Method for sensitively detecting human EGFR gene mutation based on enzyme digestion and reagent kit

Abstract

In order to overcome defects of existing methods and reagent kits, the invention provides a method for sensitively detecting human EGFR gene mutation based on enzyme digestion and a reagent kit thereof, and belongs to the field of gene mutation detection. The method comprises the steps that heat-resisting DNA ligase and a specific connection primer set for a mutation site are introduced into an EGFR gene segment amplification system, a wild type sample can be directly determined on the basis of an amplified segment strip, or the proportion of a final PCR product of a low-mutation-content sample is made to be higher than 50%; then, a DNA segment hybridization sample containing a mismatched structure at the mutation site is prepared through denaturation and annealing, and therefore a heterozygosis gene segment sample can be effectively detected by means of a mismatched digestion enzyme. The method and the reagent kit can detect EGFR gene mutation with the content as small as 1% in the sample through enzyme digestion, and are high in accuracy rate, short in detection time, low in cost and suitable for clinical detection.

Description

technical field [0001] The invention belongs to the field of gene mutation detection, in particular to a method and a kit for sensitive detection of human EGFR gene mutation based on enzyme digestion. 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

Sanger sequencing method is the gold standard for gene mutation / diversity detection, which has the advantages of mature technology and platform, accurate and comprehensive results; however, the main disadvantage of Sanger sequencing method is that the detection sensitivity is only about 10-20% (that is, the content of mutant genes needs to be More than 10% or even 20% can be reliably detected), and the detection process is cumbersome and the detection time is long (about 2 days), which restricts its clinical application

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, and rapid detection Advantages; however, the detection cost of such imported kits is too high (3000-5000 yuan per specimen) which limits its clinical promotion and application

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