Study And Application Of Polymerase Chain Reaction And Gene Chip In The Quarantine And Surveillance Of Aquatic Viral Diseases

The twelve aquatic animal diseases in this study have caused great economic loss in aquaculture throughout the world, which caused by viral pathogens, including Infectious haematopoietic necrosis virus (IHNV), Spring viraemia of carp virus (SVCV), Epizootic haematopoietic necrosis virus (EHNV), Viral Haemorrhagic Septicaemia virus (VHSV), Infectious pancreatic necrosis virus (IPNV), Grass carp haemorrhagic virus (GCHV), Red sea-bream Irodivirus (RSIV), Viral nervous necrosis virus (VNNV), Koi herpesvirus (KHV), Taura syndrome virus (TSV), Infectious hypedermal and haematopoietic necrosis virus (IHHNV) and White spot syndrome virus (WSSV). These viral diseases were prevalent in some countries and spread to other countries along with the commercial activities.These diseased were listed in the important aquatic diseases by World Organisation for Animal Health (OIE), Network of Aquaculture Centres in Asia-Pacific (NACA) and the aquaculture department of each country and are quarantined as in the trades of aquatic animals. Polymerase chain reaction methods (PCR) and Reverse-trancription polymerase chain reaction methods were studied and practised in the quarantine and surveillance of these 12 diseased in aquatic animls cultured, imported or exported. The sequences of amplified products were analyzed with bioinformatical methods. The specific nucleotide fragments were selected, amplified and cloned. They were spotted on glassslides as probes and the gene chip were prepared, with which the 12 viral pathogens were detected from two smples. The main results in this study were described as follow.1. RT-PCR and nested-PCR for IHNV and sequence analysisRT-PCR and nested-PCR for detection of IHNV were developed and the nucleotide fragments of 786 bp and 323 bp coding part of the viral nucleoprotein were amplified. IHNV were detected positively from cultured turbot and rainbow trout with significant clinical signs and high mortalities. The similarity of nucleotide was 98.1% between the amplified products of samples and IHNV reference strain, with the similarity of 97% and 99% separately between the induced peptides. The samples from imported Mississippi paddlefish Polyodon spathala was found to be infected by IHNV and the similarities of nucleotide and peptide were 92.5% and 93% separately compared with reference strain of IHNV.2. RT-PCR and nested-PCR for SVCV and sequence analysisRT-PCR and nested-PCR for detection of SVCV were developed and the nucleotide fragment of 714 bp and 606 bp coding part of the viral nucleoprotein were amplified. Viral strains were isolated from culured koi and common carp and SVCV specific nucleotides were amplified, with the similities of nucleotide 98% between the two samples and 88% with otherSVCV strains. The two strains had closer relationship with the strains of SVCV la sub-genogroup based on phylogenetic analysis.3. PCR for EHNV and sequence analysisPCR for detection of EHNV were developed and the nucleotide fragment of 410 bp coding part of the viral main capsid protein (MCP) was amplified. The specific nucleotide fragment was amplified from the virus isolated from diseased turtle with the clinical sign of red neck. High homology was found beteen the strain and other ranavirus and it was suggested that the strain belonged to ranavirus.4. RT-PCR and nested-PCR for VHSVRT-PCR and nested-PCR for detection of VHSV were developed and the nucleotide fragments of 1131 bp and 811 bp coding part of the viral nucleoprotein were amplified. None of all the samples from cultured fish or imported fish were found to be infected by VHSV.5. RT-PCR for IPNV and sequence analysisRT-PCR for detection of GCHV was developed and the nucleotide fragment of 304 bp coding part of the RNA-dependant RNA polymerase of segment B was amplified. The specific nucleotide was detected from the viral strain isolated from diseased larvae of rainbow trout, with the similarity of nucleotide 100% with the IPNV strain Sp.6. RT-PCR for GCHVRT-PCR for detection of GCHV was developed and the nucleotide frag...