盖塔病毒TaqMan探针实时荧光定量RT-PCR检测方法的建立  

Development of a TaqMan-Based Real-Time RT-PCR Method for Detection of Getah Virus

作　　者：顾志刚 郭洁真 王子涵 孙彤 陈鸿军[2] 孙竹筠 陈丹清 GU Zhigang;GUO Jiezhen;WANG Zihan;SUN Tong;CHEN Hongjun;SUN Zhuyun;CHEN Danqing(Husbandry and Veterinary Center of Wujin District,Changzhou 213161,China;Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]江苏省常州市武进区畜牧兽医管理服务中心,常州213161 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《中国动物传染病学报》2025年第1期112-118,共7页Chinese Journal of Animal Infectious Diseases

基　　金：上海市2020年度“科技创新行动计划”实验动物研究领域项目(20140900300);2021—2022年增补常州市现代农业产业技术体系建设项目。

摘　　要：盖塔病毒(GETV)是一种在我国广泛分布的虫媒病毒,可通过蚊虫传播感染人畜,导致猪、马等动物出现发热、出疹及淋巴结肿大等症状,可致妊娠母猪流产或死胎。目前尚无特效治疗方式。本研究根据GenBank公布的盖塔病毒nsP1基因序列,设计3组引物探针,筛选出最佳的引物探针,建立一种灵敏度高、特异性强的针对盖塔病毒的TaqMan探针实时荧光定量RTPCR检测方法。对该方法进行敏感性、特异性、重复性试验和临床样品检测。标准曲线Ct值与相应质粒标准品浓度存在良好的线性关系,相关系数(R^(2))为0.9989;对含GETV基因的重组质粒pCDNA3.1-nsP1体外转录RNA进行敏感性试验,最低检出限达10 copies,是普通RT-PCR方法的1000倍,敏感性高;与PRRSV、CSFV与PEDV的核酸不发生交叉反应,特异性好;变异系数小于2%,重复性好。本研究建立了一种敏感、特异的GETV检测方法,对GETV的快速诊断具有重要意义。Getah virus(GETV)is an arbovirus that is widely distributed in China.The virus infects humans and animals through mosquito transmission,causing fever,rash,lymphadenectasis and other disorders in pigs,horses and other animals.It also causes abortion or stillbirth in pregnant sows.Currently,there is no specific treatment.In this study,three pairs of primers and probes were designed based on the GETV nsp1 gene sequence published by GenBank.The optimal primers and probes were screened to develop a highlysensitive and strongly specific TaqMan probe real-time RT-PCR method for detection of Getah virus.The results showed a good linear relationship between Ct value and logarithm of copy numbers.The correlation coefficient R^(2)was 0.9994.The lowest limit of detection was 10 copies,demonstrating 1000 times more sensitivity than RT-PCR.There was no cross-reaction with PRRSV,CSFV and PEDV,indicating its good specificity.The coefficient of variation was less than 2%,showing its good repeatability.In conclusion,a sensitive and specific detection method for GETV was developed in this study,which was of great significance for the rapid diagnosis for GETV.

关 键 词：盖塔病毒 nsP1 TAQMAN探针 实时荧光定量RT-PCR 

分 类 号：R373[医药卫生—病原生物学]