Sanger测序技术在检测肺癌吉西他滨化疗耐药敏感基因CDA中的应用

Application of Sanger sequencing technology in detection of gemcitabine resistance sensitive gene CDA

目的分析吉西他滨耐药敏感基因胞苷脱氨酶(CDA)多态性位点突变,以实时荧光定量聚合酶链反应(qPCR)法为参照,探讨Sanger测序技术应用于临床样本检测的适用性。方法随机选取255例ⅢB和Ⅳ期晚期非小细胞肺癌(NSCLC)外周血样本,提取DNA,分别采用qPCR法和Sanger测序法检测CDA基因G208A和A79C突变情况,评价Sanger测序法检测的敏感度和特异度。结果 255例ⅢB和Ⅳ期晚期NSCLC外周血样本Sanger测序法和qPCR法检测CDA基因突变的成功率分别为98.04%(250/255)和97.25%(248/255)。以qPCR法为参照,Sanger测序法检测的敏感度和特异度分别为92.50%和99.52%,两种方法检测出的突变分型完全吻合。结论采用Sanger测序法可有效检测耐药基因CDA突变,实现了突变分型,可准确、高通量、高效检测CDA突变位点,为临床NSCLC对吉西他滨用药敏感提供一种有效检测手段。

Objective To analyze the polymorphic site mutation of gemcitabine resistant sensitive gene cytidine deaminase（CDA）,and to investigate the applicability of the Sanger detection technology in clinical sample detection with the real-time fluorescence quantitative PCR as the reference.Methods The peripheral blood samples from 255 cases of stageⅢB to Ⅳ non small-cell lung cancer（NSCLC）were randomly selected for extracting DNA.The G208 Aand A79C mutation situation of CDA gene was respectively detected by qPCR and Sanger sequencing technology.Results The success rate for detecting the CDA gene mutation in peripheral blood sample in 255 cases of stage ⅢB and Ⅳ NSCLC were 98.04%（250/255）by the Sanger sequencing method and97.25%（248/255）by qPCR.With the qPCR as the reference,the sensitivity and specificity of Sanger sequencing method was92.50% and 99.52% respectively.All mutation types detected by two methods were fully consistent.Conclusion Adopting the Sanger sequencing method can effectively detect drug resistance gene CDA mutation,realizes the mutation typing,can accurately,high throughput and efficiently detect CDA mutation site,which provides an effective detection method for gemcitabine sensitive medication of NSCLC.