctDNA is very low in content and there is a lot of interference from other sources of cfDNA, it is especially important to develop a DNA mutation detection technology that can be highly sensitive, highly accurate and highly reliable.

At present, the prior art can generally detect the V600E mutation of the human B-raf gene by adopting ARMS-PCR fluorescent probe technology, oligonucleotide modification technology and TspR I restriction enzyme technology, but the above method is not suitable for the detection of cfDNA. In addition, the Sanger sequencing method is an international gold standard for all gene detection at present, the specific performance of the Sanger sequencing method reaches 100 percent, and the Sanger sequencing method is a classical technical platform in the aspect of molecular diagnosis; however, Sanger sequencing has low sensitivity and cannot be detected for samples with low mutation frequency. Therefore, there is a need for a highly sensitive BRAF gene mutation method that can be used to detect cfDNA samples with low mutation frequency.

Disclosure of Invention

The invention aims to provide a primer group, a kit and a method for detecting BRAF gene mutation, so as to solve the problems in the background technology.

In order to achieve the above purpose, the embodiments of the present invention provide the following technical solutions:

a primer group for detecting BRAF gene mutation comprises a BRAF upstream primer and a BRAF downstream primer; the nucleotide sequence of the BRAF upstream primer is shown as a sequence table SEQ ID NO 1; the nucleotide sequence of the BRAF downstream primer is shown in a sequence table SEQ ID NO. 2.

The kit comprises a PCR amplification reaction reagent, a positive quality control product, a negative quality control product and a standard curve equation, and further comprises a primer mixed solution and a primer probe mixed solution corresponding to the primer mixed solution.

Preferably, the primer probe mixture comprises the BRAF upstream primer, the BRAF downstream primer and the BRAF probe; the nucleotide sequence of the BRAF probe is shown in a sequence table SEQ ID NO. 3.

Preferably, the molar concentration of the BRAF upstream primer and the BRAF downstream primer in the primer mixture is 2-100 mu mol/L.

Preferably, the molar concentration of the BRAF upstream primer and the BRAF downstream primer in the primer probe mixed solution is 2-100 mu mol/L, and the molar concentration of the BRAF probe is 10-1000 mu mol/L.

Preferably, the positive quality control product comprises mutant plasmids of mutation sites of BRAF gene V600E and healthy human genome DNA; the nucleotide sequence of the mutant plasmid is shown in a sequence table SEQ ID NO. 4.

Preferably, the standard curve equation is obtained by the difference between the first CT value and the second CT value of the standard substance obtained by diluting the mutant plasmid and the healthy human genome DNA according to BRAF V600 position with the molecular number ratio of 50%, 10%, 5%, 1%, 0.1% and 0%, and specifically comprises the following steps:

mixing the primer probe mixed solution, the PCR amplification reaction reagent and the 6 mutation frequency standard products together for PCR amplification reaction to obtain first CT values of the 6 mutation frequency standard products;

respectively mixing the primer mixed solution, the PCR amplification reaction reagent and the 6 mutation frequency standard products together for PCR amplification reaction to obtain second CT values of the 6 mutation frequency standard products;

and obtaining a standard curve equation according to the difference value of the first CT value and the second CT value of the 6 standard products.

Another objective of the embodiments of the present invention is to provide a method for detecting BRAF gene mutation, in which a test sample is analyzed simultaneously with a positive quality control substance and a negative quality control substance in each detection reaction, comprising the following steps:

obtaining genome DNA or plasma free DNA (cell-free DNA) of a sample to be detected as a template;

mixing the primer probe mixed solution, the PCR amplification reaction reagent and the template together for PCR amplification reaction to obtain a first PCR product and a first CT value;

mixing the primer mixed solution, the PCR amplification reaction reagent and the template together for PCR amplification reaction to obtain a second PCR product and a second CT value;

mixing the primer probe mixed solution, the PCR amplification reaction reagent and the negative quality control product together to perform PCR amplification reaction to obtain a first negative reference CT value;

mixing the primer mixed solution, the PCR amplification reaction reagent and the negative quality control product together for PCR amplification reaction to obtain a second negative reference CT value;

mixing the primer probe mixed solution, the PCR amplification reaction reagent and the positive quality control product together to perform PCR amplification reaction to obtain a first positive reference CT value;

mixing the primer mixed solution, the PCR amplification reaction reagent and the positive quality control product together for PCR amplification reaction to obtain a second positive reference CT value;

and calculating the difference value of the first CT value and the second CT value, the negative reference CT difference value and the positive reference CT difference value, and judging the positive and negative of the sample.

