致病性副溶血性弧菌菌落杂交检测体系的优化

Colony Hybridization Optimization to Detect Pathogenic Vibrio parahaemolyticus

对常见食源性致病菌副溶血性弧菌的杂交条件进行了优化。基于副溶血性弧菌的tdh,trh,toxR基因选用了3种探针序列,从菌落杂交样品的预处理方法、杂交时间和显色检测等方面对基于副溶血性弧菌毒力基因的原位杂交实验进行了条件优化。结果表明,采用80℃热固定2 h,37℃预杂交10 min后杂交8 h以及封闭1 h的改良方法可获得高特异性、低背景且着色清晰的实验结果。优化的菌落杂交技术检测副溶血性弧菌与传统检测方法相比,具有高效、准确、可区分致病性菌株的优点,为水产品中副溶血性弧菌致病菌株和非致病菌株的同步检测和筛选提供参考。

Based on selected 3 reported probe sequences of tdh,trh,and toxR,hybridization conditions of common foodborne pathogenic V. parahaemolyticus（ Vp） have been optimized,including pretreatment of colony hybridization samples,time,and developing and other aspects were carried out conditions optimization based on gene hybridization experiment in situ of Vp virulence. The results showed that a high specific,low background,and distinctly colored results have been acquired adopting an improved methods of 80 ℃ heat fixation for 2 h,hybridization for 8 h after 37℃ pre-hybridization for 30 min as well as sealing for 1 h. As compared with traditional detection method the optimized colony hybridization technology to detect Vp possessing the advantages of high efficiency,accuracy,being able to distinguish. This study provides a technical support for the detection of pathogenic and nonpathogenic Vp in aquaproducts synchronistic detection and screening.