马疱疹病毒8型TaqMan实时荧光定量PCR检测方法的建立  

Establishment of TaqMan Real-time Fluorescence Quantitative PCR Detection Method for Equine Herpesvirus Type 8

作　　者：纪言霏 齐来勇 张健鹏 许丹丹 张敬文 赵霞 刘文强[1] JI Yan-fei;QI Lai-yong;ZHANG Jian-peng;XU Dan-dan;ZHANG Jing-wen;ZHAO Xia;LIU Wen-qiang(School of Agriculture Science and Engineering,Liaocheng University,Liaocheng 252000,China;Liaocheng Dongchangfu District Agricultural and Rural Bureau,Liaocheng 252000,China)

机构地区：[1]聊城大学农学与农业工程学院,山东聊城252000 [2]聊城市东昌府区农业农村局,山东聊城252000

出　　处：《中国兽医杂志》2023年第8期24-28,共5页Chinese Journal of Veterinary Medicine

基　　金：山东省驴产业技术体系基金(SDAIT-27-11);聊城大学理工科研项目(318011701)。

摘　　要：为了建立一种能够快速检测马疱疹病毒8型(EHV-8)的方法,本试验以EHV-8的全基因组序列为模板,针对糖蛋白B(gB)基因的保守序列,进行对比分析,设计EHV-8的特异性引物和探针,优化反应条件,建立EHV-8 TaqMan实时荧光定量PCR检测方法,并对该方法的敏感性、特异性和重复性进行验证,使用该方法进行临床样本检测。结果显示,建立的EHV-8 TaqMan实时荧光定量PCR检测方法对EHV-8 DNA模板的最低检测限为1.1×10^(2)copies/μL,敏感性高;EHV-8与EHV-1、EHV-4、马流产沙门氏菌和肠产毒性大肠杆菌均无交叉反应,特异性强;批内重复性试验和批间重复性试验均表明该方法重复性好。对132份临床样本的检测结果显示,阳性检出率为10.61%,基因测序正确。由此可见,本试验建立的EHV-8 TaqMan实时荧光定量PCR检测方法能够满足EHV-8的检测需求,且该方法敏感性高、特异性强、重复性好,为EHV-8的进一步研究提供了有效的辅助检测手段。In order to develop a rapid detection method for equine herpesvirus type 8(EHV-8),this study used the whole genome sequence of EHV-8 as a template and performed comparative analysis targeting the conserved sequence of glycoprotein B(gB)gene.Specific primers and probes for EHV-8 were designed,and reaction conditions were optimized to establish the TaqMan real-time fluorescence quantitative PCR detection method for EHV-8.The sensitivity,specificity,and repeatability of this method were verified,and clinical samples were tested using this method.The results showed that the established TaqMan real-time fluorescence quantitative PCR method had a minimum detection limit of 1.1×10^(2) copies/μL for EHV-8 DNA template,indicating high sensitivity.No cross-reaction was observed with EHV-1,EHV-4,Salmonella abortus equi,and enterotoxigenic Escherichia coli(ETEC),indicating strong specificity.Both intra-batch and inter-batch repeatability tests demonstrated good repeatability of the method.Among the 132 clinical samples tested,the positive detection rate was 10.61%,which was fully confirmed by gene sequencing.Therefore,the established TaqMan real-time fluorescence quantitative PCR detection method can meet the demands for EHV-8 detection,with high sensitivity,strong specificity,and good repeatability,providing an effective auxiliary detection tool for further research on EHV-8.

关 键 词：驴 马疱疹病毒8型 糖蛋白B(gB)基因 实时荧光定量PCR TAQMAN探针 

分 类 号：S822[农业科学—畜牧学]