Preferably, the method for detecting a mutation in the BRAF gene further comprises:

sequencing the first PCR product and the second PCR product of the sample with positive judgment results (such as Sanger sequencing, next-generation high-throughput sequencing, single-molecule sequencing, nanopore sequencing and pyrosequencing) to obtain sequencing results;

if Sanger sequencing is used, the universal primer M13RP can be used for direct sequencing, and a sequencing peak image can be obtained;

analyzing a sequencing result, and identifying the BRAF gene mutation type of the sample to be detected;

and substituting the difference value of the first CT value and the second CT value into the standard curve equation corresponding to the mutation type according to the identified BRAF gene mutation type to obtain the mutation frequency corresponding to the BRAF gene mutation type.

Compared with the prior art, the embodiment of the invention has the beneficial effects that:

the invention discloses a polymerase chain reaction for BRAF gene mutation detection, which can selectively amplify a variant sequence, and can ensure that the amplification efficiency of the variant sequence is 1000 times or more than that of a wild type sequence in about 20 nucleotide window regions. According to known gene mutation sites, specific primers and probes are designed, multiple mutation modes of the same site can be detected at the same annealing temperature, and the gene mutation frequency can be quantitatively analyzed. After the polymerase chain reaction selectively amplifies the variant sequence disclosed by the patent, the sensitivity of sanger sequencing can be improved from 10% to 0.1%. Wherein:

(1) the invention can directly sequence the PCR amplification product during detection by adopting the M13RP universal primer, and carry out mutation analysis by software.

(2) The method for detecting BRAF gene mutation provided by the invention has high sensitivity, can detect the mutation frequency as low as 0.1%, and can enrich the mutant template so as to meet the problem of low sensitivity of Sanger sequencing.

(3) The method for detecting the BRAF gene mutation has the characteristics of high efficiency, simplicity, convenience, intuition, high sensitivity and the like, and can be used for carrying out quantitative analysis on the mutation frequency of the BRAF gene.

Drawings

Fig. 1 is a standard graph of mutation site V600E of BRAF gene obtained in example 3.

Fig. 2 is a graph of PCR amplification curves of BRAF V600E for different mutation frequencies.

FIG. 3 is a sanger sequencing peak plot of 5% standard primer probe set products in example 3.

FIG. 4 is a graph showing the sanger sequencing peaks of the primer set products of 5% standard in example 3.

FIG. 5 is a sanger sequencing peak plot of 1% standard primer probe set products in example 3.

FIG. 6 is a graph showing the sanger sequencing peaks of the primer set products of 1% standard in example 3.

FIG. 7 is a sanger sequencing peak plot of 0.1% standard primer probe set products in example 3.

FIG. 8 is a graph showing the sanger sequencing peaks of 0.1% standard primer set products in example 3.

FIG. 9 is a sanger sequencing peak plot of 0% standard primer probe set products in example 3.

FIG. 10 is a graph showing the sequencing peaks of sanger for 0% of the standard primer set in example 3.

FIG. 11 is a sanger sequencing peak plot of the primer probe set products for detecting cfDNA of melanoma patients in example 4.

Fig. 12 is a sanger sequencing peak plot of cfDNA primer set products for melanoma patients detected in example 4.

FIG. 13 is a graph of sanger sequencing peaks of cfDNA primer probe set products for patients with thyroid cancer in example 4.

FIG. 14 is a graph of sanger sequencing peaks of cfDNA primer set products of patients with thyroid cancer detected in example 4.

FIG. 15 is a graph of sanger sequencing peaks of cfDNA primer probe set products of colorectal cancer patients detected in example 4.

FIG. 16 is a graph of sanger sequencing peaks of cfDNA primer set products of colorectal cancer patients detected in example 4.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. 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. In addition, the apparatus and reagents used in the following examples are commercially available ones unless otherwise specified.

Example 1

The embodiment provides a primer group for detecting BRAF gene mutation, which comprises a BRAF upstream primer and a BRAF downstream primer; the nucleotide sequence of the BRAF upstream primer is shown as a sequence table SEQ ID NO 1; the nucleotide sequence of the BRAF downstream primer is shown in a sequence table SEQ ID NO. 2.

