TaqMan—MGB探针实时荧光聚合酶链反应快速检测非小细胞肺癌表皮生长因子受体基因突变  

Detection of epidermal growth factor receptor mutations in non-small cell lung cancer by real-time PCR using TaqMan-MGB probes

作　　者：周彩存[1] 周崧雯[1] 潘虹[1] 粟波[1] 高志强[1] 

机构地区：[1]同济大学附属上海市肺科医院肿瘤科,上海200433

出　　处：《中华肿瘤杂志》2007年第2期119-123,共5页Chinese Journal of Oncology

基　　金：上海市科委项目资助（04DZ19109）

摘　　要：目的探讨非小细胞肺癌（NSCLC）瘤组织表皮生长因子受体（EGFR）基因突变及TaqMan MGB探针实时荧光PCR快速检测EGFR突变的诊断价值。方法应用聚合酶链（PCR）反应，对80例手术切除NSCLC瘤组织EGFR基因的第18、19和21外显子片段进行扩增和测序，Chromas软件分析基因突变。设计EGFR突变位点的TaqMan MGB探针，采用实时PCR检测瘤组织EGFR突变，并与测序结果比较。实时PCR的敏感性与特异性评价，用不同混合数量的PC-9细胞（19外显子缺失）为阳性参照。结果21例NSCLC瘤组织存在EGFR基因突变，总体突变率为26．3％。其中13例为EGFR第19外显子阅读框内多核苷酸的缺失，8例为第21外显子2573位核苷酸点突变。诊断的特异性与敏感性均为100％。当PC-9突变型细胞仅占10％时或PC-9细胞数低达50只时，PCR仍然检测到EGFR基因突变的存在。女性、不吸烟和肺腺癌患者EGFR基因突变率显著高于男性、吸烟和非腺癌患者（P＜0．05）。EGFR基因突变与患者年龄、TNM分期等因素无关。结论NSCLC存在EGFR基因的突变或缺失，其中以女性、腺癌和不吸烟患者突变率较高。TaqMan MGB探针联合实时PCR可有效地检测出EGFR基因突变，操作简便，易于临床推广。Objective To investigate mutations of EGFR TK gene in non-small cell lung cancer （NSCLC） and the diagnostic value of the mutations assayed by real-time PCR using TaqMan-MGB probes. Methods Tyresine kinase genes of EGFR （exons 18, 19 and 21） were amplified by PCR technology, and sequenced and analyzed by Chromas software in 80 NSCLC patients. Based on the results of sequencing, TaqMan-MGB probes were designed and used to detect the mutations of EGFR by real-time PCR. The results of detected mutations were compared between real-time PCR and direct sequencing. The sensitivity and specificity of real-time PCR using TaqMan-MGB probes were analyzed by adding different number of PC-9 cells （exon 19 deleted EGFR） into A549 cells （Wild-type EGFR）. Results Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in 21 of 80 NSCLC patients with an incidence rate of 26.3%. In-frame deletions of exon 19 occurred in 13 patients and point mutation occurred in eodon 858 （exon 21 ） in 8 patients. Real-time PCR with the TaqMan MGB probes could detect all the mutations of EGFR found by sequencing. The sensitivity and specificity of the detection of EGFR mutations were both 100%. Real-time PCR with TagMan MGB probes could detect EGFR mutation in as rare as 50 EGFR mutant cells and in a proportion of 10% of mutant cells in a cell population. The mutations were significantly higher in the adenoeareinoma than in non-adenoeareinoma （ 16/38 vs. 5/42, X^2 = 9. 702, P 〈 0.01 ）, in the female patients than in the male patients （14/29 vs. 7/51, X^2 = 11.4, P 〈0.01） and in non-smokers than in smokers （16/40 vs. 5/40, X^2 = 7.812, P 〈 0.01 ）. The mutations were not related to patients＇age, TNM staging, etc. Conclusion Somatic mutations of EGFR gene develop in NSCLC and are more common in female, non-smoker and adenoeareinoma patients. Real-time PCR using TaqMan-MGB, which can be used to detect the EGFR gene mutations,is easy to operate and deserves widespread application.

关 键 词：非小细胞肺癌 表皮生长因子受体基因 TAQMAN MGB探针 聚合酶链反应 外显子 

分 类 号：R686[医药卫生—骨科学]