梅迪-维斯纳病毒实时荧光RPA检测方法的建立  

Development of a Real-time Fluorescence RPA for Detection of Maedi-visna Virus

作　　者：刘然 张琳 陈思旭 史晓娜[1] 刘淑英[1] Liu Ran;Zhang Lin;Chen Sixu;Shi Xiaona;Liu Shuying(School of Veterinary Medicine,Inner Mongolia Agricultural University,Key Laboratory of Clinical Diagnosis and Treatment of Animal Diseases,Ministry of Agriculture and Rural Affairs,Key Laboratory of Basic Veterinary Medicine,Hohhot,Inner Mongolia Autonomous Region 010018,China)

机构地区：[1]内蒙古农业大学兽医学院,农业农村部动物临床诊疗技术重点实验室,内蒙古自治区基础兽医学重点实验室,内蒙古呼和浩特010018

出　　处：《中国动物检疫》2023年第3期90-96,130,共8页China Animal Health Inspection

基　　金：国家自然基金面上项目(32072819);内蒙古科技重大专项计划项目(2021ZD0010);牛羊疫病防控与发育工程创新团队项目(2200001130);内蒙古草原英才创新团队项目(20151031)。

摘　　要：为实现对梅迪-维斯纳病毒(MVV)的快速检测,根据MVV gag基因保守序列设计特异性引物和探针,通过优化反应条件和反应体系,建立了MVV的实时荧光RPA检测方法,并通过灵敏度、特异性、重复性试验及样品复核检测进行评价。结果显示:该方法最佳反应温度为39℃,用时短,20 min即可完成检测;灵敏度高,检测下限可达10-1pg/μL,比常规PCR检测结果灵敏约104倍;特异性强,与小反刍兽疫病毒、羊败血性链球菌、牛肠道病毒、传染性鼻气管炎病毒、牛副流感病毒3型、肺炎支原体等继发呼吸系统症状病原无交叉反应;重复性好,对于相同质量浓度的模板DNA检测变异系数均小于10%;可有效检测出MVV,与PCR结果一致。综上所述,本研究建立的MVV荧光RPA检测方法适用于MVV的临床快速检测,可为该病的诊断及防控提供依据。In order to rapidly detect Maedi-visna virus(MVV),specific primers and probes were designed according to gag gene of MVV conserved sequence,followed by optimization of the reaction conditions and reaction system,a real-time fluorescent RPA for MVV was established and evaluated through the tests of sensitivity,specificity,repeatability and sample recheck.The results showed that,by the established method,the tests could be finished within 20 min at an optimal reaction temperature of 39℃;its lowest detection limit could reach 10-1pg/μL with high sensitivity,which was 10~4 times more sensitive than the routine PCR;failed to crossly react with Peste des petits ruminants virus(PPRV),Streptococcus septicaemia(OSS),bovine enterovirus(BEV),infectious rhinotracheitis virus(IBRV),bovine parainfluenza virus type 3(BPIV3),Mycoplasma pneumoniae(MO)and other pathogens with secondary respiratory symptoms due to its strong specificity;the coefficient of variation(CV)for detecting template DNA with the same mass concentration was less than 10%due to its good repeatability;MVV could be effectively detected,which was consistent with the results by PCR.In conclusion,the established method could be used to rapidly detect MVV in practice,and supported the diagnosis,prevention and control of the disease.

关 键 词：梅迪-维斯纳病毒 荧光RPA检测 快速检测 性能评价 

分 类 号：S855.3[农业科学—临床兽医学]