Specifically, the sequences of the primer probes are shown in the following table 1:

TABLE 1

The primer probe set designed by the embodiment of the invention mainly aims at the hotspot mutation of a V600 locus of a BRAF gene, and can detect various mutation modes of the V600 locus, including BRAF p.V600E, BRAF p.V600E2, BRAF p.V600K, BRAF p.V600K 601> E and the like.

Example 2

The embodiment provides a kit for detecting BRAF gene mutation, which comprises a PCR amplification reaction reagent, a positive quality control product, a negative quality control product and a standard curve equation, and also comprises a primer mixed solution and a primer probe mixed solution corresponding to the primer mixed solution; the primer mixture comprises the BRAF upstream primer and the BRAF downstream primer provided in example 1. Specifically, the PCR amplification reaction reagent is ZoomBDA Master Mix; the molar concentration of the BRAF upstream primer and the BRAF downstream primer in the primer mixed solution is 2-100 mu mol/L; the primer probe mixed solution comprises the BRAF upstream primer, the BRAF downstream primer and a BRAF probe; the nucleotide sequence of the BRAF probe is shown as a sequence table SEQ ID NO. 3, specifically, the BRAF probe and a specific BRAF upstream primer of a corresponding gene have an overlapping sequence of 10bp, and C3 Spacer modification is carried out at the 3' end of the probe, so that when the probe is combined with a wild type template, amplification of the wild type template can be competitively inhibited. The molar concentration of the BRAF upstream primer and the BRAF downstream primer in the primer probe mixed solution is 2-100 mu mol/L, and the molar concentration of the BRAF probe is 10-1000 mu mol/L.

In addition, the negative quality control product is healthy human whole blood genome DNA (DeoxyriboNucleic Acid); the positive quality control product is mixed liquid of mutant plasmids of BRAF gene V600E and healthy human genome DNA, and the concentration of the mutant plasmids is 50%; the nucleotide sequence of the mutant plasmid is shown in a sequence table SEQ ID NO. 4.

Example 3

This example provides a method for detecting BRAF gene mutation using the kit provided in example 2 above, where the sample should be analyzed simultaneously with the positive and negative quality controls in each detection reaction. Which comprises the following steps:

(1) obtaining cfDNA of a sample to be detected as a template; specifically, the OD260/OD280 value of the obtained cfDNA should be controlled to be 1.8-2.0; in addition, agarose gel electrophoresis is used for observing the range of cfDNA fragments, which is less than 1000bp, and subsequent sample detection is immediately carried out or stored at-20 ℃ for later use.

(2) Mixing the primer probe mixed solution, the PCR amplification reaction reagent and the template together for PCR amplification reaction to obtain a first PCR product and a first CT value; specifically, 6 μ L of primer probe mixed liquor and 25 μ L of PCR amplification reaction reagent are mixed together to obtain reaction liquid, the reaction liquid is placed in a 96-well plate or a hole corresponding to 8-connecting pipes, the template obtained in the step (1) is added into the reaction liquid, wherein the addition amount of the cfDNA template is 1/2 of the elution volume, the maximum volume is not more than 19ul, and then nuclease-free water is used for supplementing the total volume to 50 ul.

(3) Mixing the primer mixed solution, the PCR amplification reaction reagent and the template together for PCR amplification reaction to obtain a second PCR product and a second CT value; specifically, firstly, mixing 4 μ L of primer mixed solution and 25 μ L of PCR amplification reaction reagent together to obtain reaction solution, and placing the reaction solution in a 96-well plate or a hole corresponding to the 8-connecting pipe which is the same as the step (2); and (2) adding the template obtained in the step (1) into the reaction solution, wherein the addition amount of the cfDNA template is 1/2 of the elution volume, the maximum volume is not more than 19ul, and then supplementing the total volume to 50ul by using nuclease-free water.

(4) Mixing the primer probe mixed solution, the PCR amplification reaction reagent and the negative quality control product together, and placing the mixture in a 96-well plate or 8-connecting pipes for PCR amplification reaction to obtain a first negative reference CT value; specifically, the template in step (2) was replaced with a negative quality control, wherein the volume of the negative quality control added was 10ul, and then supplemented with nuclease-free water to a total volume of 50 ul. The rest components, the using amount and the reaction conditions are the same as those of the step (2).

