Method and kit for sensitively detecting human EGFR (epidermal growth factor receptor) gene mutation on basis of Sanger sequencing

Abstract

The invention belongs to the field of gene mutation detection and particularly relates to a method and a kit for sensitively detecting human EGFR (epidermal growth factor receptor) gene mutation on the basis of Sanger sequencing. In order to overcome the defects of a conventional human EGFR gene mutation detection method and kit, the method and the kit for flexibly detecting the human EGFR gene mutation on the basis of the Sanger sequencing are provided; heatproof DNA ligase and a specific connecting primer system aiming at a mutation site are introduced into an EGFR gene segment amplification system, so that amplification of wild type EGFR gene segments in a sample is greatly inhibited, and a wild type sample can be directly determined on the basis of amplified segment bands; or the proportion of mutation segments in a final PCR (polymerase chain reaction) product of a sample with low mutation content is enabled to be higher than 50%, accordingly, the accurate identification of the Sanger sequencing on a heterozygous gene segment system is guaranteed; the method and the kit can detect the EGFR gene mutation with the content as low as 1% in the sample on the basis of the Sanger sequencing, the accuracy rate is very high, the detection cost is low, and new unknown mutation can be possibly detected.

Description

technical field [0001] The invention belongs to the field of gene mutation detection, in particular to a method and a kit for sensitively detecting human EGFR gene mutation by using Sanger sequencing. Background technique [0002] Human epidermal growth factor receptor (Epidermal Growth Factor Receptor, EGFR) is a transmembrane glycoprotein with tyrosine kinase activity encoded by the proto-oncogene C-erbB-1 (HER-1), widely expressed in most normal epithelial cells middle. However, in many solid tumors (such as non-small cell lung cancer), there is often high or abnormal expression of EGFR, which is related to the inhibition of tumor cell proliferation, angiogenesis, invasion, metastasis and apoptosis, so EGFR has become a relevant tumor. (especially non-small cell lung cancer) targeted therapy is an important target. EGFR-targeted drugs currently used in clinical practice include: EGFR tyrosine kinase inhibitors (TKIs, such as gefitinib (Iressa) and erlotinib (Tarceva), a...

AI Technical Summary

Problems solved by technology

The EGFR detection kit developed based on the Scorpions-ARMS method, such as the EGFR detection kit from Qiagen, Germany, can detect 29 common mutations of the EGFR gene at the same time, with high sensitivity (can detect mutations as low as 1%), simple operation, rapid detection, etc. Advantages; however, the detection cost of such imported kits is too high (3000-5000 yuan per specimen) which limits its clinical promotion and application

The Sanger sequencing method is the gold standard for gene mutation / diversity detection. It has the advantages of mature technology and platform, accurate and comprehensive results, etc. Compared with the Scorpions-ARMS method, the detection cost is low and unknown mutations can be detected; however, the Sanger sequencing method The main disadvantage is that the detection sensitivity is about 10-20% (that is, the mutation gene content must be greater than 10% or even 20% to be reliably detected), which restricts its clinical application

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