猪流行性腹泻病毒RPA-LFD检测方法的建立  

Establishment of RPA-LFD method for detection of porcine epidemic diarrhea virus

作　　者：袁嘉康 李林岳 庞俊增 范国英[1] 姜金庆[1] 王自良[1] 李任峰[1] YUAN Jiakang;LI Linyue;PANG Junzeng;FANN Guoying;JIANG Jinqing;WANG Ziliang;LI Renfeng(College of Animal Science and Technology,Henan Institute of Science and Technology,Xinxiang,Henan 453003,China)

机构地区：[1]河南科技学院动物科技学院,河南新乡453003

出　　处：《中国兽医学报》2023年第6期1127-1132,共6页Chinese Journal of Veterinary Science

基　　金：河南省重点研发与推广专项(科技攻关)农业领域基金资助项目(212102110098)。

摘　　要：将重组酶聚合酶等温扩增(recombinase polymerase amplification,RPA)与侧流层析试纸(lateral flow dipstick,LFD)技术相结合,根据猪流行性腹泻病毒(porcine epidemic diarrhea virus,PEDV)M基因保守序列设计特异性引物,上、下游引物的5′端分别进行6-羧基荧光素(FAM)和生物素(biotin)标记,通过优化RPA反应温度和时间条件,建立基于RPA-LFD的PEDV快速检测方法,并对该方法的灵敏性、特异性和临床应用进行评价。结果显示,该方法在40.8℃恒温反应21 min即可完成对PEDV核酸的扩增,RPA-LFD的最低检测限为1.27×10^(2)拷贝/μL,且与猪传染性胃肠炎病毒、猪德尔塔冠状病毒、猪繁殖与呼吸综合征病毒等均无交叉反应;22份临床样本检测结果显示RPA-LFD与RT-PCR检测结果完全一致。结果表明,本试验建立的PEDV RPA-LFD检测方法具有操作方便、检测速度快、灵敏度高、特异性强等优点,为PEDV的现场快速检测和流行病学调查提供了新的技术手段。This study aims to establish a rapid,sensitive and simple method for porcine epidemic diarrhea virus(PEDV)detection.Specific primers based on the conserved sequence of PEDV M gene were designed,and the 5'ends of the forward and reverse primers were labelled with 6-carboxyfluorescein(FAM)and biotin,respectively.By combining recombinase polymerase amplification(RPA)with lateral flow chromatography(LFD)technique,we developed RPA-LFD detection method for PEDV by optimizing the RPA reaction temperature and time,and the sensitivity,specificity and clinical application of the RPA-LFD were evaluated.The results showed that the RPA-LFD could detect PEDV nucleic acid at 40.8℃for 21 min,and the lowest detection limit of RPA-LFD was 1.27×10^(2)copies/μL,and there was no cross-reactivity with swine transmissible gastroenteritis virus,porcine delta coronavirus and porcine reproductive and respiratory syndrome virus.The results of 22 clinical samples showed that RPA-LFD was fully consistent with RT-PCR.The PEDV RPA-LFD has the advantages of easy operation,fast detection,high sensitivity and good specificity,and provided a new technical approach for rapid on-site detection and epidemiological investigation of PEDV.

关 键 词：猪流行性腹泻病毒 重组酶聚合酶等温扩增 侧流层析试纸 M基因 

分 类 号：S852.65[农业科学—基础兽医学]