(5) Mixing the primer mixed solution, the PCR amplification reaction reagent and the negative quality control product together, and placing the mixture in a 96-well plate or 8-connecting pipes for PCR amplification reaction to obtain a second negative reference CT value; specifically, the template in step (3) was replaced with a negative quality control, wherein the volume of the negative quality control added was 10ul, and then supplemented with nuclease-free water to a total volume of 50 ul. The rest components, the using amount and the reaction conditions are the same as those of the step (3).

(6) Mixing the primer probe mixed solution, the PCR amplification reaction reagent and the positive quality control product together, and placing the mixture in a 96-well plate or 8-connecting pipes for PCR amplification reaction to obtain a first positive reference CT value; specifically, the template in step (2) is replaced by a positive quality control substance, wherein the adding volume of the positive quality control substance is 10ul, and then nuclease-free water is used for supplementing until the total volume is 50 ul. The rest components, the using amount and the reaction conditions are the same as those of the step (2).

(7) Mixing the primer mixed solution, the PCR amplification reaction reagent and the negative quality control product together, and placing the mixture in a 96-well plate or 8-connecting pipes for PCR amplification reaction to obtain a second positive reference CT value; specifically, the template in step (3) is replaced by a positive quality control substance, wherein the adding volume of the positive quality control substance is 10ul, and then nuclease-free water is used for supplementing until the total volume is 50 ul. The rest components, the using amount and the reaction conditions are the same as those of the step (3).

(8) And (3) centrifuging the added 96-well plate or 8-connecting tube at a low speed for several seconds to ensure that all liquid is placed at the bottom of the tube without bubbles, and then placing the tube in an ABI 7500PCR instrument to perform Realtime PCR reaction.

(9) And comparing the difference value of the first CT value and the second CT value with the negative reference CT difference value and the positive reference CT difference value, and judging whether the BRAF gene has mutation according to the comparison result. Specifically, if the difference is between the positive reference difference and the negative reference difference, the detection result of the sample is positive or weakly positive, and a corresponding gene mutation may exist.

(10) Respectively carrying out Sanger sequencing on the first PCR product and the second PCR product obtained in the steps (2) and (3) to obtain a Sanger sequencing peak diagram; specifically, the products in the reaction tubes with probes and without probes of each sample are collected separately, and each hole corresponds to one collection tube, so that Sanger sequencing can be carried out by a sequencing company, and M13RP can be directly used for sequencing.

(11) Analyzing a sanger sequencing peak map, and identifying the BRAF gene mutation type of a sample to be detected; specifically, software such as sequencing analysis, Bioedit and Snapgene is used for sequence comparison analysis, a sequence obtained by sequencing is compared with a detection gene amplification sequence, and the gene mutation type is identified.

(11) And substituting the difference value of the first CT value and the second CT value into the standard curve equation corresponding to the mutation type according to the identified BRAF gene mutation type to obtain the mutation frequency corresponding to the BRAF gene mutation type. Specifically, the difference between the first CT value and the second CT value is taken as the Y value and is brought into a standard curve equation, as shown in fig. 1, the obtained X is the logarithm (log (vaf)) of the mutation frequency of the corresponding BRAF gene mutation type, and then 10 is calculated^XNamely the mutation frequency of the mutation type of the BRAF gene to be detected.

The standard curve includes a standard product obtained by mixing the mutant plasmid and the healthy human genomic DNA at concentrations of 0%, 0.1%, 1%, 5%, 10%, and 50% of the mutant plasmid, respectively, and PCR reactions are performed according to steps (4) and (5), and 6 sets of PCR amplification curves are shown in fig. 2, in which a to F correspond to concentrations of 0%, 0.1%, 1%, 5%, 10%, and 50%, respectively. It can be known from the figure that the sensitivity of the method for detecting the mutation of the V600 site of the BRAF gene provided by the embodiment of the invention is 0.1%. Meanwhile, Sanger sequencing is carried out on qPCR amplification products of 5%, 1%, 0.1% and 0% of four standard primer probe sets and primer sets, and Sanger sequencing peak maps are shown in accompanying figures 3-10, wherein the Sanger sequencing peak maps of the primer probe sets and the primer set products of 5% of the standard are shown in figures 3 and 4 respectively; FIGS. 5 and 6 are sanger sequencing peak plots of 1% standard primer probe set and primer set product, respectively; FIGS. 7 and 8 are sanger sequencing peak plots of 0.1% standard primer probe set and primer set product, respectively; FIG. 9 and FIG. 10 are sanger sequencing peak plots of 0% standard primer probe set and primer set product, respectively.

