猪轮状病毒RT-RAA-LFD快速诊断方法的建立及应用  

Establishment and application of RT-RAA-LDF method for rapid detection of porcine rotavirus

作　　者：罗晓莹 林福忠 林秋娟 曾彦钦 陈震 施文豪 LUO Xiaoying;LIN Fuzhong;LIN Qiujuan;ZENG Yanqin;CHEN Zhen;SHI Wenhao(Fuzhou Institute of Agricultural Sciences,Fuzhou 350018,China;Agricultural and Rural Bureau of Yongtai Country,Fuzhou 350700,China)

机构地区：[1]福州市农业科学研究所,福建福州350018 [2]永泰县农业农村局,福建福州350700

出　　处：《中国兽医学报》2024年第12期2534-2539,共6页Chinese Journal of Veterinary Science

基　　金：福州市科技计划市属农科院所科研专项资助项目(2023-N-008)。

摘　　要：为实现猪轮状病毒(porcine rotavirus,PoRV)的快速检测,本研究将重组酶聚合酶等温扩增(recombinase aided amplification,RAA)与侧流层析试纸条(lateral flow dipstick,LFD)技术结合,根据PoRV VP6基因保守序列,设计特异性引物和探针,构建并优化扩增体系,建立了基于RAA-LFD的PoRV一步法快速检测方法,并对检验该方法的特异性、敏感性、重复性及临床应用进行了评价。结果显示,该方法在37℃恒温反应15min即可实现对PoRV核酸的扩增,最低检出限为22.3拷贝/μL,与其他常见病毒性腹泻病毒均无交叉反应,RT-RAA-LFD与RT-PCR检测方法总符合率为97.0%,Kappa系数为0.93(K>0.75)。本试验建立的RT-RAA-LFD检测方法具有良好的特异性、敏感性和稳定性,并具备快速、可视化和适合现场快检等特点,为PoRV快速诊断和流行病学调查提供了新的技术手段。To rapidly detect porcine rotavirus(PoRV),a one-step detection method was developed based on recombinase-aided amplification(RAA)combined with lateral flow dipstick(LFD)technology.Specific primers and probes were designed according to the conserved sequence of the PoRV VP6gene,and the amplification system was optimized.The specificity,sensitivity,and repeatability of the method were thoroughly tested,and its clinical application was evaluated.The results demonstrated that the method could amplify PoRV nucleic acid at 37℃in just 15min with a minimal detection limit of 22.3copies/μL.No cross-reactivity was observed with other common diarrhea viruses.The total coincidence rate between RT-RAA-LFD and RT-PCR was 97.0%,with a Kappa coefficient of 0.93(K>0.75).In summary,the RT-RAA-LFD detection method established in this study exhibits excellent specificity,sensitivity,and stability,characterized by rapid detection speed and visualization,making it suitable for on-site rapid detection.This method provides a novel technical approach for the rapid diagnosis and epidemiological investigation of PoRV.

关 键 词：猪轮状病毒 重组酶聚合酶等温扩增 侧流层析试纸条 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]