三基因PCR检测志贺菌、副溶血弧菌和霍乱弧菌的研究

Simultaneous detection of two virulence genes in food by using rapid three-gene PCR

目的探索一种快速、经济、敏感的诊断方法,对常见的食品中的腹泻致病菌进行基因检测研究。方法建立一种快速标本处理和多基因PCR反应(multiplePCR)诊断方法,在一次PCR反应中同时扩增3种菌的致病基因:志贺菌的侵袭性(ipaH)基因、副溶血弧菌的热稳定直接溶血素(tdh)基因和霍乱弧菌Ο139的霍乱毒素(ctx)基因。扩增产物经电泳,出现与已知阳性对照细菌条带大小一致的条带即判断为阳性标本;另外对这些标准菌株进行Southernblot杂交鉴定。结果对标准志贺菌、副溶血弧菌和霍乱弧菌检测,多基因PCR法检测为阳性;PCR作为诊断方法较培养法阳性率高。结论三基因PCR具有简便、快速、特异、经济和不需要培养等特点,适用于食品检测。

Objective To explore the rapid ,economical and sensitive procedures for diagnosis of polluted bacterial food. Methods A method of using rapid multiple gene PCR was established to simultaneously detect three virulence genes. In single tube, the following virulence genes were amplified: ipah gene of Shigella, thermostable direct hemolysin (tdh)of Vibrio parahaemolyticus and ctx gene of Vibrio cholera. By the DNA marker or known size fragments, infection or coinfection of the bacteria was determined if the same size bands were seen via electrophoresis,then the result of Southern blot hybridization as the last standard. Results Shigella, Vibrio parahaemolyticus and Vibrio cholera were detected, all of them were positive by PCR; the hybridization has the same result as PCR. The gene detection of ipah, tdh and ctx was more sensitive than the conventional procedures. Conclusion This method is highly specific, sensitive, universal rapid, simple as well as economical, and can be used for detection of food, water and other products in life.