Enzyme digestion enriched PCR method used in noninvasive detection of epidermal growth factor receptor T790M mutation

Abstract

The invention relates to an enzyme digestion enriched PCR method used in noninvasive detection of epidermal growth factor receptor T790M mutation, and belongs to the field of molecular biology. The invention aims at solving a clinical application bottleneck problem of NSCLC patient EGFR gene T790M mutation detection. According to the nucleotide differences between EGFR gene T790M mutant and wild-type sequences, proper PCR primers are designed, and are introduced into specific restriction endonuclease site during a PCR amplification process, and the EGFR gene T790M mutation enzyme digestion enriched PCR method is established. The method provided by the invention is mainly advantages in that: time required by the detection method is greatly shorter than that of common sequencing technologies; target sequence PCR amplification is carried out by using an enzyme digestion enriching method, and is used in detection, such that the interference caused by a large amount of wild-type sequence in the product to the detection result is avoided; the detection method steps are simple; a second-round PCR product is analyzed through mutant gene fragment specific restriction endonuclease digestion, such that mutant gene sequence can be accurately detected, and detection accuracy can be greatly improved; sampling is convenient, patient pain is low, and dynamic detection can be carried out.

Description

Technical field [0001] The invention belongs to the field of molecular biology, relates to medicine and biotechnology, in particular to an enzyme-cut enrichment PCR method for non-invasive detection of epidermal growth factor receptor T790M mutation. Background technique [0002] Non-small cell lung cancer (NSCLC) is a common malignant tumor, the incidence of which is increasing year by year worldwide, and has become one of the main causes of death from tumor-related diseases in my country and even in the world. one. NSCLC is not sensitive to conventional radiotherapy and chemotherapy. Although the second and third generation chemotherapy drugs represented by platinum and taxane have been proved to have improved curative effect compared with the previous ones, compared with most advanced patients, the overall survival benefit Not big. In recent years, with the continuous elucidation of abnormal signal transduction pathways in NSCLC cells, epidermal growth factor receptor (E...

AI Technical Summary

Problems solved by technology

At present, the methods used in the detection of EGFR gene T790M mutation have been reported to include direct sequencing of PCR amplification products, Taqman probe, PCR-SSCP, and amplification block mutation detection system. The main reason for the detection is that it is difficult to achieve a convenient sampling method, a simple, sensitive and specific method, and no need for special instruments and equipment.

Therefore, if the free mutant nucleic acid in peripheral blood can be effectively enriched, the clinical application bottleneck of EGFR gene T790M mutation detection in NSCLC patients can be solved.

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