西藏环状病毒TaqMan反转录聚合酶链式反应方法的建立  

Establishment of TaqMan RT-PCR for detection of TIBOV virus

作　　者：冯盼盼 殷启凯 赵洁荣 付士红[1] 李樊 何英[1] 许松涛[1] 梁国栋[1] 聂凯[1] 王环宇[1] Feng Panpan;Yin Qikai;Zhao Jierong;Fu Shihong;Li Fan;He Ying;Xu Songtao;Liang Guodong;Nie Kai;Wang Huanyu(National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心病毒病预防控制所,北京102206

出　　处：《中华实验和临床病毒学杂志》2021年第2期209-212,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：国家科技重大专项(2018ZX10711001);国家重点研发(2017YFC1200505);传染病预防控制国家重点实验室重点项目(2015SKLID505)。

摘　　要：目的建立一种灵敏、特异的实时荧光定量TaqMan反转录聚合酶链式反应(RT-PCR),用于快速、准确地检测西藏环状病毒(Tibet orbivirus, TIBOV)。方法收集GenBank数据库中全部TIBOV基因序列,利用Clustal X 2.1软件进行比对,根据分析结果选择TIBOV VP4基因保守区域设计特异性引物、探针;以体外转录的RNA为标准品,建立检测TIBOV的TaqMan RT-PCR方法,绘制荧光定量标准曲线;对该方法的特异性、灵敏度、重复性进行评价。结果引物与探针具有良好的特异性,对多种虫媒病毒无交叉反应。检测反应灵敏度为1.0×10^(2)拷贝/反应,在1.0×10^(2~8)拷贝/反应模板范围内具有良好的扩增曲线,同一样品重复检测循环阈值(Ct)的变异系数均小于1.5%。对西藏自治区、云南省、湖南省和广东省采集的蚊虫样本进行筛查,结果均为阴性。TIBOV模拟样本检出率为100%。结论该方法灵敏度高、特异性强,可用于TIBOV的常规监测。Objective A highly sensitive and specific real-time quantitative TaqMan reverse transcription-polymerase chain reaction(RT-PCR)assay was developed for rapid and accurate detection of Tibet orbivirus(TIBOV).Methods The TIBOV genomic sequences from GenBank were analyzed by Clustal X 2.1 and the specific primers and probe were designed in the conserved segment of VP4 gene.RNA standard was obtained from in vitro transcription and a TaqMan RT-PCR assay for TIBOV was established.The sensitivity,specificity and repeatability of this method were evaluated.Results The assay showed a good amplification curve within the range of 1.0×102~8 copies/reaction template,the detection limit was 1.0×102 copies/reaction,the coefficients of variation of Ct values in repeat detections were all less than 1.5%.No cross-reaction was found in this assay.Variable mosquito samples were screened by this assay and the result showed TIBOV negative.The prepared TIBOV simulated positive samples were 100%detected.Conclusions The assay developed in this study is specific and sensitive for detection of TIBOV and can be used for laboratory detection and routine surveillance.

关 键 词：西藏环状病毒 实时荧光定量RT-PCR 分子检测 

分 类 号：R373[医药卫生—病原生物学]