牛源新型环状病毒RT-qPCR方法的建立和应用  

Establishment and Application of Real-time Quantitative PCR Method for Detection a Novel Orbivirus Isolated from Cattle

作　　者：杨振兴[1] 何于雯 谢佳芮 朱建波[1] YANG Zhen-xing;HE Yu-wen;XIE Jia-rui;ZHU Jian-bo(Yunnan Tropical and Subtropical Animal Virus Disease Laboratory,Key Laboratory of Transboundary Animal Diseases Prevention and Control(Co-construction by Ministry and Province),Yunnan Veterinary and Animal Science Institute,Kunming,Yunnan 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,农业农村部跨境动物疫病防控重点实验室(部省共建),云南昆明650224

出　　处：《动物医学进展》2023年第11期8-14,共7页Progress In Veterinary Medicine

基　　金：2019年云南省"万人计划"产业技术领军人才项目;云南省热带亚热带动物病毒病重点实验室自主研究课题(2022RW001);国家公益性行业(农业)科研专项(201303035)。

摘　　要：目前世界范围内仅分别从日本西南的与那国岛(2015年)及中国云南省景洪市勐罕镇(2019年)的哨兵动物牛上分离到过新型环状病毒Yonaguni orbivirus(YONOV),但尚缺乏该病毒核酸的特异性检测方法。该研究对比我国分离的YONOV毒株JH2019C603和日本毒株(ON-7/E/15)的基因序列,选择编码非结构蛋白3(Non-structural protein 3,NS3)的Segment-10(Seg-10)节段的保守区设计引物和探针,并分别进行了特异性、灵敏度、重复性和临床血液样品的测试,建立了牛YONOV的RT-qPCR和RT-PCR检测方法。结果显示,两种方法能特异性扩增YONOV核酸片段,对其他环状病毒无有效扩增;RT-qPCR和RT-PCR检出YONOV核酸浓度的下限分别为11.5 copies/μL和115.0 copies/μL;两种方法均能检测出毒株或动物临床血样中的YONOV核酸,并且能在动物感染并产生中和抗体前1周检测出病毒核酸,因此更适用于病毒感染动物的早期诊断。RT-qPCR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RT-PCR的扩增产物可以进行测序,获取待测毒株的基因信息,两种检测方法相互辅助,可以为YONOV的核酸检测及动物感染该病毒的诊断及流行病学研究提供技术支撑。At present,a novel orbivirus(Yonaguni orbivirus,YONOV)has only been isolated from sentinel cattle in subtropical islands of Japan(2015)and Menghan town of Jinghong city,Yunnan province,China(2019),but there is no specific nucleic acid detection method for the virus.Therefore,according to the comparison of the gene sequences of YONOV(JH2019C603)strain isolated in China and Japan's strain(ON-7/E/15),we selected the conserved region of Segment-10(Seg-10)gene encoding non-structural protein 3(NS3)to design primers and probes,and developed a real-time quantitative PCR(RT-qPCR)and RT-PCR method.Then specificity,sensitivity and clinical samples of the above two methods were tested.The results showed that the two methods could detect YONOV specifically,but did not cross-react with other orbivirus.The sensitivity test results showed that the detection limit of YONOV by RT-qPCR was 11.5 copies/μL,and that of routine RT-PCR was 115.0 copies/μL.Clinical sample detection showed that both methods can detect the YONOV nucleic acid in blood samples of virus infected animals.However,compared with the neutralization test,two methods can detect the viral nucleic acid one week before the neutralization antibody turns positive,so they are more suitable for the early diagnosis of animal infection with virus.RT-qPCR 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.Therefore,the two methods can provide rapid,convenient and reliable technical support for detection,diagnosis and epidemiological survey of YONOV.

关 键 词：环状病毒 实时荧光定量PCR 常规RT-PCR 病毒检测 

分 类 号：S852.65[农业科学—基础兽医学] S858.23[农业科学—兽医学]