新型十万目六边形微腔芯片数字PCR对肺癌基因突变的绝对定量分析  

Novel 100000 hexagonal microchamber chip digital PCR for absolute quantification of gene mutations in lung cancer

作　　者：冯攀 郭铭静 李刚[2] 谢腾宝 张立群 刘飞 蒲晓允 FENG Pan;GUO Mingjing;LI Gang;XIE Tengbao;ZHANG Liqun;LIU Fei;PU Xiaoyun(Center of Clinical Laboratory,Second Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400037;Key Laboratory of Optoelectronic Technology and Systems of Ministry of Education,Defense Key Disciplines Lab of Novel Micro-Nano Devices and System Technology,Chongqing University,Chongqing,400044,China)

机构地区：[1]陆军军医大学(第三军医大学)第二附属医院检验医学中心,重庆400037 [2]重庆大学光电技术及系统教育部重点实验室,新型微纳器件与系统技术国防重点学科实验室,重庆400044

出　　处：《第三军医大学学报》2021年第8期675-682,共8页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(81772284);重庆市技术创新应用示范项目(CSTC2018jscx-mszdX0073)。

摘　　要：目的以表皮生长因子受体(epidermal growth factor receptor,EGFR)G719S突变为对象,建立用于肺癌基因突变绝对定量分析的新型十万目六边形微腔芯片(novel 100000 hexagonal microchamber chip,NHMC)数字PCR(digital PCR,dPCR)平台。方法首先构建并优化适用于EGFR G719S突变的PCR检测体系;然后用NHMC-dPCR平台检测8个浓度梯度的EGFR G719S标准品质粒和4种干扰物质,评估该方法的灵敏度、特异性和抗干扰能力;最后分别用NHMC-dPCR、液滴式数字PCR(droplet digital PCR,ddPCR)和DNA测序法检测6例EGFR G719X突变的肺癌组织样本,验证NHMC-dPCR在临床样本检测中的可靠性。结果NHMC-dPCR对EGFR G719S的最低检测浓度为3.01 copies/μL,检测值与期望值的线性方程为Y=0.725X-0.581,相关系数R^(2)=0.984。该平台检测4种干扰物未发现阳性拷贝,检测G719S突变及其与4种干扰物混合的结果分别为23194.4 copies和22095.7 copies。NHMC-dPCR、ddPCR和DNA测序3种方法检测6例临床样本的定性结果符合率为100%,其中2例由NHMC-dPCR定量的结果为1822.4 copies和451.7 copies,其对应由ddPCR定量的结果为1581.8 copies和218.3 copies。结论本研究建立的核酸绝对定量技术——NHMC-dPCR具有成本低、操作简单、速度快、抗干扰能力强等优点,可用于肺癌基因突变的绝对定量分析。ObjectiveTo construct a novel 100000 hexagonal microchamber chip(NHMC)digital PCR(dPCR)platform for absolute quantification analysis of gene mutations in lung cancer based on epidermal growth factor receptor(EGFR)G719S mutation as an object.MethodsFirst,a PCR detection system for EGFR G719S mutation was constructed and optimized.And then 8 concentration gradients of EGFR G719S standard plasmids and 4 interfering substances were detected by the NHMC-dPCR platform,and the sensitivity,specificity and anti-interference ability of the platform were evaluated.Finally,6 tissue samples of lung cancer with EGFR G719X mutation were detected by NHMC-dPCR,droplet digital PCR(ddPCR)and DNA sequencing,and the reliability of NHMC-dPCR in clinical sample testing was verified.ResultsThe minimum detection concentration of EGFR G719S detected by NHMC-dPCR is 3.01 copies/μL.The linear equation between the detected value and the expected value is Y=0.725X-0.581,and the correlation coefficient is R^(2)=0.984.Four interfering substances were tested by this platform and no positive holes were found.The results of the G719S mutant plasmids and the mixture of 4 interferences were 23194.4 copies and 22095.7 copies,respectively.The qualitative results of 6 clinical samples tested by NHMC-dPCR,ddPCR and DNA sequencing had a coincidence rate of 100%.Among them,the quantitative results of 2 cases by NHMC-dPCR were 1822.4 copies and 451.7 copies,and their corresponding quantitative results by ddPCR were 1581.8 copies and 218.3 copies,respectively.ConclusionThe nucleic acid absolute quantification technology established in this study,namely NHMC-dPCR,has the advantages of low cost,simple operation,fast speed,and strong anti-interference ability,and can be used for absolute quantification analysis of gene mutations in lung cancer.

关 键 词：微流控芯片 数字PCR 肺癌 基因突变 核酸检测 

分 类 号：R446.61[医药卫生—诊断学] R730.43[医药卫生—临床医学]