As can be seen from FIGS. 3-4, for the standard with a mutation frequency of 5%, there is a single peak in the primer probe set product, and it can be clearly seen that T is mutated to A at V600 site; likewise, there is a single peak in the primer set product, but the wild-type sequence.

As can be seen from FIGS. 5-6, for the standard with a mutation frequency of 1%, there is a single peak in the primer probe set product and it is shown as the V600E mutant; also unimodal in the primer set product, but shown as wild-type sequence.

As can be seen from FIGS. 7 to 8, for the standard with a mutation frequency of 0.1%, the sanger sequencing peak pattern differs from those of other mutation frequency standards, and is a set peak in the primer probe set products and is shown as V600E mutant and wild type; a clear wild type single peak is shown in the primer set product, indicating that the mutant sequences were greatly enriched in the primer probe set and could be detected by Sanger.

As can be seen from FIGS. 9 to 10, for the standard with a mutation frequency of 0%, the sanger sequencing peak patterns of the primer probe set and the primer set product are both single peaks and are both shown as wild-type sequences.

As can be seen from the sanger sequencing peak maps of different concentration gradients, qPCR products in the primer probe set and the primer set are single, and the peak maps are clear, which indicates that the primers and the probes have higher specificity; meanwhile, mutant type and wild type samples with mutation frequency of more than 0.1 percent can be clearly distinguished, so that the method has higher sensitivity and specificity.

When the ABI 7500PCR instrument is used, the "SYBR Green Reagents" mode is selected, and the PCR program shown in table 2 is set:

TABLE 2

Example 4

The embodiment is a detection experiment of clinical samples, and specifically, a part of clinical samples which are detected and determined to have BRAF V600E mutation are taken firstly, wherein the samples comprise cfDNA of melanoma patients, cfDNA of thyroid cancer patients and cfDNA of colorectal cancer patients; then, the above three samples were detected according to the detection method provided in example 3, and the detected sequencing peaks are shown in FIGS. 11-16. Wherein, fig. 11 and fig. 12 are sequencing peak diagrams of cfDNA plus BRAF probe (primer probe set) of melanoma patients and cfDNA without probe (primer set) of melanoma patients, respectively; FIGS. 13 and 14 are sequencing peak plots of cfDNA of patients with thyroid cancer plus a BRAF probe (primer probe set) and cfDNA of patients with thyroid cancer without a probe (primer set), respectively; fig. 15 and fig. 16 are sequencing peak diagrams of cfDNA of colorectal cancer patients plus BRAF probe (primer probe set) and cfDNA of colorectal cancer patients without probe (primer set), respectively.

As can be seen from FIGS. 11-12, the mutation from T to A was clearly detected in the probe set with BRAF, but not detected without any probe, indicating that the mutation rate was low, and △ CT values (difference between the first CT value and the second CT value) detected from the sample were substituted into the equation of the curve provided in FIG. 1, and the mutation frequency was calculated to be 13% because the mutation rate was low in the conventional Realtime PCR amplification process, but the proportion of the mutation was low compared with the wild type.

It can be seen from FIGS. 13-14 that the mutation of T to A was clearly detected in the probe set with BRAF and remained wild-type in the probe set without BRAF, and the mutation frequency was calculated to be 3% by substituting △ CT value detected from this sample into the equation of the standard curve provided in FIG. 1.

From the attached drawings 15-16, T mutation is obviously detected in the probe set with the BRAF to A, the mutation is a double peak at the same time, the mutation type and the wild type exist at the same time, no mutation is detected in the probe set without the BRAF, the mutation frequency of the BRAF V600E in the sample is very low, after the mutation sequence is amplified by the method provided by the invention, a large amount of enrichment is carried out on the mutation sequence, the △ CT value detected by the sample is substituted into the equation of the standard curve provided by the figure 1, and the mutation frequency is calculated to be 0.2%.

It should be noted that the mutation frequency obtained by the detection method provided by the present invention can provide a reference for the clinician to the prognostic treatment data, but cannot be directly used as the only basis for clinical diagnosis and treatment.

