传染性造血器官坏死病毒(IHNV)实时荧光环介导等温扩增快速检测方法的建立  

Establishment of a real-time fluorescence LAMP assay for detection of infectious hematopoietic necrosis virus

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

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12期53-58,共6页China Animal Health Inspection

基　　金：2011年东莞市科技计划资助项目(2011108102047)

摘　　要：建立了一套基于环介导等温扩增技术(LAMP)的实时荧光检测方法,用于IHNV的检测。根据IHNV的保守基因glyG,设计合成3套LAMP引物,LAMP反应在63℃下进行,引入环引物后,第3套LAMP引物显示出良好的扩增效率。以ESE-Quant tube scanner为恒温反应及检测平台,对IHNV进行实时荧光检测,反应在40 min内可得出结果。本研究建立的IHNV实时荧光LAMP法检测限达到3.6 pg。特异性试验表明仅IHNV发生特异性扩增,而SVCV、VHSV、HRV、PFRV以及IPNV均不发生反应。所建立的恒温实时荧光检测法操作简单、反应迅速,同时具有较高的灵敏度和特异性。引入的ESE-Quant tube scanner平台设备要求低,同时使扩增过程数字化和自动化,适合IHNV的现场检测和大规模疫病的监控。A highly specific and sensitive real-time loop-mediated isothermal amplification （LAMP） assay was developed for detection of infectious hematopoietic necrosis virus （IHNV） . Three sets of LAMP primers were designed based on the IHNV restrictive gene glyG. After the introduction of loop primers, the third set primers showed better amplification efficiency at 63 ℃.The ESE-Quant tube scanner system was used in the assay to evaluate the specific amplification. Multiple parallel tests demonstrated that the amplification could be finished within 40 min at 63 ~ C. The developed real-time LAMP assay was capable to detect the IHNV with no cross action with SVCV, VHSV, HRV, PFRV and IPNV. The detection limit of the assay was 3.6pg. The ESE-Quant tube scanner introduced in this study made the amplification processes digitalized and automated. The result showed that the real-time LAMP assay was suitable for field detection of IHNV and convenient handling of mass samples.

关 键 词：传染性造血器官坏死病毒 环介导等温扩增 恒温实时荧光 ESE Quanttube SCANNER 

分 类 号：S941.414[农业科学—水产养殖]