Development Of Membrane Chip Technology About Identification And Virulence Genes Detection Of Marine Fish Vibrios

Vibriosis is fish disease responsible for considerable economic hardship to mariculture operations worldwide. Both pathogenic and non-pathogenic strains exist in the marine fish, detection of the virulence genes will help in differentiation of pathogenic strains. Conventional microbiological methods identifying these organisms are often limited by the length of time required to complete the assays. The requirement for multiplex detection of a wide range of potential pathogens has never been greater. Recently, huge potential of microarray generates considerable interest for its utility as a diagnostic tool. Thus, in this study we develope this technology included the design and evaluation of PCR coupled with a low-density oligonuleotide membrane chip for the detection and identification the presence of specific markers in bacterial genomes associated with pathogenesis of selected marine fish pathogens.A total of more than 160 strains bacteria, isolated from marine fish, shrimp and aquatic environment, are examined for their inhibitory activities to pathogenic bacteria by using cross-streaking method, dot-inoculating method and scrip diffusion assay on 2216E plates. Two pathogenic vibrios(vibrio anguillarum and Vibrio harveyi) are used as indicating strains. using preparatory selection methods, 2004103101 is selected by its inhibitory activities to the indicating strains. the evident clear zones are observed around the pathogenic bacteria. Then the indicating strains are inoculated in sterile cultivated supernatant of the inhibitor respectively, sterile cultivated supernatant is maked by percolation. After incubation for period of time, measuring the value of their OD600 to verifying it's restraining function. The result shows that antagonistic bacterium 2004103101 really has inhibitory activities to the indicating strains.Strain 2004103101 is a halophilic, gram-negative, curve-shaped bacterium. It can make TCBS culture medium yellow, and it has strong haemolytic activity. The clone is round, semitransparent and wettish, According to the clone character, physiological, biochemical results and the sequence of 16S ribosomal DNA (16S rRNA), it's identified as vibrio anguillarum. Turbots are challenged with strain 2004103101 and vibrio anguillarum. Strain 2004103101 is attenuated sharply virulent by intraperitoneal injection than the Vibrio anguillarum. In order to explore the reason of this v.anguillarum's virulence loss, Seven sets of primers are designed for six virulence genes vah, empA, virC, virA, flaA, angM and 16S rRNA, the genome DNA of vibrio anguillarum and 2004103101 are using as templates to amplified respectively, the result of 1% agarose gel electrophoresis show that virC of 2004103101 was absent. VirC is a gene related to synthesizing major surface antigen, the virC mutant showed a loss in virulence. The amplified fragment of angM of 2004103101 is different from vibrio anguillarum in quantity, and angM is including by virulent plasmid pJM1 and pEIB1 of vibrio anguillarum, encoding a 78-kDa nonribosomal peptide synthetase protein.Members of the vibrio group carry genes encoding several important virulence factors. Since 16S rDNA sequences of group members are very similar, it's taxing to differentiate them, and vibrio virulence factors are very important for pathogenesis, a oligonucleotide membrane-array system be specifically developed for strain identification and determinate virulence gene detection. The 16S rRNA and hsp60 of partial strain sequences are used as identification reference. Total 29 oligonuleotides probes specific for each sequence are synthesized and apply to nylon membranes. Genomic DNA is extracted from fresh bacterium cultures using a phenol-choroform extraction and are used as template. Digoxigenin(DIG)-labeled target genes are amplified by gene specific primers and DIG-labeled dUTP, the amplified fragments are hybridized to the membrane-array. The quantity of PCR products and the time of hybridization is evaluated by serials grads assay. Hybridization signals are read by NBT/BCIP color developed. 25 out of 29 sequences ready for detection are amplified specifically, twelve virulence related genes of 25 have exact color development, ten 16S rRNA and hsp60 products have cross reaction with 1 to 3 probes of other 16S rDNA and hsp60, and three fragments, vibrio alginolyticus omp, vibrio parahaemolyticus trh, vibrio fischeri ampC, can be detected by 1% agarose gel electrophoresis but not membrane-array, reveal that the strains attending assay are not the strains harboring this genes. The membrane array can detect 2.56ng target fragments, and equal to 12.8pg DNA of bacteria. The results of this work indicate that the membrane-array is a reliable method to detection vibrios, and it can offer a powerful evidence to pathogenicity of pathogenic germs.