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

发布时间：2018-02-14 13:14

本文关键词： EGFR热点突变等位基因PCR 脱氧次黄嘌呤肺癌　出处：《中国现代医学杂志》2017年22期 　论文类型：期刊论文

【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。
[Abstract]:Objective to establish a modified DNA hypoxanthine allele specific polymerase chain reaction (D I-AS-PCR) method, based on the detection of epidermal growth factor receptor (EGFR) gene mutations L858R and 19del (2235~22492236~2250). The design method includes specific primers for amplification of EGFR gene of L858R and 19del hot point mutations, fluorescent probe and reference system and specific; detection of gene mutation allele PCR primer 3 '- end n-1 or n-2 position using hypoxanthine (D I) - modified. To establish standards and quality control samples, application of fluorescence PCR detection, and the results of the analysis. The D I-AS-PCR is able to detect less than 0.1% mutations in the wild-type background, detection of clinical samples of 50 cases lung cancer, 9 cases (18%) of EGFR gene L858R mutation, 14 (28%) cases of 19del mutation in.EGFR gene mutation rate was 46%, the detection results and DNA sequencing were identical. Conclusion based on D I-AS-PCR technology specific fluorescence PCR detection of EGFR gene hotspot mutation, high sensitivity, specificity, can be applied to clinical EGFR gene mutation detection, to guide personalized drug therapy and tyrosine kinase inhibitor (TKI) for drug resistance monitoring.

【作者单位】：河北省胸科医院(河北省肺癌防治研究中心);
【基金】：河北省卫生和计划生育委员会重点跟踪项目(No:GL2014061)
【分类号】：R735.35

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