上皮生长因子受体外显子21基因突变L858R的实时定量PCR检测  

Detection of the exon 21 gene mutation L858R of epidermal growth factor receptor by real-time quantitative PCR

作　　者：董强刚[1,2] 李建璋 黎飒 刘刚 苏建中 贯剑 

机构地区：[1]上海交通大学肿瘤研究所 [2]上海奇诺肿瘤生物高新技术有限公司,上海200032 [3]上海奇诺肿瘤生物高新技术有限公司

出　　处：《肿瘤》2007年第2期150-154,共5页Tumor

基　　金：2005年上海市引进技术的吸收与创新计划(编号:05PTYY-12)

摘　　要：目的应用实时定量PCR技术建立上皮生长因子受体(epidermal growth factor receptor,EGFR)外显子21基因突变L858R的检测新方法。方法根据Taqman-MGB原理设计野生型外显子21和L858R特异性探针,经过优化制备成试剂盒检测171例肺癌组织中的L858R基因突变,并将结果与基因测序进行对比分析。结果171例肺癌组织经基因测序共检出L858R突变15例,突变率为8.8%。突变多见于女性、肺腺癌及腺鳞癌。定量PCR检测显示,15例突变型组织的R值平均为0.840±0.223,而156例野生型组织的R值平均为1.552±0.278,两组间差异具有统计学显著意义(P<0.001)。此外,以突变型为主的肺癌组织其R值(0.613±0.137)显著低于野生型占优势组织(0.992±0.103),两组间差异极为显著(P<0.001)。实验证明,定量PCR检测方法对L858R基因突变具有高度特异性,其最低检测限度为突变型DNA占野生型DNA含量的12.5%。结论本方法检测EGFR基因L858R突变具有敏感性高、特异性好、简便快速等优点,应用本方法不仅可以对野生型EGFR外显子21或突变型L858R进行诊断,而且还可以根据R值判断样本中突变型基因的比例。Objective：To establish a novel method for detecting the mutation of epidermal growth factor receptor （EGFR）, L858R in exon 21, by using real-time quantitative polymerase chain reaction （RQ-PCR） technique. Methods： The primers and probes specific for wild type exon 21 and mutant L858R were designed according to Taqman-MGB principle. The assay was developed after optimization and applied for detecting L858R mutation in 171 cases with lung cancer. The results were compared with the direct sequencing data. Results： The sequencing assay identified 15 cases with L858R mutation in a series of 171 lung cancer tissues, with the mutation rate of 8.8 %. The mutation was detected predominantly in females, adenocarcinomas, and adenosquamous carcinomas. Real-time PCR showed that the average R value for the 15 cases with L858R mutation was 0.840 ±0.223 and that for the 156 cases with wild type exon 21 was 1. 552 ±0. 278. The difference between the two groups was statistically significant （P 〈0.001 ）. Meanwhile, the R value for L858R predominant lung cancer tissues （ 0. 613 ± 0. 137 ） was much lower than that for wild type predominant ones （ 0. 992 ± 0. 103 ）. The difference between the two groups was statistically significant （P 〈 0.001 ）. The data presented here demonstrated that real-time PCR was highly specific for detecting L858R mutation. The detection limit was 12.5 % （ the ratio of mutated DNA to wild type DNA）. Conclusion：The real-time PCR assay has higher specificity and sensitivity in detecting L858R mutation and is easy to perform. It can be used not only in the diagnosis of wild type of EGFR exon 21 or L858R mutation, but also in the determination of the mutation ratio in the samples according to the R value.

关 键 词：肺肿瘤 癌 非小细胞肺 受体 表皮生长因子 DNA突变分析 试剂盒 诊断 分子诊断技术 

分 类 号：R734.2[医药卫生—肿瘤]