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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: To compare the consistency and diversity of epidermal growth factor receptor (EGFR) gene mutation by PCR-direct sequencing and PCR-pyrosequencing, and to find a suitable method for clinical detection of EGFR gene mutation in somatic cells. For non-small cell cancer patients to provide a theoretical basis for individual medication. Methods Double-blind PCR-direct sequencing and PCR-pyrosequencing were used to detect the mutations of EGFR exons 18, 19, 20 and 21 in DNA samples with known mutation of 13 cases (17 mutation points) 100 cases of non-small cell lung cancer tissue samples collected from the laboratory were selected and detected by direct sequencing and pyrosequencing method respectively to detect the expression of EGFR exon 18, 19,20 and 21 of the mutation, the results were compared using the chi-square test. Results The results of two kinds of detection methods for detecting EGFR18, 19, 20 and 21 were statistically different, but the pyrosequencing method had higher accuracy and sensitivity and easier operation. Conclusion Compared with direct sequencing, PCR-pyrosequencing is more suitable for the clinical detection of somatic mutations, which can better identify the tissue samples with lower mutation rate, Predict the efficacy of their drugs are of great significance, it is worth further clinical application.