title: Development of simple, rapid and sensitive detection assay for grouper nervous necrosis virus using real-time loop-mediated isothermal amplification.
abstract: A quantitative rapid detection method based on loop-mediated isothermal amplification has been developed for red-spotted grouper nervous necrosis virus (RGNNV). The nested polymerase chain reaction (PCR) assay is the mainstream inspection of the brooder in the hatchery. In this study, a real-time loop-mediated isothermal amplification (LAMP) method has been applied for RGNNV detection, known as a high-speed gene amplification procedure. Of the three temperatures (60 °C, 63 °C and 65 °C) attempted, it has been found that 63 °C is giving higher amplification from 11th minute onwards. Sensitivity analysis performed in comparison with real-time polymerase chain reaction, reverse transcriptase PCR and nested RT-PCR using various concentrations of template revealed that real-time LAMP method is efficient in terms of cost and time consumption. Specificity analysis revealed that the method developed is specific to RGNNV, whereas it has sequence cross-match with tiger puffer NNV giving advantage in detecting both the viruses. This method could be much efficient in analysing RGNNV in combination with TPNNV.
authors: Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, Saiki, Oita, Japan.
authors: T
authors: T
authors: Mekata
authors: Aquatic Animal Health Division, National Research Institute of Aquaculture, Fisheries Research Agency, Saiki, Oita, Japan.
authors: J
authors: J
authors: Satoh
authors: Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan.
authors: M
authors: M
authors: Inada
authors: Aquaculture Biotechnology Laboratory, School of Biosciences and Technology, VIT University, Vellore, Tamilnadu, India.
authors: S
authors: S
authors: Dinesh
authors: Aquaculture Biotechnology Laboratory, School of Biosciences and Technology, VIT University, Vellore, Tamilnadu, India.
authors: P
authors: P
authors: Harsha
authors: Faculty of Agriculture, University of Miyazaki, Miyazaki, Japan.
authors: T
authors: T
authors: Itami
authors: Aquaculture Biotechnology Laboratory, School of Biosciences and Technology, VIT University, Vellore, Tamilnadu, India.
authors: R
authors: R
authors: Sudhakaran
keywords: 
  GNNV
  ; RT-LAMP; detection; virus