作　　者： ; ; ;

机构地区： 华南理工大学食品与生物工程学院

出　　处： 《微生物学报》 2004年第3期399-401,共3页

摘　　要： 建立一套原位PCR检测方法 ,联合流式细胞仪作为检测工具 ,作为基因水平转移研究中的基因监控手段。通过常规PCR反应以确定靶基因的基本扩增参数 ;细菌菌体经过多聚甲醛PBS液固定和溶菌酶处理后进行原位PCR扩增 ,产物洗涤后迅速用流式细胞仪进行荧光检测 ,并辅以荧光显微镜镜检。扩增样品在荧光显微镜的蓝光激发下发出明亮的黄绿色荧光 ,与空白对照中的无扩增菌体的自发荧光可明显区分。流式细胞仪检测结果也显示 ,阴性菌与阳性菌的荧光强度有明显区别。完成了对目标细菌的原位PCR扩增 ,并成功地应用流式细胞仪对原位PCR扩增菌体实施了检测。由此表明isPCR 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.

关 键 词： 原位聚合酶链式反应 流式细胞仪 副溶血弧菌 耐热的直接溶血素基因

领　　域： [生物学]