马疱疹病毒8型荧光定量PCR检测方法的建立及初步应用  

Establishment and Application of Real-time PCR for Equine Herpesvirus Type 8

作　　者：李亮亮 于跃[1] 王天娇 丁相丹 胡乐玉 王长法 刘文强[1] 刘桂芹[1] 任慧英[2] 王彤彤 LI Liang-liang;YU Yue;WANG Tian-jiao;DING Xiang-dan;HU Le-yu;WANG Chang-fa;LIU Wen-qiang;LIU Gui-qin;REN Hui-ying;WANG Tong-tong(Liaocheng Research Institute of Donkey High-efficiency Breeding and Ecological Feeding,Liaocheng University,Liaocheng,Shandong,252000,China;College of Veterinary Medicine,Qingdao Agricultural University,Qingdao,Shandong,266109,China)

机构地区：[1]聊城大学毛驴高效繁育与生态饲养研究院,山东聊城252000 [2]青岛农业大学动物医学院,山东青岛266109

出　　处：《动物医学进展》2022年第12期15-19,共5页Progress In Veterinary Medicine

基　　金：聊城大学博士启动基金项目(318051918,318051919);聊城大学畜牧学高水平学科建设项目(319312101-23,319312101-22)。

摘　　要：马疱疹病毒8型(EHV-8)是近年来在规模化养驴场发现的一种病毒,其主要导致马属动物呼吸困难、繁殖障碍(流产、产弱驹等),严重危害养驴业的健康发展。为建立EHV-8的分子生物学检测方法,用于该病原感染的流行病学调查和致病机制研究,根据NCBI数据库中EHV-8参考毒株ORF 70基因序列的高度保守区域,设计特异性引物,优化程序,建立检测EHV-8的荧光定量PCR方法。结果表明,该方法灵敏度高,最小检测浓度为100拷贝/μL,灵敏度是常规PCR的100倍;特异性强,与EHV-1、EHV-4均无交叉反应;重复性好,组内和组间重复试验变异系数分别为0.15%~0.83%和0.06%~0.47%。利用新建立的方法对120份临床驴血样本进行检测,阳性检出率为32.5%,研究结果为EHV-8的检测和致病性研究提供了可靠的技术支撑。Equine herpesvirus 8(EHV-8)is a newly found virus from large-scale donkey farm,which induces respiratory disease,reproductive failure(abortion,weak foal)in equids,and endangers the development of donkey industry.In order to establish a detection method for the epidemiological investigation and pathogenesis study of EHV-8,in this present study,a real-time quantitative PCR method was established for the detection of EHV-8.The primers were designed according to the highly conservative region of ORF 70 gene sequence of EHV-8 and the PCR program was optimized.The results showed that the minimum detection concentration of the established qPCR was 100 copies/μL,which was 100-fold lower than conventional PCR.The real-time PCR is highly specific to EHV-8,but without cross-reaction with other equine herpesvirus type(such as EHV-1 and EHV-4).The real-time PCR is also repeatable,the intra-assay and inter-assay coefficients of variation were 0.15%-0.83%and 0.06%-0.47%,respectively.The EHV-8 positive rate of 120 clinical samples collected from donkey farm was 32.5%using the real-time PCR method.This study provides a reliable method for EHV-8 diagnosis and basic researches of virus-related pathogenicity.

关 键 词：马疱疹病毒8型 ORF70 实时荧光定量PCR 检测 

分 类 号：S852.659.1[农业科学—基础兽医学] S858.21[农业科学—兽医学]