Primer and probe system as well as method and kit for detecting twenty-nine mutations of human EGFR gene

Abstract

The invention relates to a primer and probe system for detecting twenty-nine mutations of a human EGFR gene, which comprises nucleotide sequences shown as SEQ ID No: 1-26. The invention further relates to a method for detecting twenty-nine mutations of the human EGFR gene. The method comprises the following steps: synthesizing a primer and a probe, introducing deoxyinosine to a second or third position from the 3'-terminal of the primer, carrying out fluorescence PCR reaction to collect fluorescence signals FAM and HEX, and carrying out result determination; the invention further relates to a kit comprising at least one of the primer and / or the probe. The primer and probe system has very high sensitivity, and can meet the detection for the sample with low mutation abundance, namely, under the background of the wild genome DNA, the primer and probe system can complete the detection for the relatively low mutant gene content, and accurately distinguish the types of the sample with high sensibility and specificity, so as to exert the greatest technical advantage.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a primer and probe system, method and kit for detecting 29 mutations of human EGFR gene. Background technique [0002] A large number of studies have shown that about 40% of non-small cell lung cancer (NSCLC) patients carry somatic mutations in the epidermal growth factor receptor (EGFR) gene, and these mutations have a clear relationship with the efficacy of tyrosine kinase inhibitors (TKIs). Correlation, such as drugs such as Iressa (Iressa) and Tarceva (Tarceva). Among them, NSCLC patients without T790M mutation but carrying mutations in other sites of EGFR gene have significant curative effect when receiving Iressa and Tarceva. The study found that the T790M site on EGFR exon 20 is a drug resistance site , will inhibit the efficacy of drugs such as Iressa and Tarceva. [0003] The EGFR gene is located on chromosome 7 and consists of 28 exons. Its tyrosine...

AI Technical Summary

Problems solved by technology

[0004] Current gene mutation detection methods such as mutation amplification retardation system (amplification refractory mutation system, ARMS), TaqMan technology, etc., although widely used in clinical detection work, its polymerase chain reaction (Polymerase Chain Reaction, PCR) primers have the following disadvantages: (1 ) The specificity is not good, and the primers designed for the mutation site of the diagnostic gene are prone to non-specific amplification, which affects the detection efficiency and accuracy; (2) The selectivity is not good, and the ability to detect low copies is poor under the background of high concentration wild type; (3) Poor sensitivity and resolution, limited by primer design, resulting in poor detection sensitivity and low resolution

At present, the gold standard of SNP mutation detection technology is "DNA sequencing", but the sensitivity of DNA sequencing is relatively low. The operability is better than sequencing, and it has been successfully promoted to the clinic for the detection of SNP mutations. However, the development of this technology is limited by the nucleic acid sequence adjacent to the SNP, which greatly limits the wide application of ARMS, and it is difficult to meet the increasing clinical needs. SNP and gene mutation detection needs