高致病性猪繁殖与呼吸综合征RPA-LFD诊断方法的建立及初步应用

Establishment and primary application of RPA-LFD method for detecting HP-PRRSV

高致病性猪繁殖与呼吸综合征(HP-PRRS)给我国养猪业带来了巨大的损失.为快速、便捷诊断该病,建立重组酶聚合酶扩增结合侧流层析技术(RPA-LFD)的HP-PRRS诊断方法.针对HP-PRRSV Nsp2基因特异序列,设计生物素(Biotin)标记的特异性引物和FAM荧光素标记的探针,对RPA-LFD的反应体系和条件进行优化,结果显示,HP-PRRSV RPA-LFD最佳反应体系为:上下游引物(0.42 pmol/μL)各2.1μL,探针(0.3 pmol/μL)0.6μL,Rehydration buffer 29.5μL,cDNA模板1μL,ddH_(2)O 12.2μL,MgOAc 2.5μL,共50μL;最佳反应条件为:37℃,20 min.该方法同时检测高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)、猪繁殖与呼吸综合征病毒VR2332株(PRRSV VR2332)和NADC30-like株等10种常见感染猪的病原,结果显示,该方法除对HP-PRRSV检测呈阳性外,对PRRSV VR2332株、NADC30-like和其它9种感染猪的病原检测均为阴性,特异性强;该方法对10倍系列稀释的质粒标准品检测结果显示,该方法对质粒标准品的最低检测限为4.05×10^(1)拷贝/μL,灵敏度高;该方法与HP-PRRSV商品化荧光检测试剂盒同时检测50份临床样品,二者符合率为100%.本研究首次建立了一种特异、灵敏、方便、快速、现场化的HP-PRRSV RPA-LFD检测方法,为HP-PRRS的快速诊断和流行病学调查提供了新的技术手段.

Highly pathogenic porcine reproductive and respiratory syndrome(HP-PRRS)has caused great losses to the pig industry in China.In order to diagnose the disease more quickly and conveniently,a diagnostic method for HP-PRRS based on recombinant enzyme polymerase amplification combined with lateral flow dipstick(RPA-LFD)was established.In this study,specific primers containing biotin and FAM-fluoresce in labeled probes were designed according to the specific sequence of Nsp2 gene of HP-PRRSV.The reaction system and conditions of RPA-LFD method were optimized,and the results showed that the optimal reaction system of HP-PRRSV RPA-LFD was as follows:Upstream and downstream primers(0.42 pmol/μL)2.1μL,probe(0.3 pmol/μL)0.6μL,Rehydration buffer 29.5μL,cDNA template 1μL,ddH_(2)O 12.2μL,MgOAc 2.5μL,total 50μL.And the optimized reaction conditions were amplified for 20 min under 37℃.HP-PRRSV,PRRSV VR2332,NADC30-like,PEDV,TGEV,PRV,PCV-2,CSFV,S.Suis and E.coli were detected simultaneously by this method.The results showed that only HP-PRRSV was detected positively showing strong specificity of this method.The method was used to detect the 10 times series diluted standard plasmids.The results showed that the minimum detection limit of the standard plasmid was 4.05×10^(1)copies perμL,showing good sensitivity.The RPA-LFD method and HP-PRRSV commercial fluorescence detection kit were used to detect 50 clinical samples simultaneously,and the coincidence rate was 100%.This study established for the first time a specific,sensitive,convenient,rapid and on-site RPA-LFD detection method for HP-PRRSV.