Method and kit thereof for detecting BRAF gene mutation

Abstract

The invention discloses a method and kit thereof for detecting BARF gene mutation. The method comprises the following steps: (1) carrying out reactions in tube A, B, and C at the same time, carrying out BARF gene V600E and V600K mutation realtime quantitative PCR detection on a plasmid standard with a known mutation amount so as to obtain standard curves; (2) extracting and purifying the DNA of a sample, measuring the concentration, carrying out realtime quantitative PCR detection on the sample according to the tube A, B, and C reaction systems in the step (1), judging whether the gene mutation exists or not and the mutation amount according to the standard curves and the amplification fluorescence signals. The kit comprises a primer pair for detecting the No.15 exon mutation V600 E and V600K of BRAF gene, a probe, and a PNA repressing sequence. The provided method and kit can more precisely detect the No.15 exon mutation V600 E and V600K of BRAF gene, and the detection sensitivity is greatly improved.

Description

technical field [0001] The invention relates to a method for detecting gene mutation and its kit, in particular to a method for detecting BRAF gene mutation and its kit. Background technique [0002] The incidence of colorectal cancer is increasing year by year, and patients with advanced and postoperative recurrence need biological targeted therapy. The U.S. FDA and my country’s postoperative treatment plan for colorectal cancer clearly point out that when implementing first-line and second-line chemotherapy regimens, when it is planned to add targeted drugs targeting EGFR receptor antibodies (cetuximab and panitumumab), Drug susceptibility gene mutation detection is required. International large-sample multi-center studies have shown that simultaneous detection of KRAS, BRAF, NRAS, and PIK3CA gene mutations, followed by tandem diagnosis in this order, can maximize and accurately predict patients’ sensitivity to antibody-targeted drugs (see literature Roock WD, Claes B, Be...

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: 1) Each specific primer or probe can only be identified for a certain type of gene mutation; 2) False positives due to single base mismatches may occur

2) For mismatched PNA / DNA, even if there is only one base mismatch, its melting temperature will drop by about 9-10°C

[0009] To sum up, the frequent V600E and V600K mutations of BRAF have clinical diagnostic significance, but the high-sensitivity PCR detection method reported so far lacks certain specificity, sensitivity and accuracy

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