Primer set, kit and method for detecting braf gene mutation
A kit and mutation frequency technology, applied in the field of primer sets for detecting BRAF gene mutations, can solve the problems of low sensitivity of Sanger sequencing, undetectable samples with low mutation frequency, unsuitable cfDNA detection, etc., achieving high sensitivity and meeting low sensitivity Effect
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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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