The Establishment And Application Of On-site And Rapid Detection Methods For Red-spotted Grouper Nervous Necrosis Virus

Nervous necrosis virus( NNV) is a major pathogen of viral nervous necrosis disease in a variety of marine aquaculture animals. It was susceptible to juvenile fish and larva fish and with high mortality after infection. The disease brought huge economic losses to aquaculture. However, there were no effective drugs and vaccines for the disease, so the development of a simple, high sensitivity and specificity detection method for NNV is very important.In this study, the primers specifically targeting RGNNV were designed and cross-priming isothermal amplification coupled with lateral flow dipstick(CPA-LFD) system was established. We optimized magnesium ion concentration, d NTPs concentration, reaction temperature in the reaction system. Optimized reaction system was 25mL, including 5 mmol/L Mg CI2, 1.2 mol/L Betaine, 1.2 mmol/L d NTPs, 1.6μmol/L primer 1a, 0.8 μmol/L primer 2a/3a, 0.2 μmol/L primer 4 s/5 a, 10 × Thermo Pol Buffer 2.5μL, Warmstart Bst DNA polymerase 8 U, M-MLV reverse transcriptase 28 U and appropriate amount of RNA template. The reaction system was settled in a metal water bath and incubated at 69 oC for 60 min. The reaction was terminated by incubation at 80 oC for 5 min. The product amplified by CPA was detected through visualization with lateral flow dipstick(LFD). The result showed that the sensitivity of this method was 101 copies/μL, similar with real-time RT-PCR and 10 times higher than conventional RT-PCR.We proved that CPA-LFD method was highly specific for many kinds of fish pathogens, such as Viral haemorrhagic septicaemia virus(VHSV), Infectious pancreatic necrosis virus(IPNV),turbot reddish body iridovirus(TRBIV), Lymphocystis disease virus(LCDV), vibrio anguillarum, Vibrio harveyi and so on. We used this method and LAMP kit to test actual samples and contrasted the two methods. CPA-LFD method appeared to be more accurate and easier to read. This study established a simple, rapid, high sensitivity and specificity CPA-LFD method to detect red-spotted grouper nervous necrosis virus, can be widely used in the on-site rapid detection of disease.This also study produced and characterized a nanoprobe(Ag-NNV) for detecting fish nervous necrosis virus. The morphology and spectroscic properties of Ag-NNV nanoprobe were evaluated by transmission electron microscopy, automatic microplate reader and surface-enhanced Raman microscope. The results showed that the Ag-NNV nanoprobe was sphere-shaped with average diameter of 90 nm and dispersed well in the solution. The number of hairpin probe attached on a silver nanoparticle was estimated to be about 100 and the coverage density was 0.56 pmol/cm2. With easy preparation, the Ag-NNV nanoprobe can be used for quick detection of fish nervous necrosis virus by surface enhanced Raman scattering.