一种 EGFR基因突变检测方法的建立及初步应用

摘要

目的：建立一种表皮生长因子受体（EGFR）基因实时荧光定量 PCR-Sanger测序突变检测方法，并初步探讨其临床应用价值。方法以EGFR基因热点突变区域19、21外显子为研究位点设计特异性扩增、测序引物，利用已知野生型、突变型样品，以T A克隆技术构建相应质粒作为标准品，建立EGFR基因实时荧光定量PCR-Sanger测序突变检测方法，并进行方法学和应用评估。结果成功构建了EGFR基因19、21外显子野生型、突变型质粒。建立了EGFR基因实时荧光定量PCR-Sanger测序突变检测方法，该方法灵敏度高（101 copy/μL ），重复性好（19、21外显子实时荧光定量 PCR部分批内、批间变异系数分别为1．42％、3．52％和0．97％、2．44％）。该法与传统Sanger测序法同时检测20份临床样品，结果完全相符。结论成功建立了可用于临床样品检测的EGFR基因实时荧光定量PCR-Sanger测序突变检测方法。%Objective To establish a method for detecting the EGFR gene mutations by the real-time fluorescence quantification PCR combined with Sanger sequencing and to preliminarily explore its clinical application value .Methods With EGFR gene hotspot mutations region exon 19 and 21 as the research locus ,the specific amplification and the sequencing primer were designed ,the known wild-type and mutant samples were utilized to construct the corresponding plasmid as the standard substance by the TA clone technique .Then the EGFR gene mutation detection method by the real-time fluorescence quantification PCR combined with Sanger sequencing was established and the methodological and the application evaluation were performed .Results The wild-type and mutant standard plasmids of the EGFR gene exon 19 and 21 were constructed successfully .The EGFR gene mutations detection method of the real-time fluorescence quantification PCR combined with Sanger sequencing was established ,which had high sensitivi-ty(101copies/μL)andgoodrepeatability(intra-assayCVandinter-assayCVofthereal-timefluorescencequantificationPCRofex-on 19 and 21 were 1 .42% /3 .52% and 0 .97% /2 .44% ,respectively ) .20 clinical samples were simultaneously detected by this method and the traditional Sanger sequencing ,the results were completely consistent .Conclusion The EGFR gene mutations detec-tion method of the real-time fluorescence quantification PCR combined with Sanger sequencing is successfully established ,which can be used in the clinical sample detection .