| Vibrio parahaemolyticus (VP) is a halophilic gram-negative bacterium disseminated in marine and estuarine environments worldwide,especially in fishes, shellfish and other seafood products. It is one of the most important pathogens causing seafood-borne gastroenteritis associated with the consumption of raw or partially cooked seafood. Additionally, V. parahaemolyticus has shown great harm to the development of the aquaculture. Molecular distribution characteristics of Vibrio parahaemolyticus and rapid detection of this organism are of great importance.The major contributions of the research were described as follows:1. Establishment and evaluation of multiplex PCR for the detection of Vibrio parahaemolyticusOne new species-specific target gene (aadS), encoding D-amino acid dehydrogenase, small subunit of Vibrio parahaemolyticus was obtained by bioinformatics analysis, and primers were designed for target gene PCR detection. Because its pathogenicity was strongly correlated to two well-characterized hemolysins, the thermostable direct hemolysin (tdh) and the tdh-related hemolysin (trh), multiplex PCR were established and evaluated based on the target genes aadS, tdhand trh. The detection specificity of this method was 100%, with the sensitivity for pure genomic DNA at 20 fg per reaction. Application of the method was involved in detecting 55 naturally contaminated seafood samples (shrimp, fish, and oysters) compared with conventional assays (microbiological and biochemical tests). It turned out that only one sample was identified negative by the multiplex PCR assay but positive by the conventional method. Therefore, the multiplex PCR method established in this study is accurate, sensitive, specific and applicable for the detection of V. parahaemolyticus. It can be a supplement of conventional method to be applied to seafood detection and clinical analysis to increase detection efficiency and determine pathogenicity of the isolates.2. Development of a single base extension-tag microarray for the detection of pathogenic Vibrio species in seafoodA single base extension-tag array on glass slides (SBE-TAGS) microarray based on the multiplex PCR was established to detect the seven leading seafood-borne pathogens, including Vibrio parahaemolyticus, V. cholerae, V. vulnificus, V. mimicus, V. alginolyticus, V. anguillarum, and V. harveyi. Three multiplex PCR systems were developed to specifically target the following species with individual gene markers, which are aadS, tdh, and trh for V. parahaemolyticus; col, toxR, and vvh for V. alginolyticus, V. mimicus and V. vulnificus; and empA, vhh1, and tcpA for V. anguillarum, V. harveyi and V. cholera respectively. The purified PCR products were used as template DNA for single base extension-tag reactions, labeled with Cy3 fluorescent dye and hybridized to DNA microarrays. The detection specificity of this microarray method was 100%, with the sensitivity for pure genomic DNA at 200 fg to 2 pg per reaction. Application of the DNA microarray methodology to 55 naturally contaminated seafood samples (shrimp, fish, and oysters) revealed the presence of V. parahaemolyticus at 50.9% and V. alginolyticus at 32.7%, of all samples, there were 18 samples which contained V. parahaemolyticus and V. alginolyticus. This corresponds with traditional assays (microbiological and biochemical tests) except one sample which was identified as negative in V. parahaemolyticus by the microarray assay but as positive by the conventional method. Therefore, a combination of multiplex PCR with DNA microarray hybridization based on SBE-TAGS ensures rapid and accurate detection of pathogenic Vibrio species in seafood, thereby providing safer seafood products for consumers at a low financial burden to the aquaculture industry.3. Distribution characteristics and diversity of V. parahaemolyticus in seafoodThere were 368 seafood samples including 215 from Shanghai area and 153 from the other countries, which were taken from the wholesale markets, retail markets and the supermarkets in Shanghai area between April 2006 and June 2009. V. parahaemolyticus was isolated by combining the method of national standard with PCR. There were 47 V. parahaemolyticus strains which were isolated from Shanghi area samples, and the isolation rate was 21%. There were 56 V. parahaemolyticus strains which were isolated from the samples which originly came from the other countries, and isolation rate was 37%. The V. parahaemolyticus isolates from Shanghai area with the hemolysin genes were low, and only two isolates (2/47,4%) carried the the thermostable direct hemolysin (tdh) without the tdh-related hemolysin (trh), and the urease experiment was negative for all of the isolates from Shanghai area. The isolates from the other countries with the hemolysin genes were also low, and there were only four isolates (4/56,7%) with tdh gene and only one isolate from above four isolates carried both tdh gene and trh gene with positive result for the urease experiment. Serotypes of all the isolates were measured by agglutination using a commercial kit of O-Antigen according to the manufacturer’s specifications, fourteen isolates from Shanghai area consisted of O1 (14/47, 30%), ten isolates consisted of O2 (10/47, 22%). On the other hand, thirteen isolates from the other countries consisted of O10 (14/56, 23%), and ten isolates consisted of O2 (10/56, 18%). All 103 isolates from the seafood samples and 5 major clinial isolates were typed by enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and there were 44 patterns possessed by applying the cluster analysis of genetic profiles from ERIC-PCR typing to all strains based on the similarity value of more than 70% using BioNumerics version 5.0 by the unweighted-pair group method using average linkages. Genetic profiles of cluster analysis from ERIC-PCR clearly showed that four isolates (SJTUF30004, SJTUF30013, SJTUF30025 and SJTUF30037) from Shanghai area and six isolates (SJTUF30083, SJTUF30086, SJTUF30089 ,SJTUF30090,SJTUF30094 and SJTUF30102) from the other countries that originated from seafood were clustered together which demonstrated that they were potentially pathogenic.4. Genetyping and the relatedness among the V. parahaemolyticus isolatesThe outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlight the need for strain characterization and differentiation of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China between 2006 and 2008. The isolates were characterized using four various molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly by the unweighted-pair group method using average linkages showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpson’s Index of Diversity based on the similarity value more than 0.76. The discriminatory index of ERIC–PCR typing was maximal (D﹦0.942), while that of sequence analysis of the gyrB gene was minimal (D﹦0.702), and the discriminatory index of PFGE and ribotyping was 0.907 and 0.756. Therefore, ribotyping exhibited a discriminatory ability much lower than PFGE and ERIC-PCR, and may have limited application in V. parahaemolyticus subtyping and outbreak investigation. In conclusion, our results suggest that ERIC-PCR used in conjunction with gyrB sequence analysis would provide a reliable and accurate typing strategy for V. parahaemolyticus. This combination of techniques exhibits an excellent discrimination (D=0.966) and can be used as a rapid means of comparing V. parahaemolyticus isolates for epidemiological investigations. |
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