Detection Of Vibrio Alginolyticus And Its Virulence-related Genes

The genus Vibrio is a group of gram-negative bacteria which widely distributes in estuarineand marine habitats. Some of them are pathogenic to marine animals such as shrimps, shellfish,and fish. Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae have been recognized asthe important foodborne pathogens causing human diseases. Remarkably, Vibrio alginolyticushas been reported to be human pathogen in1979which caused endophthalmitis, otitis media andfood poisoning. However, recent attentions were put on its hazards to aquaculture animalrecently, while it seems to be easy to overlook the clinical detection and related food riskassessment in human beings. This study focused on development of high-throughput PCRtechniques to differentiate Vibrio species, particularly to detect Vibrio alginolyticus and itsvirulence-related genes. Reuslts were summerized as the following:1, Species-specific PCR primers were designed based on gyrB gene for Vibrio alginolyticus,Collagenase gene for Vibrio parahaemolyticus, vvhA gene for Vibrio vulnificus and ompW genefor Vibrio cholerae.16S rRNA gene of bacteria as an internal amplification control (IAC)primers was used to indicate false-negative results. Multiple PCR method wass developed afteroptimization reaction condition. The detection limit of this multiplex PCR was10CFU/mL for V.alginolyticus, V. parahaemolyticus and V. vulnificus and105CFU/mL for V. cholerae in mixedsample condition. The multiplex PCR was then applied to identify63suspicious Vibrio strainsisolated from21samples. The PCR results were consistent with physiological and biochemicaltests.2, Two sets of multiplex PCR systems were developed to detect11main virulence-relatedgenes of Vibrio spp., containing toxR, Collagenase, toxS, trh, tdh, tlh, UreR, FlaA, ompW, AspAand fur gene. This method was used to investigate the virulence-related gene distribution ofenvironmental isolated V. alginolyticus and V. parahaemolyticus isolated from the environment.The results showed that these virulence-related genes distribute widely in these Vibrio strains.3, A multiplex tandem PCR method was developed which can not only simultaneouslydetect V. alginolyticus, V. parahaemolyticus, V. vulnificus and V. cholerae, but also can detecteight virulence-related genes and16S rRNA gene as a reference gene used for relativequantification. The multiplex tandem PCR method was confirmed to be rapid, high-throughput, sensitive, and specific in diagnosis and detecting the gene expression levels.