Study On The Detection Methods Of Viruses In Fish Viral Infectious Diseases

Spring Viremia of Carp Virus(SVCV)is a rhabdovirus that occurs in the spring mainly and can cause large-scale outbreaks of Cyprinus carpulans(SVC)in fry or adult fish.Infectious Pancreatic Necrosis Virus(IPNV)is a double-fragment RNA virus that causes infectious pancreatic necrosis(IPN).It is a worldwide disease that threatens Rainbow trout,Atlantic salmon,Turbot Atlantic Cod and so on,more than ten kinds of common breeding fry and juvenile fish.Both viruses are infectious viruses that not only can seriously compromise fisheries even cause devastating blows.In this study,FHM cells were first used to amplify the purified Cyprinus viresensum virus and infectious pancreatic necrosis virus,and showed typical CPE.Then,the two viruses were amplified by the designed primers to get the fragments were 714bp for SVCV and 224bp for IPNV.The two of genes connected to pMD18-T carrier to construct recombinant plasmid and sequenced.Sequencings were analyzed by NCBI online nucleotide blast.the results showed the similarity of SVCV and the glycoproteinsequence was as highas 99%,IPNV nucleotide homology was 79.9%-98.2%.The healthy carp fry were infected by SVCV and IPNV,the infected ways were feed and injected into the venom.Three kinds of molecular biology detection methods were used to detect the carp fry that occurred typical symptoms,they were RT-PCR,fluorescence quantitative RT-PCR and in situ hybridization(ISH).RT-PCR:Two pairs of specific primers were used to detect the infected samples.A fragment of 714bp was obtained that proved the SVCV infection was successfully but the IPNV was not amplified,it showed that the IPNV infection failed.Fluorescence quantitative RT-PCR:Develop a quantitative RT-PCRassay using a TaqMan probe to detect and quantify SVCV and IPNV.The primers and probes were designed according to the glycoprotein genes.In specificityexperiment,the control group of VHSV,IHNV and EHNV were not amplified that demonstrated the designed primers specific to the SVCV and IPNV.Theassay had a detection limit less than 10 copies showed higher sensitivity.The four repeat amplification curves for each dilution are basically consistent and are parallel to each other,indicating that this method has good repeatability.Regression analysis showed a significant correlation.The standard curve of SVCV was CP =-3.331gX +32.00,amplification efficiency was 1.882,error is 0.0297,and IPNV standard curve was CP =-3.21 lgX +30.68,efficiency was 1.910,the error was 0.0182.According to the standard curve,it can count the concentration of infected carp fiy,the SVCV were 104.14and 103 21.The infection IPNV samples had no amplification curve,which was consistent with the RT-PCR results.In Situ Hybridization(ISH):In this study,SVCV and IPNV were amplified with specific primers designed by glycoprotein genes then purified DNA,two nucleic acid probes labeled with digoxigenin2dUTP were prepared by random priming named D1G-SVCV and DIG-IPNV.The amount of digoxigenin-labeled DNA was calculated by comparing the intensity difference of the point produced by the control labeling reaction.The comparing results were that the probes diluted to 25 ng/mL with the hybridization solution in Southern blot,and diluted to 1 ng/μL in the tissue section hybridization experiment.After the specific experiment the two probes were only able to label the corresponding virus,and the control group did not cross-react showing good specificity of the probe.In the artificial infection experiment,the infected carp fry of SVCV had typical lesion features:deep body color,abdominal swelling,the skin has a clear bleeding,but IPNV had no morbid characteristic.Using the DIG-SVCV to detect artificial carp fry,the results showed that a small amount of virus was detected in the viscera,eye,brains and muscle sections.This experiment established the in situ hybridizationdetection method of SVCV that were high specific,easy to operate,and helpfiul to get the location of virus and study pathogenesis.