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

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（dI-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（dI）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dI-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。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dI-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 method to test the mutations of gene hotspots L858R and 19 del(2235-2249, 2236-2250) based on deoxyinosine-modified allele-specific PCR (dI-AS-PCR). Methods A dI-ASPCR primer system was designed to detect the mutations of EGFR gene hotspots (L858R 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 dI-AS-PCR system. Results The detection limit of dI-AS-PCR was less than 0.1% under the background of wild type template. The dI-AS-PCR system was used to screen 50 lung cancer samples, in which 9 samples (18%) had L858R 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 dI-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.