人类EGFR基因突变核酸标准物质的研制

Development of nucleic acid RM of EGFR gene mutations of human

目的:研制人类表皮生长因子受体(EGFR)基因突变核酸标准物质,为国内体外诊断产品生产厂家、行业检验机构和临床用户提供检验标准和参考依据。方法:利用数字聚合酶链反应(PCR)方法,对多种含有EGFR基因突变核酸混合后进行定量,制备EGFR基因4种常见的突变类型(包括p.G719S、p.E746_A750delELREA、p.T790M和p.L858R)预期值为20%、10%、5.0%、2.5%、1.0%和0.5%的6种不同突变频率的标准物质。结果:研制出人类EGFR基因突变核酸标准物质一套,共含有RM-1、RM-2、RM-3、RM-4、RM-5和RM-6共6支,每支含有p.G719S、p.E746_A750delELREA(p.Ex19del)、p.T790M和p.L858R的4个特性值,同一个标准物的特性值之间具有相似的突变频率水平;两种数字PCR平台的定值表现出良好的一致性,且具有较好的均匀性和稳定性。结论:研制的人类EGFR基因突变核酸标准物质已基本具备了溯源的属性特征,经不同实验室的努力合作和市场产品的测试反馈,将对我国的分子诊断市场提供重要的指导和借鉴意义。

Objective:To develop nucleic acid reference material(RM)of human epidermal growth factor receptor(EGFR)gene mutation so as to provide inspection standard and reference for domestic manufacturers of in-vitro diagnosis(IVD)products,inspection institute of industry and clinical users.Methods:Using digital PCR method to implement quantification for various mixed nucleic acid with EGFR gene mutants,and to prepare 4 general mutant types(contained p.G719S,p.E746_A750delELREA,p.T790M and p.L858R)of EGFR gene.The predicted values of 6 RMs with different mutation frequencies were 20%,10%,5.0%,2.5%,1.0%and 0.5%,respectively.Results:We developed a set of nucleic acid RM of human EGFR gene mutation,and it contained 6RMs included RM-1,RM-2,RM-3,RM-4,RM-5 and RM-6.And each one of them contained 4 characteristic values included p.G719S,p.E746_A750delELREA(p.Ex19del),p.T790M and p.L858R.There was similar mutation frequency level among characteristic value of same RM.The data of two kinds of digital PCR platforms appeared favorable consistency,better homogeneity and stability.Conclusion:The developed nucleic acid RM of EGFR gene mutation of human has basically possessed the attributive character of traceability.In the future,through the hard cooperation of different laboratories and feedback of market products,it will provide important guidance and reference significance for the molecular diagnostic market in China.