Development And Application Of EGFR Gene Mutation Detection Kit

Epidermal growth factor receptor EGFR protein is a transmembrane protein ubiquitous in cell membrane.It is related to cell signal transduction.Studies have shown that high expression or abnormal expression of EGFR is present in many solid tumors.EGFR is an important target in the treatment of non-small cell lung cancer.EGFR gene mutation will lead to changes in EGFR protein structure,which causes small molecule targeting drugs such as Tarceva and EGFR protein receptor binding changes,increased binding or weakened will directly affect the efficacy of Tarceva.Therefore,patients with non-small cell lung cancer in the use of targeted drugs before the need for EGFR gene mutation diagnosis.This study aims to develop a real-time quantitative assay kit for the detection of mutations in EGFR,to help patients determine EGFR mutations and the doctor choose the right medicines.In this study,wo used ARMS primer technology and Taqman probe detection technology to achieve EGFR gene mutation detection.The twenty nine mutant plasmids associated with non-small cell lung cancer were constructed by overlapping extension method,which was mainly due to the fact that most of the mutants were very difficult.We used each of the mutant plasmids to mix the extracted normal human cell genomic DNA in a proportion,using this mixture to express a specific EGFR mutant to simulate the pathologic specimens of patients with non-small cell lung cancer.We designed the EGFR genotype corresponding to the specificity of the Taqman probe by designing the EGFR genotype corresponding to the different characteristic ARMS primer corresponding to the EGFR genotype.In order to facilitate the detection,we divided twenty nine mutations into E18,E19,E20,E21,T790,S768I six tubes reaction.We successfully constructed the EGFR mutant detection kit,which can detect twenty nine mutations in EGFR genes,accounting for more than 99%of the total EGFR mutations.The detection sensitivity of the EGFR mutant kit reaches 1%(under 10~4copies of the background genome)and It can detect samples with a minimum of 10 copies.We tested our kits with 109 clinical specimens.It can well distinguish EGFR mutations from positive and negative,and the specificity was good.Compared with the sequencing method(detection sensitivity 10%)the detection sensitivity reach 1%,and its operation is simple,time-consuming,just two hours to complete the detection and analysis of results,It is more consistent with clinical testing requirements.