塞内卡病毒实时荧光定量RT-PCR检测方法的建立及应用  

Establishment and Application of the Real-time Fluorescent Quantitative RT-PCR Method for Detection of Senecavirus A

作　　者：周霞 温肖会[1] 赵亮 翟颀 吕殿红 罗胜军 贾春玲 周秀蓉 牛佳伟 陈天宝 贺东生 翟少伦[1,4] ZHOU Xia;WEN Xiao-hui;ZHAO Liang;ZHAI Qi;LV Dian-hong;LUO Sheng-jun;JIA Chun-ling;ZHOU Xiu-rong;NIU Jia-wei;CHEN Tian-bao;HE Dong-sheng;ZHAI Shao-lun(Institute of Animal Health,Guangdong Academy of Agricultural Sciences,Scientific Observation and Experiment Station of Veterinary Drugs and Diagnostic Techniques of Guangdong Province,Ministry of Agriculture of Rural Affairs,and Key Laboratory of Animal Disease Prevention of Guangdong Province,Guangzhou,Guangdong,510640,China;Key Laboratory of Zoonosis Prevention and Control of Guangdong Province,College of Veterinary Medicine,South China Agricultural University,Guangzhou,Guangdong,510642,China;College of Animal Science,Tibet Agriculture&Animal Husbandryy University,Nyingchi,Tibet,860000,China;Maoming Branch Center of Guangdong Laboratory forLingnan Modern Agricultural Science and Technology,Maoming,Guangdong,525099,China)

机构地区：[1]广东省农业科学院动物卫生研究所/广东省畜禽疫病防治研究重点实验室/农业农村部兽用药物与诊断技术广东科学观测实验站,广东广州510640 [2]华南农业大学兽医学院/广东省人兽共患病预防与控制重点实验室,广东广州510642 [3]西藏农牧学院动物科学学院,西藏林芝860000 [4]岭南现代农业科学与技术广东省实验室茂名分中心,广东茂名525099

出　　处：《动物医学进展》2023年第6期1-6,共6页Progress In Veterinary Medicine

基　　金：广东省农业科学院协同创新中心项目(XTXM202202和XT202208);广东省科技计划项目(2021B1212050021);广东省现代农业岗位体系专家项目(2021KJ114和2021KJ119)。

摘　　要：为加强塞内卡病毒(SVA)在不同宿主的诊断及流行病学监测与防控,根据GenBank中发表的SVA VP1基因区域设计一对特异性引物和带有TaqMan发光基团标记的探针,构建重组阳性质粒并用作建立实时荧光定量RT-PCR方法的模板,对反应体系和条件进行优化,构建标准曲线并验证该方法的特异性、敏感性和重复性。结果显示,该方法的最适退火温度为55℃,最佳引物和探针浓度分别为0.2μmol/L和0.4μmol/L,最佳反应循环数为45,Ct值与标准品浓度的对数线性关系好。对猪源、鼠源及牛源SVA和其他动物病毒(O型口蹄疫病毒、水疱性口炎病毒、牛病毒性腹泻病毒、猪伪狂犬病病毒、边界病毒、猪库布病毒、牛库布病毒、山羊鼻内肿瘤病毒、牛传染性鼻气管炎病毒、牛结节性皮肤病病毒、牛冠状病毒、D型流感病毒、蓝舌病毒)的核酸检测结果显示,除猪源、鼠源及牛源SVA为阳性外,其他病毒核酸为阴性。该方法最低检测限为2.66×10~1拷贝/μL,重复试验中组内和组间变异系数均小于0.1%。建立的检测多宿主SVA TaqMan RT-qPCR方法具有良好的特异性和稳定性,为SVA在不同宿主间的疾病诊断与流行病学调查或监控提供了快捷的方法。In order to strengthen the diagnosis and epidemiological surveillance of Senecavirus A(SVA)in different hosts,a pair of specific primers and one Taq Man probe were designed according to the VP1 gene of SVA published in GenBank database,the positive plasmids were also constructed and used to be templates for establishing real-time fluorescence quantitative RT-PCR methods.The optimal reaction system and condition were optimized.The standard curve was constructed,and the specificity,sensitivity and reproducibility of the method were verified.The results showed that the optimal annealing temperature was 55℃,the optimal primer and probe concentrations were 0.2μmol/L and 0.4μmol/L respectively,the best reaction cycle was 45.The standard curve showed a good linear relationship between cycle threshold(Ct)value and the concentration of standard samples.The RT-qPCR method had good specificity for porcine-origin,rat-origin and buffalo-origin SVA,and had no cross reaction with other animal viruses(FMDV type O,VSV,BVDV,PRV,BDV,PKV,BKV,ENTV-2,IBRV,LSDV,BCoV,IDV,BTV).The sensitivity of RT-qPCR method was 2.66×101 copies/μL.The reproducibility test results proved that all coefficients of variation(CV)of Ct values were lower than 0.1%in both intra-assay and inter-assay.The RT-qPCR method established in this study was of good specificity and stability,which can be used for the detection of multi-host SVA,and provide a rapid tool for the diagnosis and the epidemiological investigations of SVA.

关 键 词：塞内卡病毒 探针 实时荧光定量PCR 检测 

分 类 号：S852.659.6[农业科学—基础兽医学] S854.43[农业科学—兽医学]