EGFR等体细胞基因突变检测技术最新研究进展.ppt

JY Zhao zhaojy526@ ; Characterizing early and posttreatment tumor status in cancer patients drug-resistant strains detection from clinical samples of infectious disease patients ;IT is difficult! Why?;; To detect low-level early mutations (e.g., 10e–3 to 10e–6 mutant to wild-type DNA), both high selectivity and enrichment of minority alleles are required for successful detection and identification For a particular approach to be used as a routine diagnostic tool, it must achieve a balance of high selectivity and enrichment while maintaining accuracy, convenience, and low cost ;Mainstay methods for the somatic mutations and minority alles in clinical tests ;PCR-Sequencing (敏感性20%);PCR-MASS Sensitivity (5％）;ARMS PCR (1%); ; How to successful detect and identify these low-level mutations Some novel methods have been developed by us ;The novel methods for the low-level mutations detection by us;基因突变和B-raf基因点突变V600E的一步检测方法及试剂盒(CN102154480) 上皮生长因子受体外显子19缺失突变和外显子21点一步突变富集ARMS检测法(CN102220413) 一种基于热稳定性内切酶检测基因突变和SNP位点的方法级其试剂盒 一种基于Blocker引物和ARMS引物检测基因突变的方法和试剂盒 检测结核分枝杆菌(TB)的耐药基因突变型的方法和试剂盒（201110448201.4） 一种核酸检测方法及其专用试剂盒(CN101845511);检测范围： G719X 3 19 Del 19 20 T790M 1 20 Ins 3 20 S768I 1 L858R 1 21 L861Q 1 合计 29; 采用改进的ARMS技术与Taqman探针相结合的策略，研发一种拥有自主知识产权并且能够快速、敏感而且简便的检测EGFR基因突变的方法 通过优化酶切反应体系，将酶切体系与ARMS方法整合，建立更加敏感的突变富集检测方法 进一步通过一轮PCR扩增放大待测靶区含量，并通过温度差与延伸效率将扩增放大与ARMS荧光PCR方法整合，提高检测的灵敏度;改进型ARMS特异性引物、Taqman探针的设计;ARMS引物+Taqman探针反应体系的检测灵敏度;ARMS引物+Taqman探针反应体系的检测灵敏度;ARMS引物+Taqman探针反应体系的检测灵敏度;检测分辨率结果;检测分辨率结果;检测分辨率结果;检测分辨率结果;检测分辨率结果;检测分辨率结果;特异性试验以及Cut-offΔCt值确定;Cut-offΔCt值及特异性验证结果;Cut-offΔCt值及特异性验证结果; 采用改进的ARMS技术与Taqman探针相结合的策略，研发一种拥有自主知识产权并且能够快速、敏感而且简便的检测EGFR基因突变的方法 通过优化酶切反应体系，将酶切体系与ARMS方法整合，建立更加敏感的突变检测方法 进一步通过一轮PCR扩增放大待测靶区含量，并通过温度差与延伸效率将扩增放大与ARMS荧光PCR方法整合，提高检测的灵敏度;Fig. 1. The comparison of mutation-enriched PCR sequencing and