重组酶聚合酶扩增技术结合侧向流动试纸条快速检测锦鲤疱疹病毒  

RAPID VISUAL DETECTION OF KHV BY RECOMBINASE POLYMERASE AMPLIFICATION(RPA)COMBINED WITH A LATERAL FLOW DIPSTICK(LFD)

作　　者：楚馨 冯梓钊 姜有声[1,2,3] 王浩[1,2,3] 吕利群[1,2,3] 许丹[1,2,3] CHU Xin;FENG Zi-Zhao;JIANG You-Sheng;WANG Hao;LÜLi-Qun;XU Dan(National Pathogen Collection Center for Aquatic Animals,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Freshwater Aquatic Genetic Resources,Ministry of Agriculture and Rural Affairs,Shanghai Ocean University,Shanghai 201306,China;National Demonstration Center for Experimental Fisheries Science Education,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学国家水生动物病原库,上海201306 [2]上海海洋大学农业农村部淡水水产种质资源重点实验室,上海201306 [3]上海海洋大学水产科学国家级实验教学示范中心,上海201306

出　　处：《水生生物学报》2023年第6期866-873,共8页Acta Hydrobiologica Sinica

基　　金：国家重点研发计划“蓝色粮仓科技创新”重点专项子课题(2019YFD0900101);上海市自然科学基金面上基金项目(22ZR1427200);上海海洋大学未来生物技术学院基金资助。

摘　　要：为建立一种快速、灵敏并适用于临床检测Ⅲ型鲤疱疹病毒(CyHV-3,KHV)的方法,实验根据KHV SphI-5基因的保守序列片段设计引物及探针,采用重组酶聚合酶扩增技术结合侧流层析试纸条(RPA-LFD)检测KHV。重组酶聚合酶扩增技术(RPA)具恒温扩增及高灵敏度特点,简化了设备要求的同时又能做到高效检测病毒,再结合侧向流动试纸条(LFD)将RPA结果快速地可视化,提高了该疾病的检测效率。结果表明,在38℃的最适反应温度下,采用RPA-AGE技术仅需10min便可检测出病原的目标片段,结合LFD方法仅需5min便可将RPA结果通过试纸条可视化呈现。研究研发的KHV RPA-LFD检测方法简单、快捷,可为实验条件有限的养殖场快速诊断需求提供技术支撑。Cyprinus carpio and Cyprinus carpio kio are important aquaculture varieties in China.Highly pathogenic and infectious of Cyprinid herpesvirus 3(CyHV-3,KHV)has caused great losses in the culture of Cyprinus carpio and Cyprinus carpio kio.Therefore,the rapid and convenient detection technology is desperately needed to be used for nonlaboratory and fast detection.Recombinase polymerase amplification(RPA)is such an Isothermal amplification technology which can amplify DNA within 30min under low reaction temperature.Then we combined RPA with lateral flow dipstick(LFD)and established a rapid and sensitive method which are suitable for field clinical detection of KHV.In this study,primers and probe sequences were designed according to the conserved fragment of SphI-5 gene of KHV.The experimental results show that the RPA technique can detect target fragment by agarose gel electrophoresis within 30min at the optimal temperature of 38℃and the RPA results can be visualized in only 5min in combination with the LFD and the entire RPA-LFD assay takes 50min which are a lot faster than PCR method.The method established in this experiment for the detection of KHV by RPA-LFD is so simple and sensitivity that can be useful for rapid diagnosis in aquaculture with limited resources.

关 键 词：快速检测 重组酶聚合酶扩增技术 侧向流动试纸条 锦鲤疱疹病毒 

分 类 号：Q-33[生物学] S941.4[农业科学—水产养殖]