蓝舌病实时荧光定量RT-PCR检测方法的建立

Establishment of Real-time Fluorescent Quantitative RT-PCR for Detection of Bluetongue

为建立一种快速、灵敏、特异的检测蓝舌病的实时荧光定量RT-PCR方法,本研究参考2022年世界动物卫生组织《陆生动物诊断试验和疫苗手册》,合成一对针对蓝舌病病毒(Bluetongue virus,BTV)NS3基因的特异性引物和探针,建立TaqMan荧光定量RT-PCR方法.结果显示,建立的方法与流行性出血热病毒、牛病毒性腹泻病毒、水泡性口炎病毒、小反刍兽疫病毒不存在交叉反应,具有良好的特异性,最低检测限为6.685 copies/μL,批内、批间重复性试验的变异系数分别在0.9%、2.46%以下,具有较好的重复性.结果表明,建立的TaqMan荧光定量RT-PCR检测方法特异性强、灵敏性高、重复性好,适用于BTV的快速检测.

In order to establish a rapid,sensitive and specific real-time fluorescence quantitative RT-PCR method for the detection of bluetongue virus(BTV),a pair of specific primers and probes targeting the Bluetongue virus NS3 gene were synthesized with reference to WOAH Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2022,and a TaqMan fluorescence quantitative RT-PCR method was established.The results showed that the established method had no cross reaction with epidemic hemorrhagic fever virus,bovine viral diarrhea virus,vesicular stomatitis virus,and peste des petits ruminants virus,with good specificity and a minimum detection limit of 6.685 copies/μL.The coefficient of variation of intra batch and inter batch repeatability tests were less than 0.9%and 2.46%,respectively,indicating good repeatability.The results showed that the established TaqMan fluorescence quantitative RT-PCR detection method had strong specificity,high sensitivity and good repeatability,and was suitable for the rapid detection of BTV.