Development And Application Of Detection Methods For Detection Of VHSV

Viral haemorrhagic septicaemia is caused by a slug virus,can cause serious infections diseases such as a variety of fish,lead to high mortality rate.The pathogen is Viral haemorrhagic septicaemia virus. Initially popular in North America and Europe. In recent years, with the increase of import trade of aquatic animals, viral hemorrhagic septicemia virus has been introduced into our country and is popular in some areas, caused serious economic losses. At present, the methods used for detection of VHSV has achieved accurate diagnosis,but high cost and time-consuming. Therefore, this thesis attempts to explore more rapid, accurate, high flux integrated detection means, for monitoring and prevention the viral hemorrhagic septicemia virus.In this study,using a purified viral hemorrhagic septicemia virus strain stored at-80℃,culture proliferation in EPC cell. recovery the cell that appeared typical cytopathic. Determination of physical and chemical testing the virus suspension., using a pair of of VHSV generic type-specific primers amplified a811bp fragment, ligated to pMD18-T vector to construct recombinant plasmid and sequenced. Compare the sequencing result with the nucleotide sequence published on GENBANK.Results show that the amplified fragment have homologous to rate to99.2%with gene sequence (FN665788.1). Then artificial infection the healthy Zebrafish dorsal by injected the VHSV virus. Detection of the Zebrafish that appeared typical symptoms. Design and the establishment of four kinds of detection methods for diseased fish samples infected by VHSV, namely:.reverse transcriptase polymerase chain reaction (RT-PCR), PCR-DHPLC, Real-time PCR,Fluorescent reverse transcriptase loop-mediated isothermal amplification.Reverse transcriptase polymerase chain reaction (RT-PCR):According to highly conserved regions of nucleoprontein gene of VHSV,design a couple specific primers, use of the optimal reaction system and reaction conditions to amplify the nucleic acid extracted from diseased fish samples infected by VHSV. A gene of811bp fragment was amplified.PCR-DHPLC:A rapid method of detecting the viral haemorrhagic septicaemia(VHSV)was established by using denaturing high performance liquid chromatography (DHPLC) combined with PCR. Designed one pair of primer according to highly conserved regions of nucleoprontein gene of VHSV. The lower limit of detection for PCR-DHPLC was3pg. The results indicated that PCR-DHPLC was a rapid and sensitive assay in detection of VHSV.Real-time PCR:Using the sequence of PCR amplification product results, enter the NCBI BLAST to carry on the sequence homologous comparison. A couple specific primers and taqman MGB probe were designed from highly conserved regions of nucleoprontein gene of VHSV,.Optimize the reaction system and conditions, The lower limit of detection for PCR-DHPLC was confirmed to3pg. Results show that the method is a specific, sensitive and high automation for VHSV detection.Fluorescent reverse transcriptase loop-mediated isothermal amplification:Using GenieⅡ real-time fluorescence isothermal amplification detection system, establish fluorescent real-time reverse transcription loop-mediated isothermal amplification mediated detection methods. According to highly conserved regions of nucleoprontein gene of VHSV,design of three specific primers, add the dsDNA mixed dye, monitoring the nucleic acid amplification process in real-time fluorescent. This method has high specificity, and could not amplify other slug virus of fish. The detection limit of this assay was20fg VHSV RNA. The experimental results show that this detect method is a kind of simple operation, with the characteristic of rapidity and efficiency, high specificity and sensitivity.