The Pilot Study Of The Horizontal Gene Transfer In Vibrio Cholerae At Abalone Aquiculture Setting

Many toxin genes of Vibrio cholerae are carried as part of phage, for example cholera-toxin gene carried by CTX F . By bacteriophage transduction, the toxin genes could be transferred horizontally. So, the horizontal gene transfer is very important step during the evolution of toxigenic Vibrio cholerae. And with the notion about the toxigenic Vibrio cholerae autochthonous to the aquatic environment established, the researchers are paid more attention to the environmental factors impacting on the emergence of novel strains of toxigenic Vibrio cholerae.The toxigenic Vibrio Cholerae 0l and 0139 strains were firstly analyzed to study the lysogency status and the phages carried. The electron microscopy examination and PCR analysis revealed that 18.75% (3 out of 16) of the strains produced CTX 4) in the culture supernatants wheri induced with mitomycin C, whereas none spontaneously produced particles. And a filamentous phage from 0139 strains was also induced, which characters were still in the dark. We failed to show the filamentous phage VPI F which was considered to transfer the TCP pathogenicity island.Vibrio cholera was chosen as the model species and its toxigenic gene ctxAB as the target. Firstly conventional PCR was conducted to optimize the PCR parameters. Bacteria cells were fixed and treated with lysozyme, then cells subjected to in situ PCR using FAM-labeled primer as a label, showed the presence of ctxAB by the epifluorescence microscopy examination. The analysis from flow cytometer showed that 100% ctxAB+ bacterial was amplified. To confirm the ctxAB gene, the amplified product was analyzed by agarose gel electrophoresis detection and sequencing. After many timesrepeating, there had the same results. And the sensitivity of the method was 104/ml. So, our results successfully demonstrated that the newly developed detection method was established.When the new method was used to detect the bacteria in aquaculture seawater, the results showed that there are a number of challenges ahead, including the effects of interfering agents such as planktons, suspended solids, natural inhibitors and so on. For this new method, how to improve its sensitivities and the flow cytometer's maneuverability for sorting are still the big problem we face now.