鲤浮肿病病毒环介导等温扩增(LAMP)检测方法的建立  

Establishment of a loop-mediated isothermal amplification assay for detection of carp edema virus

作　　者：翟立文 刘文枝[2] 许晨 范玉顶[2] 江南[2] 周勇[2] 曾令兵 ZHAI Liwen;LIU Wenzhi;XU Chen;FAN Yuding;JIANG Nan;ZHOU Yong;ZENG Lingbing(National Demonstration Center for Experimental Fisheries Science Education(Shanghai Ocean University),Shanghai 201306,China;Yangtze River Fisheries Research Institute,Chinese Academy of Fishery Sciences,Wuhan 430223,China)

机构地区：[1]水产科学国家级实验教学示范中心(上海海洋大学),上海201306 [2]中国水产科学研究院长江水产研究所,湖北武汉430223

出　　处：《中国水产科学》2019年第5期1004-1013,共10页Journal of Fishery Sciences of China

基　　金：国家大宗淡水鱼产业技术体系专项资金项目(CARS-45-16);中国水产科学研究院基本科研业务费专项(2017HYZD1005);湖北省科技支撑计划项目(2015BBA234)

摘　　要：本研究针对鲤浮肿病病毒(carp edema virus,CEV)基因组核蛋白编码基因P4a的序列,设计2对特异性引物,以克隆构建的重组质粒为标准模板,通过优化反应体系中引物浓度组合、Mg2+浓度、dNTPs浓度、反应温度和扩增时间等参数,建立了CEV环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测方法。结果表明,CEV-LAMP方法的最佳反应温度为62℃,引物浓度组合为引物F3/B3 0.2μmol/L,引物FIP/BIP 1.2μmol/L,Betaine 0.7 mol/L,Mg2+8.0 mmol/L,dNTPs 1.2 mmol/L,反应时间60 min。反应产物经凝胶电泳呈现梯型条带,添加SYBR Green I荧光染料后,呈现明显的绿色阳性反应。CEV-LAMP法灵敏度高,最低检测限为10 copies/μL,较常规PCR法灵敏度高100倍;CEV-LAMP法特异性强,与锦鲤疱疹病毒(KHV)、鲤疱疹病毒Ⅱ型(CyHV-2)、鳜传染性脾肾坏死病毒(ISKNV)及鲤春病毒血症病毒(SVCV)无交叉反应。CEV-LAMP应用于患病鲤样本检测结果准确,简便快速,可为鲤浮肿病的现场诊断与防控提供技术支撑。Common carp(Cyprinus carpio),the earliest farmed fish in the world,is one of the most important and economically significant species in freshwater aquaculture in China.In recent years,carp edema virus(CEV)disease,a newly emerged infectious disease in common carp,has been discovered in many countries,threatening the healthy development of common carp farming.Due to a lack of CEV-sensitive cell lines,detection of CEV relies on methods that use electron microscopy and polymerase chain reaction(PCR);however,these methods are not used universally for clinical diagnosis because they require expensive equipment and a long response time.Therefore,there is an urgent need to establish a simple,accurate,and rapid diagnostic method to detect CEV.Loop-mediated isothermal amplification(LAMP)is a novel amplification technique used to produce DNA copies within 1 h.Furthermore,the results can be judged based on turbidity or by the addition of SYBR Green I to the reaction products and observation of color change within the reaction system.LAMP technology has played an important role in human medicine,animal medicine,and food safety,and has gradually been developed for industrialization and commercialization.In this study,two pairs of specific primers were designed based on the conserved sequence of the gene encoding the CEV nucleus protein,P4a,and a recombinant plasmid was constructed for use as a standard template for amplification.Then,a LAMP detection method for CEV was established after optimizing the reaction conditions.The optimal LAMP reaction system for CEV detection included F3/B3 0.2μmol/L,FIP/BIP 1.2μmol/L,betaine 0.7 mol/L,Mg2+8.0 mmol/L,and dNTPs 1.2 mmol/L and was performed at 62℃for 60 min.The amplified products included a ladder band on agarose electrophoresis and green products,which were observed directly by the addition of SYBR Green I.The sensitivity of the detection method for CEV was 10 copies/μL,which is 100-fold higher than the traditional PCR method.Moreover,CEV-LAMP was specific for detecting CEV wit

关 键 词：鲤浮肿病病毒 核蛋白编码基因P4a 环介导等温扩增 检测 优化 

分 类 号：S941[农业科学—水产养殖]