焦磷酸测序法和直接测序法检测非小细胞肺癌表皮生长因子受体基因突变的比较研究  

Comparative study of EGFR mutations in non-small cell cancer patients by Sanger sequencing and pyrosequencing

作　　者：钟焱 周钢[2,3] 

机构地区：[1]长沙卫生职业学院,湖南长沙410100 [2]中南大学临床药理研究所,湖南长沙410078 [3]中南大学湘雅医院临床药理研究所,湖南长沙410078

出　　处：《现代预防医学》2017年第8期1494-1498,共5页Modern Preventive Medicine

摘　　要：目的比较PCR-直接测序法和PCR-焦磷酸测序法检测表皮生成因子受体(Epidermal Growth Factor Receptor,EGFR)基因突变的一致性和差异性,探寻适应于临床检测体细胞EGFR基因突变的方法,为非小细胞癌患者的个体化用药提供理论依据。方法采用双盲的方法应用PCR-直接测序法和PCR-焦磷酸测序法检测13例(17个突变点)已知突变比例DNA样本中EGFR外显子18、19、20和21的突变情况,分析2种检测方法对各种突变的检出率和检准率;另选择实验室收集的100例非小细胞肺癌组织标本,分别采用直接测序法和焦磷酸测序法检测EGFR外显子18、19、20和21的突变情况,结果比较采用卡方检验。结果 2种检测方法对于检测EGFR18、19、20和21的结果差异有统计学差异,但焦磷酸测序法的准确度和灵敏度更高,操作更加简便。结论与直接测序法相比,PCR-焦磷酸测序法更加适用于临床检测体细胞基因突变,能够更好地识别突变比例较低的组织样本,对临床指导非小细胞肺癌TKI类药物个体化用药与预测其药物疗效都具有重要意义,值得临床进一步推广应用。Objective In order to deplore an appropriate clinical method for detecting the EGFR somatic mutation, and provide theoretical basis for personal medicine of patients with non-small cell carcinoma, the PCR Sanger sequencing and PCR pyrosequencing were performed to compare their consistency and difference in testing EGFR mutation. Methods The Sanger sequencing and pyrosequencing were used to detect the EGFR18／lg／21~21 mutations in 13 cases （17 mutations） with known mutation rate by double blind method. The detection rate and accuracy rate were analyzed for this two methods. And 100 clinical cases of non-small cell lung cancer tissue samples were collected to detect the mutations of EGFR exon 18, 19, 20 and 21 respectively by this two methods. Result Two test methods for the detection of EGFR18, 19, 20 and 21 had statistical significance. The pyrosequencing was more sensitivity, accurate and simpler than the Sanger sequence. Conclusion Compared to Sanger sequence, the pyrosequencing was more suitable for clinical detection of somatic mutations. Pyrosequeneing can better detect mutation in a lower mutation rate for samples. And this method had clinical guidance for non-small cell lung cancer in TKI drug personal medication and important significance in predict drug efficacy. It could be widely performed in clinical tests.

关 键 词：焦磷酸测序 非小细胞肺癌 EGFR 个体化 

分 类 号：Q33[生物学—遗传学]