In situ PCR and Detection by Flow Cytometer of tdh-containing Bacteria Vibrio parahaemolyticus
结合流式细胞仪检测技术的菌体原位PCR扩增

To develop a new detection method of toxin gene thermostable direct haemolysin (tdh) by employing in situ PCR (isPCR) technique and in combination with flow cytometry, for the purpose of research in horizontal gene transfer. Parameters employed for isPCR in this study were derived from the conventional PCR test. In setting up the in situ PCR, bacteria Vibrio parahaemolyticus were first grew in nutrient medium, and then fixed by paraformaldehyde in PBS buffer. Afterwards, bacteria were further treated with lysozyme to enhance their cell wall permeabilities. The treated bacteria were then subjected to isPCR reactions and rapidly detected by flow cytometry and epifluorescence microscopy. Bacteria Vibrio parahaemolyticus carrying tdh genes showed bright yellow-green fluorescence under epifluorescence microscope at the excitation of blue light. They could be distinctly differentiated from those cells in the negative controls who displayed very dim auto-emitting fluorescence. Flow cytometry also confirmed the vast differences between the negative cells and the positive ones that had gone through in situ PCR reactions. The results successfully demonstrated the power of isPCR in combination with flow cytometry in the detection of bacterial toxin gene tdh in situ. This newly developed technique could be employed to the study of behaviour of bacterial genes, like tdh, in a microbial community.