The Establishment And Evaluation Of The EGFR Gene Mutation Rate Detection Method Based On PASS Technology

Objective:To establish a sensitive and specific PASS technology platform for the detection of EGFR gene mutation ratio,and understood the reliability and clinical significance of the PASS method,then provided a theoretical basis for the selection of appropriate treatment options for patients with advanced NSCLC.Methods:1.With molecular cloning techniques,the recombinant plasmids containing wild-type exon 19 and exon 21 of the human EGFR gene were constructed by using pQE30 as the framework plasmid,and then the other recombinant plasmids of exon19 carrying the E746-A750 deletion mutation and exon 21 carrying the L858 R mutation were also constructed,which all identified by restriction enzymes and sequencing,respectively.2.According to the references and the established method by our research group in the earlier period,the specific PASS amplification primers and sequencing primers of the corrsponding exons of EGFR were designed,then with the PASS detection procedure,using the above-mentioned plasmid containing the wild-type and mutant EGFR gene fragment as a detection template,to detect,explore and establish a PASS detection technology platform and then find out the best conditions for the detection of the EGFR gene mutation ratio in this method.We further verified its detection sensitivity,accuracy and minimum detection limit.Results:1.Our results showed the wild-type and mutant-type recombinant control plasmids containing exons 19 and 21 of the human EGFR gene were successfully constructed,respectively,whose sequences were identical to those of the NCBI database.2.Our study successfully established a reliable PASS technology platform for detecting the proportion of EGFR gene mutations.Its detection sensitivity,accuracy,and detection limit were all meeted the detection requirements.Conclusion:In this study,we have successfully established a highly sensitive and specific PASS technique for detecting the proportion of human EGFR gene mutations,which provides a good technical foundation for testing late-stage clinical specimens and choosing clinically accurate EGFR-TKI targeted therapy of patients with advanced NSCLC in the future.