Method for fast detecting BRAF gene mutation with allele RNA (ribonucleic acid) isothermal amplification method

Abstract

A method for fast detecting BRAF gene mutation with an allele RNA (ribonucleic acid) isothermal amplification method comprises the following steps: cell lysis treatment is performed; RNA reverse transcription treatment is performed; cDNA (complementary deoxyribonucleic acid) is generated and serves as an RNA amplification template; and RNA amplicon is quantified through regular monitoring for a fluorescence signal. According to the method, an isothermal reaction method is adopted for fixed-point specific detection of a V600E mutation site of the BRAF, the detection is performed at the isothermal temperature of 41 DEG C, test conditions are mild, chemical compounds for reaction are common, the operation is simple, the specificity is high, and only an instrument capable of detecting a fluorescent probe is required. A higher amplification coefficient is provided, a billion RNAs which are complementary with the target RNA are produced with two specific primers aiming at targeted RNA as well as three enzymes in 1.5 hours, and BRAF mutation information with as low as 0.1% of content is read through detection of the fluorescent probe and used for targeted drug use instruction for a tumor patient.

Description

technical field [0001] The invention relates to a method for detecting gene mutation, in particular to a method for rapidly detecting BRAF gene mutation by using an allele RNA isothermal amplification method. Background technique [0002] 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-regulated kinase (MAPK / ERK) pathway protein. It is the most efficient phosphorylation agent in the MAPK kinase family and plays an important role in apoptosis and proliferation. Function, located at the entrance of the MAPK pathway, it connects receptors and RAS proteins on the cell surface to transcription factors in the nucleus through MEK and ERK, and regulates cell growth and development. Recent studies have ...

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Problems solved by technology

[0034] The object of the present invention is to provide a method for rapidly detecting BRAF gene mutations by using the allelic RNA isothermal amplification method. The method requires simple equipment, is easy to implement, and can rapidly amplify a large number of mutated single-stranded RNAs with high specificity. High, the sensitivity can reach 0.1%, which solves the problems of difficulty and slow speed in the detection of BRAF gene mutation

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