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

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.