建立基于脱氧次黄嘌呤核苷修饰等位基因PCR方法检测EGFR基因热点突变  

Establishment of a method for detection of EGFR gene hotspot mutations based on deoxyinosine-modified allele-specific PCR

作　　者：杨永辉[1] 李辉[1] 朱桂云[1] 李秀武[1] 陈宁[1] 任雪飞[1] 

机构地区：[1]河北省胸科医院(河北省肺癌防治研究中心),河北石家庄050041

出　　处：《中国现代医学杂志》2017年第22期13-19,共7页China Journal of Modern Medicine

基　　金：河北省卫生和计划生育委员会重点跟踪项目(No:GL2014061)

摘　　要：目的建立基于脱氧次黄嘌呤修饰等位基因特异荧光聚合酶链反应(d I-AS-PCR)方法,检测表皮生长因子受体(EGFR)基因热点突变L858R和19del(2235~2249,2236~2250)。方法设计包含扩增EGFR基因L858R和19del热点突变在内的特异性引物、荧光探针和内参体系;且特异检测基因突变的等位基因PCR引物3’-末端n-1或n-2位置采用脱氧次黄嘌呤(d I)修饰。建立标准品和质控样品,应用荧光PCR检测,并分析结果。结果 d I-AS-PCR能够在野生型背景下检测低于0.1%的突变,对50例肺癌临床样本进行检测,有9例(18%)EGFR基因发生L858R突变,14(28%)例19del基因突变。EGFR基因热点突变率为46%,其检测结果与DNA测序完全一致。结论基于d I-AS-PCR技术的特异荧光PCR检测EGFR基因热点突变,敏感性高,特异性强,可以应用于临床EGFR基因突变检测,指导个性化用药及酪氨酸激酶抑制剂(TKI)治疗耐药监测。Objective To establish a method to test the mutations of EGFR gene hotspots L858 R and 19 del(2235-2249, 2236-2250) based on deoxyinosine-modified allele-specific PCR(d I-AS-PCR). Methods A d I-ASPCR primer system was designed to detect the mutations of EGFR gene hotspots(L858 R and 19 del), which included specific primers modified by deoxyinosine at the n-1 or n-2 position of the primer 3'-terminal, fluorescent probe and internal control primers. Standard substance and quality control samples were built and analyzed by the d I-AS-PCR system. Results The detection limit of d I-AS-PCR was less than 0.1% under the background of wild type template. The d I-AS-PCR system was used to screen 50 lung cancer samples, in which 9 samples(18%) had L858 R mutation of EGFR gene and 14 cases(28%) had 19 del mutation. The hotspot mutation rate of EGFR gene was 46%, which was consistent with the results of DNA sequencing(P > 0.05). Conclusions The d I-AS-PCR is a sensitive and specific assay, which could be widely used to detect EGFR mutations, guide patient-specific treatment and monitor the drug resistance of TKI-therapy in clinic.

关 键 词：EGFR热点突变 等位基因PCR 脱氧次黄嘌呤 肺癌 

分 类 号：R734[医药卫生—肿瘤]