Abstract:The reverse transcription loop-mediated isothermal amplification (RT-LAMP) method is a sensitive nucleic acid diagnostic method that can amplify rapidly a target template; it can be applied for the diagnosis of viral disease in grouper aquaculture. In this study, two outer and two inner primers were designed from nervous necrosis virus (NNV) coat protein gene sequence. The reaction temperature and time for the detection of NNV were optimized at 65 °C for 60 min. The detection limit of RT-LAMP is 10−6 NNV-RNA from infected groupers, and more sensitive than the one-step RT-PCR and nested RT-PCR. The combination of RNA rapid extraction and RT-LAMP, the process can be completed within 2 h. Thus, the RT-LMAP is a rapid, sensitive, specific and efficient method for detection of NNV in groupers.