病毒性出血性败血症病毒(VHSV)实时荧光环介导等温扩增检测方法的建立  

Establishment of Real-time Fluorescence LAMP Assay for Viral Hemorrhagic Septicemia Virus

作　　者：陈进会[1] 陈文[1] 黄伟[1] 李红梅 石磊 邱杨[1] 刘建丽[1] 叶蕾 唐大运 

机构地区：[1]东莞出入境检验检疫局,广东东莞523072 [2]广州迪澳生物科技有限公司,广东广州510012

出　　处：《中国动物检疫》2013年第5期42-46,共5页China Animal Health Inspection

基　　金：2011年东莞市科技计划资助项目(2011108102047)

摘　　要：根据VHSV的较为保守的glyG基因,设计了三套LAMP引物,筛选出扩增效率最优的第二套引物VHSV-2,引入其环引物以加快其反应。采用ESE-Quant tube scanner(德国QIAGEN公司)恒温实时荧光反应及检测平台,反应温度为63℃,恒温下30min内可得出结果。同时评价VHSV引物的灵敏度和特异性,灵敏度结果显示其检测限可达到4.5pg每反应,与几种重要的病原RNA均无交叉反应。本文建立的检测VHSV的RT-LAMP检测方法简单、易操作,同时具有较高的灵敏度和特异性。本研究建立的实时荧光RT-LAMP检测方法可作为VHSV的快速诊断工具,适合VHSV的现场快速检测和大规模监控。A real-time loop-mediated isothermal amplification （LAMP） assay was developed for detecting viral hemorrhagic septicemia virus （VHSV） . Three sets of LAMP primers were designed based on the VHSV glyG sequence, and the best one （VHSV-2） was chosen to introduce its loop primer to speed up the reaction rate.Specificity and sensitivity of the assay was evaluated using an ESE- Quant tube scanner system.The amplification temperature was 63 ℃. The results demonstrated that the amplification could be finished within 30 min.The real time LAMP assay was able to specifically detect the VHSV without reaction with other fish virus RNA. The detection limit was 4.5 pg per reaction. The results in this study suggested that the real-time LAMP method had great potential as a field molecular tool for diagnosis of VHSV. It could provide an altemative diagnostic method for field detection of VHSV.

关 键 词：病毒性出血性败血症病毒 恒温实时荧光 环介导等温扩增 

分 类 号：S941.413[农业科学—水产养殖]