| The species of genus vibrio are among the most common bacteria in marine environments. Some pathogenic species of vibrio cause vibriosis, one of the most prevalent diseases of maricultured animals. With seafood being intermediate host, pathogenic Vibrio sp. are reported to have caused human diseases such as diarrhea, bacteremia and severe wound infection. Vibriosis is sometimes mortal for human beings. Furthermore, some Vibrio sp. are opportunistic pathogens. It has been reported that the large-scale outbreak of vibriosis is not linear with the total amount of Vibrio species in water but, is determined by the amount of a certain pathogenic Vibrio sp.. However, related studies are currently based on the development of pure-culture techniques, which is increasingly known as a gigantic bottleneck in some fields like diversity research. On account of the high complexity of environmental microbial metagenome and the restricted analytic capacity of current techniques such as AFLP and sequencing, molecular diversity analysis of 16S rRNA gene based on UPPCR (universal primer PCR) can only be performed to analyze high-level taxonomy. It is difficult to depict accurately the diversity composition within a certain genus or even, a family. Consequently, it is significant in mariculture and food safeties to analyse the community structure and kinetics of Vibrio genus in environmental samples by molecular techniques.DNA extraction is the base of research on the environmental microbial metagenome. Analytic result will be reliable if the extracted DNA is representative of the true nature of the environment we studied. Firstly in this study, we introduced an effective method -alkaline-heating, for preparing PCR template DNA from marine plankton. The DNA solution prepared using this method has pH values ranging from 8 to 9, which can serve as PCR template directly for the amplification of genes like ribosomal RNA gene, mitochondrial cytochrome oxidase gene and chloroplast rbcL gene. Secondly, we designed three Vibrio genus-specific primers, VF169, VR744 and VR1150, according to 16S rRNA gene (16S rDNA) sequences ofVibrio and non-Vibrio genus in GenBank. Those sequences of non-Vibrio genus are either closely related to Vibrio in molecular phylogeny or tending to confuse the background in marine environment. In combination with VF27, the universal primer for eubacterial, the designed primers were arranged to three sets of PCR primers with which PCR were performed respectively and 16S rDNA retrieved directly from water samples were amplified. Clone libraries were then constructed and an amount of clones were randomly selected to sequence (amount to 72 sequences) . These sequences were used to construct phylogenetic tree. The stringency and specificity of designed primers were tested by BLAST program and amplification of cultured strains of both Vibrio and non- Vibrio.VR744 show highly stringent selectivity to Vibrio genus, while it distinguishes almost all known Vibrio species from environmental samples by PCR amplification in combination with VF27. Phylogenetically, all of the 72 sequences show their highest similarities to and cluster with those of diverse known Vibrio species. We can also infer that Vibrio show marked diversity and V. splendidus related group predominate in Jiaozhou Bay in autumn (occupying about 50% of the total amount of Vibrio sp.). Moreover, interspecific variation among Vibrio species might be mantled by the intraspecific mutation, as make it difficult to analyse diverse composition within Vibrio genus on the level of species or sub-species with only information of 16S rDNA.Overall, alkaline-heating is a simple and rapid method suitable for the studies of planktonic molecular diversities. The designed and optimized primer is also useful tool for monitoring kinetics of pathogenic Vibrio sp. in the chain of seafood production, processing and consumption as well as studying their pathogenic threshold further real-timely.
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