Primer for detecting BRAF gene V600E mutation of human colorectal cancer, detection method and kit thereof

Abstract

The invention provides a primer for detecting BRAF gene V600E mutation of human colorectal cancer, a detection method and a kit thereof. The detection primer provided by the invention can bind to a specific sequence of the BRAF gene, and amplifies a mutant sequence. The detection method adopts a detection fluorescent signal group to bind to an amplified fragment to emit a fluorescent signal, and uses a blocking agent specifically binding to the wild type sequence corresponding to the mutation site to inhibit wild type non-specific amplification. The method adopts a competitive allele specificPCR amplification technology to establish a real-time fluorescent PCR kit, which can accurately detect the BRAF gene V600E mutation. The method of the invention has simple operation, easy result reading, and high sensitivity, can detect a sample containing 0.001% BRAF gene V600E mutation, and realizes ultrasensitive detection of the BRAF gene V600E mutation.

Description

[0001] This application claims the Chinese patent submitted to the China Patent Office on September 08, 2017, the application number is 201710806477.2, and the invention title is "Primers, detection methods and kits for ultrasensitive detection of human colorectal cancer BRAF gene V600E mutation" The priority of the application, the entire content of which is incorporated in this application by reference. technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a primer, a detection method and a kit for detecting BRAF gene V600E mutation of human colorectal cancer. Background technique [0003] The BRAF gene was first discovered and cloned in human Ewing's sarcoma by Ikawa et al. in 1988. It is an active DNA sequence that can transfect NIH3T3 cells. Because of its high homology with CRAF and ARAF, it is Call it BRAF. The BRAF protein encoded by the BRAF oncogene is a mitogen-activated protein kinase / extracellular signal-regu...

AI Technical Summary

Problems solved by technology

The most common method is the sequencing method, which is relatively cheap, but it takes a long time to operate, and it has two obvious disadvantages: one is low sensitivity, and generally requires the abundance of mutant genes to reach more than 10% to be accurately detected

Second, due to the subsequent need to process the PCR product, it is prone to contamination and results in inaccurate results

However, due to the low content of oncogenes in early cancer samples, especially blood samples, the detection sensitivity of the above methods can only reach 0.2%, which is far from the detection of extremely small amounts of abnormal DNA molecules from a large amount of normal DNA background. Require

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