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Primer group, kit and method for detecting BRAF gene mutation

A kit and reagent technology, applied in the field of primer sets for detecting BRAF gene mutations, can solve the problems of low Sanger sequencing sensitivity, undetectable samples with low mutation frequency, unsuitable cfDNA detection, etc., and achieve high sensitivity

Active Publication Date: 2020-03-31
CARRIER GENE TECH SUZHOU CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, the existing technology can generally detect the V600E mutation of human B-raf gene by using ARMS-PCR fluorescent probe technology, oligonucleotide modification technology and TspRI restriction enzyme digestion technology, but the above methods are not applicable to cfDNA detection
In addition, Sanger sequencing is currently the international gold standard for all genetic testing, and its specificity can reach 100%, which is a classic technical platform for molecular diagnosis; however, the sensitivity of Sanger sequencing is low, and it cannot detect samples with low mutation frequency

Method used

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  • Primer group, kit and method for detecting BRAF gene mutation
  • Primer group, kit and method for detecting BRAF gene mutation
  • Primer group, kit and method for detecting BRAF gene mutation

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Embodiment 1

[0055] This embodiment provides a primer set for detecting BRAF gene mutations, which includes BRAF upstream primers and BRAF downstream primers; the nucleotide sequence of the BRAF upstream primers is shown in the sequence table SEQ ID NO: 1; the The nucleotide sequence of the BRAF downstream primer is shown in the sequence listing SEQ ID NO:2.

[0056] Specifically, the sequences of each primer probe are shown in Table 1 below:

[0057] Table 1

[0058]

[0059] The primer probe set designed in the embodiment of the present invention is mainly aimed at the hotspot mutation of the V600 site of the BRAF gene, which can detect various mutation modes of the V600 site, including BRAF p.V600E, BRAF p.V600E2, BRAF p.V600K and BRAF p.V600 K601>E et al.

Embodiment 2

[0061] This embodiment provides a kind of test kit for detecting BRAF gene mutation, and it comprises PCR amplification reaction reagent, positive quality control substance and negative quality control substance and standard curve equation, and described test kit also comprises primer mixture and A primer-probe mixture corresponding to the primer mixture; the primer mixture includes the BRAF upstream primer and BRAF downstream primer provided in Example 1 above. 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 mixture is 2-100 μmol / L; the primer probe mixture includes the above-mentioned BRAF upstream primer, the above-mentioned BRAF downstream primer and BRAF probe; the nucleotide sequence of the BRAF probe is shown in the sequence table SEQ ID NO: 3, specifically, the BRAF probe has a 10bp overlapping sequence with the specific BRAF upstream primer of th...

Embodiment 3

[0064] This embodiment provides a method for detecting BRAF gene mutation using the kit provided in the above-mentioned embodiment 2. In each round of detection reaction, the sample should be analyzed simultaneously with the positive quality control and the negative quality control. It includes the following steps:

[0065] (1) Obtain the cfDNA of the sample to be tested as a template; specifically, the OD260 / OD280 value of the obtained cfDNA should be controlled at 1.8 to 2.0; in addition, use agarose gel electrophoresis to observe the range of cfDNA fragments, which must be less than 1000bp, and proceed immediately Subsequent sample testing or storage at -20°C for later use.

[0066] (2) Mix the above-mentioned primer-probe mixture, the above-mentioned PCR amplification reaction reagent, and the above-mentioned template to carry out PCR amplification reaction to obtain the first PCR product and the first CT value; specifically, first take 6 μL of the primer-probe mixture , ...

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Abstract

The invention discloses a primer group, a kit and a method for detecting BRAF gene mutation, belonging to the technical field of gene detection. The primer group provided by the invention comprises aBRAF upstream primer and a BRAF downstream primer, wherein the BRAF upstream primer has a nucleotide sequence as shown in a sequence table SEQ ID NO: 1; and the BRAF downstream primer has a nucleotidesequence as 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 BRAF gene mutation provided by the invention has the characteristicsof high efficiency, simplicity, convenience, intuition, high sensitivity and the like, and can be used for quantitatively analyzing the mutation frequency of a BRAF gene.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a primer set, kit and method for detecting BRAF gene mutation. Background technique [0002] BRAF (v-raf murine sarcoma viral oncogene homolog B) gene encodes B-RAF protein, which is involved in cell signal regulation, growth and survival. V600E mutation of BRAF gene can lead to constitutive activation of B-RAF protein monomer and subsequent activation of MEK1 and MEK2 proteins, thereby promoting cell proliferation and inhibiting cell apoptosis. BRAF mutations account for about 8% of human tumors, mainly in melanoma, colorectal cancer, thyroid cancer and non-small cell lung cancer, most of which are BRAF V600E mutations. Studies have found that tumors with BRAF V600E mutation have a poor prognosis, are not sensitive to chemotherapy, and have a short survival period. At present, the FDA has approved different targeted therapy drugs for BRAF V600E mutations in different tu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11
CPCC12Q1/6886C12Q2600/156
Inventor 徐雪宋萍汪进平陈苗苗杨玉霞罗俊峰
Owner CARRIER GENE TECH SUZHOU CO LTD
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