Diagnosis of nervous necrosis virus in orange-spotted grouper,Epinephelus coioides, by a rapid and convenient RT-PCR method

Viral nervous necrosis(VNN)causes high mortality in marine fish,especially in the grouper,worldwide and in China.Since there is no effective vaccine or drug to deal with VNN,early detection and prevention is important to block its outbreak.In this study,a reverse transcription-polymerase chain reaction(RT-PCR)was developed for the rapid,convenient,and sensitive detection of the VNN pathogen,nervous necrosis virus(NNV),in the grouper.The whole process was completed within 3.5 h from the RNA extraction to PCR product visualization.The detection limit of this method was 200 copies of NNV RNA standard,which corresponded to 200 copies of virus particles.This RT-PCR method was specific to the NNV detection with no cross-reactivity to other fish viral disease pathogens,such as infectious pancreatic necrosis virus(IPNV),infectious hematopoietic necrosis virus(IHNV),spring viraemia of carp virus(SVCV),epizootic haematopoietic necrosis virus(EHNV),and large yellow croaker iridovirus(LYCIV).With this method,the orange-spotted grouper(Epinephelus coioides)fry from hatcheries with or without incidence of the VNN epidemic in Fujian Province were detected.The results showed that all or 93%of the fry from the two hatcheries with incidence of the epidemic were diagnosed as positive,while 40%or 25%of fry from the two hatcheries without the VNN epidemic were also detected as NNV positive,indicating that this RT-PCR method can be used for rapid,sensitive detection of NNV infection and applied in the VNN epidemic alert.