水产品中副溶血性弧菌实时荧光PCR检测方法

建立了一种特异、灵敏、稳定的副溶血性弧菌(Vibrio parahemolyticus,VP)致病基因的检测方法。对已建立的副溶血性弧菌致病基因tdh、trh和tlh荧光PCR方法的特异性、灵敏度和重复性进行检测,以验证该方法的有效性。该方法与副溶血性弧菌反应良好,与其他弧菌属和非弧菌属的6株常见食源性致病菌无交叉反应;检测了6株副溶血性弧菌标准菌株和分离株,3种致病基因检出限分别为tlh 6～43 CFU/mL,tdh 97～1 700 CFU/mL,trh 1 100～4 000 CFU/mL;3种致病基因20次重复组内变异系数在0.96%～1.50%,组间变异系数在2.70%～4.10%。该方法操作简便,特异性强,灵敏度高,能够准确、快速、灵敏地检测水产品中副溶血性弧菌。

Study on detection of Vibrio parahaemolytichus in aquatic products by real-time PCR

Established a specific,sensitive and stable detection method for pathogenic genes of Vibrio parahaemolyticus(VP).Methods The detection specificity,sensitivity and reproducibility of fluorescent PCR method established for the detection of pathogenic genes tdh,trh,and tlh of Vibrio parahaemolyticus were checked to verify the effectiveness of the method.The method was successfully applied to detect 6 standard strains and isolates of Vibrio parahaemolyticus without cross-reaction with other Vibrio and non-Vibrio strains.The detectable limits for three pathogenic genes were 6~43 CFU/mL for tlh,97~1 700 CFU/mL for tdh,1 100~4 000 CFU/mL for trh,respectively.The variation coefficient inside each of 20 repeating groups for these three genes were 0.96%~1.50%.The variation coefficient between groups was 2.70%~4.10%.The method is simple,specific,highly sensitive,and is capable of accurate,rapid,sensitive detection of Vibrio parahaemolyticus in aquatic products.