重组酶等温扩增技术快速检测对虾中的白斑综合征病毒

Rapid detection of white spot syndrome virus in Penaeus orientalis by isothermal amplification of recombinant enzyme

中文关键词:  白斑综合征病毒  重组酶聚合酶扩增  重组酶介导等温扩增  现场快速检测

英文关键词:Penaeus orientalis white spot syndrome virus  recombinase polymerase amplification  recombinase-aided amplification  on-site rapid detection

中文摘要:

      目的 针对对虾中白斑综合征病毒(white spot syndrome virus, WSSV), 分别建立基于国外专利技术的重组酶聚合酶扩增(recombinase polymerase amplification, RPA)方法, 基于国内专利技术的重组酶介导等温扩增(Recombinase-aided amplification, RAA)方法, 并对两种检测方法进行比较。方法 应用重组酶等温扩增技术在恒温(39℃)条件下完成扩增反应, 调整反应体系中模板DNA和探针浓度, 优化WSSV病毒RPA和RAA两种方法的反应体系, 建立了可靠稳定的等温扩增RPA和RAA快速检测方法, 比较了两种方法的特异性、灵敏度、扩增效率和稳定性。结果 所建立的荧光RPA和RAA方法在恒温39℃的条件下特异性检测WSSV, 检测构建质粒DNA的灵敏度可达到1.0×106 copies/μL, 检测实际感染阳性样品的灵敏度可达到1.31×101 pg/μL; RPA方法的扩增线性曲线拟合度r2=0.9970, 扩增效率为98.50%; RAA方法的扩增线性曲线拟合度r2=0.9910, 扩增效率为98.50%。结论 所建立的基于重组酶等温扩增技术的RPA和RAA方法, 特异性好, 对于质粒和阳性样品检测灵敏度一致, 扩增效率一致, 具有很好稳定性, 适用于水产品养殖及生产加工和通关口岸进口虾类产品中WSSV病毒的现场快速筛查防控。

英文摘要:

      Objective To establish a recombinase polymerase amplification (RPA) method based on foreign patented technology and a recombinase-mediated isothermal amplification (RAA) based on domestic patented technology for Penaeus orientalis white spot syndrome virus (WSSV) in shrimp, and compare the two determination methods. Methods The advantages of using recombinase isothermal amplification technology to complete the amplification reaction at a constant temperature (39℃), adjusting the concentration of template DNA and probe in the reaction system, optimizing the reaction system of the 2 methods of WSSV virus RPA and RAA, a reliable and stable isothermal amplification RPA and RAA rapid detection method was established, and the specificity, sensitivity, amplification efficiency and stability of the 2 methods were compared. Results The established fluorescent RPA and RAA methods specifically were used to detect WSSV at a constant temperature of 39℃, the sensitivity for detecting the constructed plasmid DNA could reach 1.0×106 copies/μL, and the sensitivity for detecting the actual infection positive samples could reach 1.31×101 pg/μL; the fitting degree of the amplification linear curve of the RPA method was r2=0.9970, and the amplification efficiency was 98.50%; the fitting degree of the amplification linear curve of the RAA method was r2=0.9910, and the amplification efficiency was 98.50%. Conclusion The established RPA and RAA methods based on recombinase isothermal amplification technology have good specificity, consistent detection sensitivity for plasmids and positive samples, consistent amplification efficiency, and good stability. It is suitable for on-site rapid screening and prevention of WSSV virus in shrimp products imported from aquatic product breeding, production and processing and customs clearance ports.