基于级联核酸侵入反应的real-timePCR法检测咽拭子样本中-遗传.PDF

Hereditas (Beijing) 2018 年 8 月, 40(8): 668 ―675 技术与方法 基于级联核酸侵入反应的real-time PCR 法检测 咽拭子样本中UGT1A1*6 基因多态性 张婕妤，初亚男，邹秉杰，张晏洁，封利颖 南京总医院药理科，南京 210002 摘要 : 伊立替康(irinotecan)不良反应与 UGT1A1*6 单核苷酸多态性相关，目前应用的单核苷酸多态性检测方法 具有耗时长、开盖易污染、操作繁琐等缺点，因此需建立一种操作简便且不易污染的适合临床应用的新方法。 本研究利用3 种已知基因型的DNA 样本建立基于级联核酸侵入反应的real-time PCR 法检测UGT1A1*6 多态性 的最佳反应体系，并对其检测口腔咽拭子样本的灵敏度和准确性进行了考察。结果显示，本文成功建立了基于 级联核酸侵入反应的UGT1A1*6 基因多态性检测方法，可用于咽拭子样本的分型检测，其灵敏度达到6 ng DNA ， 分型准确性达到 100%。因其取样方便对人体无创，单管闭管检测不易产生交叉污染，具有用于临床检测伊立 替康的个体化用药相关 UGT1A1*6 基因多态性的潜力。 关键词 : 核酸侵入反应；UGT1A1*6；基因多态性；伊立替康 Detection of UGT1A1*6 single nucleotide polymorphism in oral swap samples using the cascade invader assay-based real-time PCR Jieyu Zhang, Yanan Chu, Bingjie Zou, Yanjie Zhang, Liying Feng Department of Pharmacology, Nanjing General Hospital, Nanjing 210002, China Abstract : The adverse reaction to irinotecan is related to the single nucleotide polymorphism (SNP) of UGT1A1*6 genotype. The current SNP detection methods have various disadvantages, including time-consuming procedures, high- risk cross-contamination, and cumbersome operation. Hence, it is necessary to establish a new method suitable for clinical application, which is easy and simple to detect SNP with minimal risk for cross-contamination. In this study, a cascade invader assay-based real-time PCR, for UGT1A1*6 genotyping has been established by optimizing reaction conditions with DNA samples of three genotypes. The sensitivity and accuracy of the method were evaluated with DNAs derived from oral swab samples. The results showed that the method could detect the UGT1A1*6 genotypes from the oral swab samples with 收稿日期: 20 18−03−26; 修回日期: 20 18−05−17 基金项目: 南京总医院院管课题(编号：2015043 ，2015042)和国家自然科学基金项目(编号资助[Supported by Nanjing General Hospital Fund (Nos. 2015043, 2015042) and the National Natural Science