牛病毒性腹泻病毒1型RT-RAA快速诊断方法的建立  

Development of a RT-RAA Assay for Rapid Detection of Bovine Viral Diarrhea Virus Type 1

作　　者：范颖 田雪 孙淑芳[1,3] 吴发兴 尼博[1,4] 刘建柱 孙翔翔[1,4] 胡莉萍 Fan Ying;Tian Xue;Sun Shufang;Wu Faxing;Ni Bo;Liu Jianzhu;Sun Xiangxiang;Hu Liping(China Animal Health and Epidemiology Center,Qingdao,Shandong 266032,China;College of Animal Science and Technology,Shandong Agricultural University,Tai'an,Shandong 271000,China;Key Laboratory of Major Ruminant Infectious Disease Prevention and Control(Eastern China),Ministry of Agriculture and Rural Affairs,Qingdao,Shandong 266032,China;Key Laboratory of Animal Biosafety Risk Warning,Prevention and Control(Southern China),Ministry of Agriculture and Rural Affairs,Qingdao,Shandong 266032,China;Shandong Center for Animal Disease Prevention and Control,Jinan,Shandong 250100,China)

机构地区：[1]中国动物卫生与流行病学中心,山东青岛266032 [2]山东农业大学动物科技学院,山东泰安271000 [3]农业农村部反刍动物重大疫病防控重点实验室(东部),山东青岛266032 [4]农业农村部动物生物安全风险预警及防控重点实验室(南方),山东青岛266032 [5]山东省动物疫病预防与控制中心,山东济南250100

出　　处：《中国动物检疫》2023年第4期93-98,共6页China Animal Health Inspection

基　　金：现代农业(奶牛)产业技术体系建设专项(CARS-36)。

摘　　要：为解决牛病毒性腹泻病毒(BVDV)现场快速诊断难点,提升应急响应速度,根据BVDV 1型基因组5’-UTR保守区序列设计特异性引物和探针,建立了BVDV 1型重组酶聚合酶等温核酸扩增方法(reverse transcription recombinase aid amplification,RT-RAA),并对该方法的特异性、敏感性和重复性进行评估。结果显示:该方法在39℃等温条件下检测时间为20 min,对BVDV 1型核酸检测下限为1.2×10^(2) copies/μL;3个浓度梯度的核酸组内重复性试验的变异系数(CV)分别为3.24%、9.48%和8.79%,均在10.00%以内,重复性好;建立的方法与猪瘟病毒、牛冠状病毒、牛传染性鼻气管炎病毒等无特异性反应。对66份临床样品进行检测,发现所建立的方法与荧光定量PCR检测结果符合率为98.48%,Kappa值为0.881(P<0.001)。结果表明,本研究建立的RT-RAA快速诊断方法特异性强、灵敏度高、操作便捷,可用于BVDV 1型的现场快速检测。In order to solve difficulties in rapid on-site diagnosis of bovine viral diarrhea virus(BVDV)and to quicken emergency response,specific primers and probes were designed based on the conserved region of 5'-UTR of BVDV-1 gene sequence to establish a reverse transcription recombinase acid amplification(RT-RAA)method,followed by evaluation on its specificity,sensitivity and repeatability.The results showed that,by the established method,the detection time was 20 min under the isothermal condition of 39℃,and the detection limit for BVDV-1 nucleic acids was 1.2×10^(2) copies/μL;the coefficient of variation(CV)of the intra-group reproducibility tests at three concentration gradients was 3.24%,9.48%and 8.79%,respectively,all of which was within 10.00%,with good reproducibility;and no specific reaction was observed for the established method with classical swine fever virus(CSFV),bovine coronavirus,infectious bovine rhinotracheitis virus(IBRV)and other viruses.It was found that,when 66 clinical samples were detected,the results by the established method were 98.48%consistent with those by the fluorescent quantitative PCR,and the Kappa value was 0.881(P<0.001).In conclusion,the RT-RAA for rapid diagnosis established in the study could be used for on-site rapid detection of BVDV-1 with the help of its strong specificity,high sensitivity and convenient operation.

关 键 词：牛病毒性腹泻病毒 重组酶介导的等温扩增 快速诊断 

分 类 号：S855.3[农业科学—临床兽医学]