实时荧光PCR检测非小细胞肺癌EGFR基因突变方法的研究  

Research on the detection of EGFR gene mutation in non-small cell lung cancer by real-time fluorescence PCR

作　　者：杨晓娟 郭志武 马红丽 杨友锋 王浩 王奕江 李振勇 YANG Xiaojuan;GUO Zhiwu;MA Hongli;YANG Youfeng;WANG Hao;WANG Yijiang;LI Zhenyong(Suzhou Bacme Biotech Co.,Ltd.,Jiangsu,Suzhou 215400,China)

机构地区：[1]苏州华益美生物科技有限公司,江苏苏州215400

出　　处：《中国医药科学》2025年第5期146-149,167,共5页China Medicine And Pharmacy

基　　金：江苏省战略性新兴产业发展专项资金(苏发改高技发[2021]77号,苏财建[2021]2号)。

摘　　要：目的基于多重PCR、特异引物结合TaqMan荧光探针技术构建表皮生长因子受体(EGFR)基因突变检测方法,并对其性能指标及临床应用进行评价。方法使用EGFR基因序列针对21个突变位点设计引物探针,并对提取参数、反应体系、扩增程序进行优化,使用EGFR假病毒颗粒及临床样本,对构建试剂的性能指标及临床检测进行评价分析。结果本次构建的EGFR基因突变检测方法最低检测限为10 ng/反应体系可检出1.0%的突变基因,精密度?Ct的变异系数(CV)均≤5.0%。临床验证研究中研制试剂与对比试剂同时检测1087例肺癌样本,阴、阳性符合率及总符合率均为100%,Kappa系数≥0.75。结论本研究自主构建的检测方法可实现高灵敏度检测,并与已上市同类产品具有等效性,满足行业标准及市场需求。Objective To construct a detection method of epidermal growth factor receptor(EGFR)gene mutation based on multiplex PCR,specific primers and TaqMan fluorescence probe technology,and to evaluate its performance indices and clinical application.Methods Primer probes were designed for 21 mutation sites using EGFR gene sequence,and the extraction par ameters,reaction system and amplification procedure were optimized.Using EGFR pseudovirus particles and clinical samples,the performance indices and clinical detection of the constructed reagents were evaluated and analyzed.Results The lowest detection limit of EGFR gene mutation detection method constructed this time was 10 ng/reaction system 1.0%of mutant genes could be detected,and the coefficient of variation(CV)of precisionΔCt was all≤5.0%.In the clinical verification study,the developed reagent and the contrast reagent simultaneously detected 1087 cases of lung cancer samples,the coincidence rate of positive and negative and the total coincidence rate were 100%,and the Kappa coefficient was≥0.75.Conclusion The self-built detection method in this study can achieve high sensitivity detection,and it is equivalent to similar products on the market,meeting industry standards and market demand.

关 键 词：表皮生长因子受体 基因突变 荧光PCR 性能评价 临床验证 

分 类 号：R734.2[医药卫生—肿瘤]