十足目虹彩病毒(DIV1)环介导等温扩增(LAMP)检测方法的建立及应用  

Establishment and Application of the LAMP Detection Method for Decapod Iridescent Virus 1(DIV1)

作　　者：邹莹 郭晓萌 万晓媛[1] 邱亮[1] 张庆利[1,2] ZOU Ying;GUO Xiaomeng;WAN Xiaoyuan;QIU Liang;ZHANG Qingli(Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Key Laboratory of Maricultural Organism Disease Control,Ministry of Agriculture and Rural Affairs,Laboratory for Marine Fisheries Science and Food Production Processes,Pilot National Laboratory for Marine Science and Technology(Qingdao),Qingdao Key Laboratory of Mariculture Epidemiology and Biosecurity,Qingdao 266071;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306)

机构地区：[1]中国水产科学研究院黄海水产研究所,农业农村部海水养殖病害防治重点实验室,青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室,青岛市海水养殖流行病学与生物安保重点实验室,青岛266071 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《渔业科学进展》2020年第6期156-164,共9页Progress in Fishery Sciences

基　　金：国家重点研发计划项目(2017YFC1404503);中国水产科学研究院基本科研业务费(2020TD39);中国水产科学研究院黄海水产研究所级基本科研业务费项目(20603022020005)共同资助。

摘　　要：本研究以十足目虹彩病毒(Decapod iridescent virus 1,DIV1)主要衣壳蛋白基因为靶序列设计引物,建立了DIV1的环介导等温扩增(Loop-mediated isothermal amplification,LAMP)检测方法,并以pMD18-DIV1质粒标准品为模板对该方法的检测灵敏度、检测特异性等进行了评估。结果显示,此方法最适反应温度为64.4℃,优化后的25μl反应体系中包含2.5μl 10×Isothermal amplification buffer、4.0 mmol/L Mg^2+、1.2 mmol/L dNTPs、6.4 U Bst 2.0 WarmStart■DNA聚合酶、0.8μmol/L EvaGreen■和4.4μl ddH2O。该方法检测灵敏度下限为3.54×10^2拷贝/反应;与虾肝肠胞虫(EHP)、致急性肝胰腺坏死病副溶血弧菌(VpAHPND)、对虾偷死野田村病毒(CMNV)、传染性皮下及造血组织坏死病病毒(IHHNV)、白斑综合征病毒(WSSV)、桃拉综合征病毒(TSV)和黄头病毒(YHV)等主要虾类病原没有交叉反应;具有较好的重复性和稳定性。以GeneFinder■替换EvaGreen■并将其预置于反应管内,结合上述扩增方法可实现对DIV1的现场快速高灵敏检测。本研究建立的DIV1-LAMP实时荧光定量和现场检测方法具有灵敏、特异和快速等特点,为近几年新发虾类病原DIV1的定性、定量以及现场快速检测提供了新的技术选择,有利于对虾养殖业中开展DIV1的监测、预警和防控。The loop-mediated isothermal amplification(LAMP)detection method of Decapod iridescent virus 1(DIV1)was established and optimized based on primers designed from the DIV1 capsid protein gene in present study.Analytic sensitivity of the newly established method was assessed using the pMD18-DIV1 plasmid standard as a template,and the detection specificity was also determined.The results showed that the optimal reaction temperature for the DIV1-LAMP method was 64.4℃,and the optimized 25μl reaction system contained 2.5μl 10×isothermal amplification buffer,4.0 mmol/L Mg^2+,1.2 mmol/L dNTPs;6.4 U Bst 2.0 WarmStart■DNA polymerase and 0.8μmol/L EvaGreen■.The detection limit of the new method was 3.54×10^2 copies/reaction;it did not cause a cross-reaction with major shrimp pathogens such as Enterocytozoon hepatopenaei(EHP),Vibrio causing acute hepatopancreatic necrosis disease(VpAHPND),covert mortality nodavirus(CMNV),infectious hypodermal and hematopoietic necrosis virus(IHHNV),white spot syndrome virus(WSSV),Taura syndrome virus(TSV),or yellow head virus(YHV).The newly developed method showed good repeatability and stability.The on-site,rapid,highly sensitive detection method of DIV1 was established by replacing EvaGreen■with GeneFinder■which was preset in the cap of the reaction tube.The DIV1-LAMP real-time fluorescence and on-site LAMP detection method established in this study is sensitive,specific,and rapid.This novel method will provide new technical options for the qualitative,quantitative,and rapid on-site detection of the emerging DIV1,which will benefit the shrimp farming industry by facilitating the monitoring,early warning,and prevention of DIV1.

关 键 词：十足目虹彩病毒(DIV1) 虾血细胞虹彩病毒(SHIV) 环介导等温扩增(LAMP) 检测方法 

分 类 号：S945.4[农业科学—水产养殖]