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

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.