Establishment Of The Multiplex Detection Technique For White Spot Syndrome Virus (WSSV), Taura Syndrome Virus (TSV) And Hepatopancereatic Parvovirus (HPV)

The thesis includes four chapters.Chapter one is a brief review on recent advances in shrimp viral pathogens, and new techniques for the diagnosis of shrimp virus. The aim of this thesis is also included.Chapter two is about the multiplex dot blotting technique for detecting White Spot Syndrome Virus, Taura Syndrome Virus and Hepatopancreatic Parvovirus. Probes were prepared by labeling the genomic fragments (DNA or cDNA) of different viruses with digoxigenin, biotin and fiuorescein, respectively. After hybridization, the detection of virus genome was performed by conjugating anti-digoxigenin, anti-biotin and anti-fluorescein antibodies, respectively, and then revealed different colors with different combinations of naphthol-phosphate and diazonium salt substrates. The multiplex detection can be used to diagnosis two or three virus in the same system.In chapter three, a rapid and sensitive PCR-ELISA has been developed for detection of WSSV, TSV and HPV. Three biotin-labeled oligonucleotide probes, prepared from three virus genome sequences, respectively, were first immobilized on a streptavidin -coated microtiter plate, and then hybridized with PCR (RT-PCR) products incorporated with digoxingenin-labeled nucleotides during the PCR reactions. The amplicons were detected with alkaline phosphatase-conjugated anti-digoxigenin antibody and the colorimetric substrate p-nitrophenyl phosphate. No cross-reactivity was observed when nucleic acid templates from WSSV, TSV and HPV were tested. Therefore, this sensitive and specific method is promisingly useful for early detection of virus infection in broodstock and viruses carriers. A large number of samples can be analyzed simultaneously.Chapter four is focus on the cloning and expressing of TSV structure protein genes in E. coli. Three major structure protein genes (vp1, vp2 and vp3) are cloned and expressed, and will be used for preparing the monoclonal antibodies.