西藏环状病毒荧光定量RT-PCR与常规RT-PCR检测方法的建立及应用  

Establishment and application of real-time fluorescent quantitative RT-PCR and routine RT-PCR for detection of Tibet orbivirus

作　　者：杨振兴[1] 何于雯 谢佳芮 李占鸿 李卓然 廖德芳[1] 李华春[1] 李乐[1] 杨恒[1] YANG Zhen-xing;HE Yu-wen;XIE Jia-rui;LI Zhan-hong;LI Zhuo-ran;LIAO De-fang;LI Hua-chun;LI Le;YANG Heng(Yunnan Tropical and Subtropical Animal Virus Diseases Laboratory/Yunnan Animal Science and Veterinary Institute,Kunming 650224,China)

机构地区：[1]云南省畜牧兽医科学院,云南省热带亚热带动物病毒病重点实验室,云南昆明650224

出　　处：《中国兽医科学》2020年第11期1365-1372,共8页Chinese Veterinary Science

基　　金：国家重点研发计划(2016YFD0500908,2017YFC1200505);国家公益性行业(农业)科研专项(201303035);云南省中青年学术和技术带头人后备人才培养项目(2017HB055).

摘　　要：为建立西藏环状病毒(TIBOV)群特异性核酸检测方法,本研究根据云南师宗新分离的TIBOV(YNSZ/V290/2019)毒株和GenBank中登录的TIBOV毒株VP6基因序列的保守区,设计并合成特异性引物和探针,建立了荧光定量RT-PCR和常规RT-PCR检测方法,并分别进行了特异性、灵敏性和临床样品的检测。试验结果表明,两种检测方法均可特异性地扩增TIBOV及检出特异荧光信号,而对帕利亚姆血清群病毒(PALV)、蓝舌病病毒(BTV)、流行性出血病病毒(EHDV)、广西环状病毒(GXOV)、阿卡斑病毒(AKAV)、云南环状病毒(YNOV)、非洲马瘟病毒(AHSV)和牛流行热病毒(BEFV)无扩增和无有效荧光信号检出,具有较好的特异性。灵敏性试验结果显示,荧光定量RT-PCR检测TIBOV核酸的下限可达10 copies/μL,常规RT-PCR的检测下限为10~2copies/μL。临床样品检测显示,两种方法均能检测出库蠓样品中的TIBOV核酸,常规RT-PCR扩增产物经测序和BLAST比对分析显示,库蠓样品中检测到的TIBOV与NCBI数据库中TIBOV(DH13C120、XZ0906、D181/2008)毒株VP6基因序列相似度均在95%以上。荧光定量RT-PCR因其更高的灵敏度和实效性,能用于大量临床样品的快速检测,而常规RT-PCR可以对扩增产物进行胶回收测序,进一步了解待测病毒的遗传信息。两种方法相互辅助,可以为TIBOV的早期诊断、快速检测、流行病学调查和实验研究等提供有效的技术方法。To establish the assay for detecting Tibet orbivirus(TIBOV),primers and probe were designed according to the conserved region of VP6 gene of TIBOV(YNSZ/V290/2019) strain newly isolated from Shizong of Yunnan Province and other TIBOV strains registered in Gen Bank.The real-time quantitative RT-PCR and routine RT-PCR methods were established,and the specificity,sensitivity and clinical samples of the two methods were tested.The results showed that the two methods could detect TIBOV specifically,but did not cross-react with the Palyam serogroup virus,Bluetongue virus,Epizootic hemorrhagic disease virus,African horse sickness virus,Yunnan orbivirus,Guangxi orbivirus,Bovine ephemeral fever virus or Akabane virus.The sensitivity test results showed that the detection limit of TIBOV by real-time quantitative RT-PCR was 10 copies/μL,and that of routine RT-PCR was 10~2 copies/μL.The results of clinical samples detection showed that the two methods could detect TIBOV in Culicoides.Sequencing and BLAST analysis of routine RT-PCR products showed that the VP6 gene sequence identities of TIBOV detected in Culicoides with TIBOV(DH13C120,XZ0906,D181/2008) strains in NCBI database was more than 95%.Real-time quantitative RT-PCR can be used for rapid detection of a large number of clinical samples due to its higher sensitivity and effectiveness,while routine RT-PCR can sequence the amplified products to further understand the genetic information of the virus to be tested. The mutual use of the two methods can provide an effective technical method for the early diagnosis,rapid detection,epidemiological investigation and experimental research of TIBOV.

关 键 词：西藏环状病毒 荧光定量RT-PCR 常规RT-PCR 病毒检测 

分 类 号：S852.659.4[农业科学—基础兽医学]