Genotyping And DNA Microarray For Detection Of The Conventional Pathogenic Vibrio Spp.

Vibrio parahaemolyticus, Vibrio cholerae, Vibrio alginolyticus, Vibrio mimicus and other pathogenic Vibrio spp. are important foodborne pathogen, widely inhabit in seawater, marine sediment, fish, prawns, crabs, shellfish and other seafood. Once eating the food contaminated by these bacteria, people will get diarrhea, vomiting,?fever and other typical gastroenteritis reaction. What's more serious is that critically ill patients may be dehydration, shock and even death. Since 1998, reports indicated that the scale of food poisoning and exposure induced by Vibrio parahaemolyticus showing a rising trend, had exceeded salmonella, ranking the first of bacterial food poisoning of China. So, developing a set of high throughput, rapid, accurate and comprehensive detection and genotyping method for the conventional Pathogenic Vibrio spp. is critical to guarantee the food safety and cope with food poisoning derived from aquatic product.To establish ERIC-PCR technique for genotyping Vibrio parahaemolyticus, analyzed the ERIC-PCR fingerprinting of Vibrio parahaemolyticus standard strains and isolates, exploring the application value of ERIC-PCR technique for epidemiology investigation and homology tracking of Vibrio parahaemolyticus; Combining with detection of thermostable direct hemolysin gene(tdh) and TDH-related hemolysin gene(trh) of various Vibrio parahaemolyticus strains, analyzed the distribution of virulence strain, providing scientific proof for prevention and therapy of food poisoning. The results show that twenty-six Vibrio parahaemolyticus strains could generate replicable DNA?fingerprinting, grouped into 12 patterns with discriminative index of 0.926, and consistent with the genotyping result of Pulsed Field Gel Electrophoresis; The results of detection of virulence genes indicate that the tdh gene was positive in all clinical strains but no trh gene in all strains except one standard strain.According to the tlh, tdh, toxR gene coding thermolabile hemolysin, thermostable direct hemolysin, TOXR-protein respectively and 16S rDNA gene of Vibrio parahaemolyticus, the primers and captured oligonucleotide probes were designed and synthesized, then performing multiplex PCR and asymmetric PCR products were hybridized with DNA microarray. Genomic DNAs from 40 bacterial strains were detected to evaluate this method. All Vibrio parahaemolyticus were positive on matching probes locus and it only exist false positive among Vibrio alginolyticus and Vibrio mimicus of 16S rDNA gene, however, probes of tlh,tdh,toxR gene show strong specificity for Vibrio parahaemolyticus, the detection limit of this microarray assay was around 7.5 pg for genomic DNA, 200 CFU/mL for artificially contaminated food samples. At the same time, we took advantage of the mature multiplex PCR technique to detect foodborne pathogens-Escherichia coli O157 and Listeria monocytohenes, the results show that our quadruple PCR could detect Escherichia coli O157 and Listeria monocytohenes simultaneously with high specificity.By comparing the sequences of 16S rDNA of Vibrio parahaemolyticus, Vibrio cholerae, Vibrio alginolyticus and Vibrio mimicus, found the sequence variations.?According to the feature of slight differences, a pair of forward and reverse primer with fluorescence was designed in the conserved region, the four oligonucleotide probes were designed in the variable region, and genomic DNAs from 40 bacterial strains were detected to evaluate this method. The results show that this DNA microarray could distinguish four Vibrio spp. quickly with strong specificity.This study established the genotyping method based on ERIC-PCR of Vibrio parahaemolyticus and DNA microarray technique for detection of the conventional pathogenic Vibrio spp.It could provide a reliable technological measure for the diagnosis and prevention of infection caused by Vibrio parahaemolyticus and other Vibrio spp., and then provides another barrier to guarantee the food security.