蓝舌病病毒荧光定量RT-PCR检测技术

A NOVEL REAL-TIME RT-PCR FOR BLUETONGUE VIRUS DETECTION

蓝舌病是一种侵害反刍动物的虫媒传染病,被世界动物卫生组织(Office International des Epizooties,OIE)列为上报疾病,我国将其列为一类动物疫病。本研究利用计算机软件分析获取蓝舌病病毒(Bluetongue virus,BTV)核酸检测的保守靶序列,设计引物和TaqMan荧光探针,建立了BTV荧光定量RT-PCR技术,进行了特异性、敏感性、重复性等实验评价,并对动物样品进行检验。结果显示,该技术可有效检出不同血清型BTV,与流行性出血病病毒(Epizootic hemorrhagic disease virus,EHDV)无交叉反应;对RNA标准品的检测灵敏度为102copies;重复检测CV<5%,可对动物样品进行有效检测。因此,所建立的BTV TaqMan/荧光定量RT-PCR技术可为蓝舌病诊断提供一种快速、可靠的病原检测手段。

Bluetongue is a haematophagous insects-transmitted disease,which is included in the Office International des Epizooties（OIE） list of notifiable diseases.A pair of primers and a TaqMan fluorescent probe were designed based on available information of the conserved VP7 and NS1 genes of bluetongue virus（BTV）.A novel Real-time fluorescent RT-PCR was developed and assessed for its specificity,sensitivity and repeatability.The method was used to detect BTV in blood and serum samples.Results showed that BTV of various serotypes were positive and EHDV were negative in the assay.The sensitivity of the methods was 102 copies of RNA preparation.The coefficients of variation（CV） of threshold cycle（Ct） values between testing of RNA preparations were 5%.The Real-time fluorescent RT-PCR described in the present study is a rapid and reliable method for quantitatively detecting different BTV serotypes.