特异引物双扩增实时PCR法和Sanger DNA测序法检测肺癌组织中表皮生长因子受体基因突变  

Screening for EGFR mutations in lung cancer by a novel real-time PCR with double-loop probe andSanger DNA sequencing

作　　者：张海萍[1] 阮力[1] 郑立谟[1] 白冬雨[1] 张海芳[1] 廖永强[1] 丁毅[1] 

机构地区：[1]厦门大学附属第一医院病理科,361003

出　　处：《中华肿瘤杂志》2013年第1期28-32,共5页Chinese Journal of Oncology

摘　　要：目的探讨特异引物双环探针扩增实时PCR技术表皮生长因子受体（EGFR）检测方法（ADx-EGFR实时PCR法）和Sanger DNA测序法在肺癌EGFR基因体细胞突变检测中的临床价值。方法收集肺癌组织石蜡切片208例，分别采用ADx-EGFR实时PCR法和SangerDNA测序法检测肺癌组织中EGFR基因外显子18、19、20、21的突变类型，计算其突变率，分析两种检测方法检测EGFR基因突变的一致性。结果208例肺癌组织中，ADx-EGFR实时PCR法成功检测208例，检出EGFR基因突变40例，突变检出率为19．2％；SangerDNA测序法成功检测196例，检出突变22例，突变检出率为11．2％。肺癌组织中主要以外显子19缺失和外显子21上的L858R的点突变为主，分别占4．8％（10／208）和11．6％（23／208），其余的突变类型少见。结论对于甲醛固定石蜡包埋组织而言，ADx-EGFR实时PCR法检测EGFR基因成功率和突变检出率均高于SangerDNA测序法，可成为临床上检测肿瘤EGFR基因突变的方法。Objective To map the frequency and types of EGFR gene mutations present in lung cancer tissues. To evaluate the clinical applicability of a novel real-time double-loop probe PCR of which the ADx-EGFR kit is based, and to compare its performance with traditional Sanger DNA sequencing in the detection of somatic mutations of tumor genes. Methods A total of 208 formalin-fixed paraffin-embedded （FFPE） tumor samples were tested. Genomic DNA of the tissue samples was extracted and purified, and subjected to both traditional PCR amplification, Sanger sequencing of EGFR gene in exon 18, 19, 20, 21, and ADx＇s EGFR mutation detection kit. The mutation rates for EGFR gene in exon 18, 19, 20, 21, as well as the frequency of each mutation detected by the two methods, were analyzed. Results The traditional Sanger DNA sequencing technique was successfully performed in 196 out of 208 （94.2%） lung cancer samples, and 22 samples （ 11.2% ） showed EGFR gene mutations. ADx-EGFR kit was successfully used in the lung cancers of all of the 208 cases （ 100.0% ） , and 40 samples （ 19.2% ） showed mutations. In the lung cancer samples analyzed, mutations were mainly detected in the exon 19 and exon 21 L858R point mutation, i.e. 4.8% （10/208） and 11.6% （23/208） of total mutations, respectively, and the remaining mutations were rare. Conclusions The success rate of ADx-EGFR real-time PCR for formalin-fixed and paraffin-embedded tissues samples is significantly higher than that of Sanger sequencing （P 〈 0.01 ）. There are significant differences between the two methods. ADx-EGFR real-time PCR shows a much higher successful detection rate and mutation rate of lung cancer tissues compared with that of Sanger sequencing. As a result, the real-time PCR with ADx-EGFR kit is proved to have a good clinical applicability and a strong advantage over the traditional Sanger DNA sequencing. It is an effective and reliable tool for clinical screening of somatic gene mutations in tumors.

关 键 词：肺肿瘤 基因 ERBB-1 聚合酶链反应 寡核苷酸序列分析 突变 

分 类 号：R734.2[医药卫生—肿瘤]