In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the content of the specification, and must be determined according to the scope of the claims.

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Claims

1. The primer group for detecting BRAF gene mutation is characterized by comprising a BRAF upstream primer and a BRAF downstream primer; the nucleotide sequence of the BRAF upstream primer is shown as a sequence table SEQ ID NO 1; the nucleotide sequence of the BRAF downstream primer is shown in a sequence table SEQ ID NO. 2.

2. The kit for detecting BRAF gene mutation comprises a PCR amplification reaction reagent, a positive quality control product, a negative quality control product and a standard curve equation, and is characterized by also comprising a primer mixed solution and a primer probe mixed solution corresponding to the primer mixed solution.

3. The kit for detecting the mutation of BRAF gene as claimed in claim 2, wherein the primer probe mixture comprises the BRAF upstream primer and the BRAF downstream primer as claimed in claim 1 and the BRAF probe; the nucleotide sequence of the BRAF probe is shown in a sequence table SEQ ID NO. 3.

4. The kit for detecting BRAF gene mutation of claim 2, wherein the molar concentrations of the BRAF upstream primer and the BRAF downstream primer in the primer mixture are both 2-100 μmol/L.

5. The kit for detecting BRAF gene mutation of claim 2, wherein the molar concentrations of the BRAF upstream primer and the BRAF downstream primer in the primer probe mixture are both 2-100 μmol/L, and the molar concentration of the BRAF probe is 10-1000 μmol/L.

6. The kit for detecting BRAF gene mutation of claim 2, wherein the positive quality control includes BRAF gene V600E mutation site mutation plasmid and healthy human genome DNA; the nucleotide sequence of the mutant plasmid is shown in a sequence table SEQ ID NO. 4.

7. The kit for detecting a BRAF gene mutation according to claim 2, wherein the standard curve equation is obtained by the difference between the first CT value and the second CT value of the standard obtained by diluting the mutant plasmid and the healthy human genomic DNA at 50%, 10%, 5%, 1%, 0.1% and 0% in terms of the number of molecules at the BRAF V600 position, and specifically comprises the following steps:

mixing the primer probe mixture according to claim 2 or 3 or 5, the PCR amplification reaction reagent according to claim 2 and the 6 mutation frequency standards together to perform PCR amplification reaction, so as to obtain first CT values of the 6 mutation frequency standards;

mixing the primer mixture according to claim 2 or 4, the PCR amplification reaction reagent according to claim 2 and the 6 mutation frequency standards together to perform PCR amplification reaction, so as to obtain second CT values of the 6 mutation frequency standards;

8. The method for detecting the BRAF gene mutation is characterized in that in each detection reaction, a sample to be detected, a positive quality control product and a negative quality control product are analyzed simultaneously, and the method comprises the following steps:

obtaining genome DNA or plasma free DNA of a sample to be detected as a template;

mixing the primer probe mixture according to claim 2 or 3 or 5, the PCR amplification reaction reagent according to claim 2 and the template for PCR amplification reaction to obtain a first PCR product and a first CT value;

mixing the primer mixture according to claim 2 or 4, the PCR amplification reaction reagent according to claim 2 and the template for PCR amplification reaction to obtain a second PCR product and a second CT value;

mixing the primer probe mixture of claim 2 or 3 or 5, the PCR amplification reaction reagent of claim 2 and the negative quality control product of claim 2 together to perform a PCR amplification reaction to obtain a first negative reference CT value;

mixing the primer mixture solution according to claim 2 or 4, the PCR amplification reaction reagent according to claim 2 and the negative quality control product according to claim 2 together to perform a PCR amplification reaction, thereby obtaining a second negative reference CT value;

mixing the primer probe mixture of claim 2 or 3 or 5, the PCR amplification reaction reagent of claim 2 and the positive quality control product of claim 2 together to perform a PCR amplification reaction to obtain a first positive reference CT value;

mixing the primer mixture solution according to claim 2 or 4, the PCR amplification reaction reagent according to claim 2 and the positive quality control product according to claim 2 together to perform a PCR amplification reaction, thereby obtaining a second positive reference CT value;

9. The method for detecting a mutation in the BRAF gene of claim 8, further comprising:

sequencing the first PCR product and the second PCR product of the sample with positive judgment results to obtain sequencing results;