马疱疹病毒1型TaqMan实时荧光定量PCR检测方法的建立  

Establishment of TaqMan real-time PCR for detecting equid herpesvirus 1

作　　者：胡月[1] 刘建华[1] 王雪竹 车传忠 吴桂灵 加尔肯[1] 冉多良[1] HU Yue;LIU Jianhua;WANG Xuezhu;CHE Chuanzhong;WU Guiling;JARHEN;RAN Duoliang(College of Veterinary Medicine,Xinjiang Agricultural University,Urumchi 830052,China)

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052

出　　处：《中国兽医学报》2021年第12期2345-2350,共6页Chinese Journal of Veterinary Science

基　　金：新疆维吾尔自治区重大专项项目子课题资助项目(2017A01002-2-4)。

摘　　要：为快速准确地检测马疱疹病毒1型(equine herpesvirus 1,EHV-1),本研究根据EHV-1基因组序列进化分析筛选出特异性较高的ORF68基因的保守区域设计引物与探针,建立了TaqMan实时荧光定量PCR检测方法。结果显示该方法线性关系良好,相关系数为0.999;灵敏度高,最低检测量为10.2拷贝/μL,比常规PCR敏感1000倍;特异性强,检测EHV-2、EHV-4、EHV-5、马动脉炎病毒和马流感病毒均无交叉反应;重复性好,组内及组间样本检测的Ct值均小于等于2%;对2020年收集的60份伴有呼吸道症状的马匹鼻试子进行检测分析显示本方法的阳性检出率(26.67%)高于SYBR GreenⅠ荧光定量PCR(23.33%)和常规PCR(21.67%)。本研究建立了一种快速、灵敏、稳定、特异性强的TaqMan荧光定量PCR检测方法,可用于EHV-1的快速诊断和核酸定量检测,为EHV-1的防控工作提供了有力手段。In order to establish a rapid and accurate method for the detection of equid herpesvirus 1(EHV-1),primers and probes were designed and synthesized according to the conserved sequence of the gene ORF68 of EHV-1 and a real-time PCR assay based on TaqMan technology was established.The results showed that the method has a good linear relationship and the correlation was 0.999.The method had good sensitivity,the detection limit was 10.2 copies/μL which was about 1000 times higher than that of conventional PCR.There was no cross-reaction with other equine viruses included equid herpesvirus 2(EHV-2),equid herpesvirus 4(EHV-4),equid herpesvirus 5(EHV-5),Equine arteritis virus(EAV)and Equine influenza virus(EIV).The repeatability test showed that the inter-and intra-assay coefficient of variation were both less than or equal to 2%,indicating good repeatability.The detection results of 60 suspected specimens collected in 2020 indicated that the positive rate of TaqMan real-time PCR(26.67%)was higher than SYBR GreenⅠreal-time PCR(23.33%)and conventional PCR(21.67%).In conclusion,a rapid,sensitive,stable and accurate TaqMan real-time PCR assay was established,which is suitable for rapid diagnosis and quantitative detection of EHV-1,and provides a powerful means for the further research and prevention and control of EHV-1 infection.

关 键 词：马疱疹病毒1型 ORF68基因 荧光定量PCR TAQMAN探针 

分 类 号：S855.3[农业科学—临床兽医学]