七带石斑鱼神经坏死病毒的RT-LAMP检测方法  

Establishment and Application of Loop-Mediated Isothermal Amplification for Fast Detection of Sevenband Grouper Nervous Necrosis Virous

作　　者：刘滨[1,2] 刘新富[1,2] 曾霖[3] 孟振[1,2] 刘江春 

机构地区：[1]中国水产科学研究院黄海水产研究所,山东青岛266071 [2]青岛市鱼类种子工程与生物技术重点实验室,山东青岛266071 [3]浙江海洋学院,浙江舟山316000 [4]莱州明波水产有限公司,山东莱州264010

出　　处：《天津农业科学》2016年第7期23-28,共6页Tianjin Agricultural Sciences

基　　金：鳌山科技创新计划项目(2015ASKJ02-03-01);中央级公益性科研院所基本科研业务费专项资金学科发展储备项目(20603022013007);"863"计划资助项目(2012AA10A413-2)

摘　　要：报道了一种可以快速、灵敏地检测七带石斑鱼神经坏死病毒(NNV)的逆转录环介导等温扩增方法(RT-LAMP)。该方法参考赤点石斑鱼NNV病毒的主衣壳蛋白(MCP)基因的保守序列,设计1对引物克隆出七带石斑鱼NNV的基因序列,利用Primer Explorer V4软件设计了3对针对所克隆NNV基因序列的特异性引物,建立了RT-LAMP的反应体系,并分别对反应系统的引物浓度、反应温度和时间进行了优化,形成了七带石斑鱼NNV病毒RT-LAMP检测技术。实践应用结果表明,在63℃的最佳反应温度下,采用RT-LAMP检测技术经过45 min就能完成一次检测,准确率达到100%。本研究建立的RT-LAMP检测NNV技术设备要求简单,为七带石斑鱼等石斑鱼无NNV受精卵和仔稚鱼的生产现场检测和筛选提供了一种方便、灵敏的检测方法。A rapid and sensitive technique for detecting of Nervous Necrosis Virus in seven-band grouper was developedthrough reverse transcriptase loop-mediated isothermal amplification （RT-LAMP） method. A set of six specific primers were designed based on MCP gene of Red-spotted Grouper Nervous Necrosis Virus （RGNNV） using Primer Explorer V3 software. The parameters of primer concentration, reaction time and temperature were optimized.The RT-LAMP method established in this article was applied to detect nervous necrosis virus of seven-band grouper larvae in the fish farming field, the results showed that the sensitivity of RT-LAMP method was consistent with the nested RT-PCR method, as well as the detection result of RT-LAMP method could be easily determi- nate,andthe detection time was short, which can be completed within 2 hours. The RT-LAMP assay developed in this article wasvery suitable for rapid diagnosis of early NNV infection of seven-band grouper larvae in fish farming field.

关 键 词：七带石斑鱼 神经坏死病毒 检测 RT-LAMP 

分 类 号：S941[农业科学—水产养殖]