猪德尔塔冠状病毒荧光RT-RAA快速检测方法的建立及初步应用  

Establishment and Preliminary Application of Fluorescence RT-RAA for Detecting PDCoV

作　　者：赵平[1,2] 秦毅斌 何颖 卢冰霞 刘芳 陈樱[1] 韦祖樟[1] 黄伟坚[1] 赵武[2] 欧阳康 Zhao Ping;Qin Yibin;He Ying;Lu Bingxia;Liu Fang;Chen Ying;Wei Zuzhang;Huang Weijian;Zhao Wu;Ouyang Kang(University of Guangxi Nanning,Guangxi 530005,China;Key Laboratory of Veterinary Biotechnology,Guangxi Veterinary Research Institute,Nanning,Guangxi 530001,China)

机构地区：[1]广西大学,广西南宁530005 [2]广西壮族自治区兽医研究所,广西兽医生物技术重点实验室,广西南宁530001

出　　处：《中国动物检疫》2021年第8期85-89,共5页China Animal Health Inspection

基　　金：广西创新驱动发展专项资金项目(桂科AA17204057-1);广西自然科学基金项目(2017GXNSFBA198092);广西南宁市科学研究与技术开发计划项目(20202090);广西南宁市西乡塘区科学研究与技术开发计划项目(2020021605);广西柳州市科技计划项目(2020NACB080);广西大学学术骨干科研基金项目(XGZ170239);广西兽医生物技术重点实验室开放基金课题项目(16-380-45-B-3,19-50-40-B-01)。

摘　　要：近年来,猪德尔塔冠状病毒(porcine deltacoronavirus,PDCoV)给养猪业造成了较大经济损失。为快速检测PDCoV,针对病毒N基因设计特异引物,建立了PDCoV重组酶介导核酸等温扩增荧光法(RT-RAA)。通过优化引物浓度、反应温度,确定了最佳反应条件,然后进行了特异性和灵敏性试验,并与普通RT-PCR方法进行了比较。结果显示:该方法特异性较好,与猪繁殖与呼吸综合征病毒、猪轮状病毒、猪伪狂犬病病毒、猪细小病毒、猪圆环病毒、猪瘟病毒、猪捷申病毒、传染性胃肠炎病毒、猪流行性腹泻病毒等猪源病毒无明显交叉反应;反应过程均在39℃恒温条件下完成,用时30 min即可得到扩增结果;反应体系可检测的最低扩增拷贝数为10 copies/μL,敏感性较高;该方法检测68份猪源病毒样品的PDCoV阳性检出率为17.6%,高于普通RTPCR(13.2%)。结果表明,本研究建立的RT-RAA快速检测PDCoV方法具有快速、特异、灵敏的优点,可用于PDCoV的快速检测和流行病学监测。In recent years,porcine deltacoronavirus(PDCoV)has brought huge economic losses to pig industry.In order to rapidly detect PDCoV,specific primers were designed for N gene of the virus,and a recombinant transcriptase recombinase aided amplification(RT-RAA)was established.The optimal reaction conditions were determined through optimizing the concentration and reaction temperature of the primers,followed by specificity and sensitivity tests,which was then compared with conventional RT-PCR assay.It was concluded that the method,with good specificity,failed to react with porcine viruses including porcine reproductive and respiratory syndrome virus,porcine pseudorabies virus,porcine parvovirus,porcine circovirus,classical swine fever virus,infectious gastroenteritis virus and porcine epidemic diarrhea virus,etc;all processes of reaction were finished at 39℃,and the amplification results were obtained for 30 min;the minimum detection limit of PDCoV nucleic acids was 10 copies/μL,showing high sensitivity;for 68 porcine virus samples,the positive rate of PDCoV detected by the method was 17.6%,which was higher than that by normal RT-PCR(13.2%).In short,the method established in the paper,with the advantages of speediness,specificity and sensitivity,could be used for rapid detection and epidemiological surveillance for PDCoV.

关 键 词：猪德尔塔冠状病毒 RT-RAA 快速检测 

分 类 号：S851.3[农业科学—预防兽医学]