Western斑点印迹法检测致病性副溶血弧菌-食品科学.PDF

※分析检测 食品科学 2010, Vol. 31, No. 20 413 杨靖亚，张 建，赵 勇，陶 妍，陆小凡，晁若瑜 (上海海洋大学食品学院，上海 201306) 目的：对副溶血弧菌( Vibrio parahaemolyticus，VP) 的直接耐热溶血毒素(thermostable direct hemolysin ， TDH)进行分离纯化，建立 Western 斑点印迹法技术检测致病性副溶血弧菌的方法。方法：用直接耐热溶血毒素免 疫小鼠得到特异性抗血清，利用棋盘方法确定免疫血清最佳工作浓度，并对致病性与非致病性副溶血弧菌分别进行 特异性测定。结果：最佳免疫血清最佳工作浓度为 1:3200；检测致病性副溶血弧菌为阳性，非致病性副溶血弧菌 属呈阴性。结论：Western 斑点印迹法技术可以特异性检测致病性副溶血弧菌，灵敏度高、操作简单，可用肉眼 判定结果。 耐热直接溶血毒素；Western 斑点印迹；致病性副溶血弧菌；检测 Detection of Pathogenic Vibrio parahaemolyticus by Western Blotting YANG Jing-ya ，ZHANG Jian ，ZHAO Yong ，TAO Yan ，LU Xiao-fan ，CHAO Ruo-yu (College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China) Abstract: Objective: To purify thermostable direct hemolysin (TDH) from the culture of Vibrio Parahaemolyticus and to establish western blotting method for determining pathogenic Vibrio parahaemolyticus. Methods: The specific anti-serum was obtained from immune mice using TDH. The working concentration of serum samples was determined by chessboard assay. The specificities of pathogenic Vibrio parahaemolyticus and non-pathogenic Vibrioparahaemolyticus were also determined. Results: The working concentration of serum samples was 1:3200. The samples with clear dots were judged as the positive reaction. V. parahaemolyticus was positive in pathogenic Vibrio parahaemolyticus, while non-pathogenic Vibrio parahaemolyticus was negative. Conclusion: Western blotting can be used to detect pathogenic Vibrio parahaemolyticus with the characteristics of high sensitivity, simple operation and eye-observable results. Key words ：TDH ；Western blotting ；pathogenic Vibrio parahaemolyticu；detection