3种食源性致病菌的多重PCR快速检测方法研究

Study on multiplex PCR of rapid detecting three food-borne pathogenic bacteria

目的建立一种能同时检测志贺菌,副溶血性弧菌和大肠埃希菌O157的多重PCR方法。方法根据志贺菌IpaH基因^([1])、副溶血性弧菌t1基因^([2])、大肠埃希菌O157 eae A基因^([3,4])设计特异性引物,采用LB对志贺菌,副溶血性弧菌和大肠埃希菌O157进行振荡培养,通过多重PCR扩增3种病原菌的目的基因,通过优化反应体系,提高反应的检测效率和特异性。结果对志贺菌,副溶血性弧菌和大肠埃希菌O157振荡培10h,经过PCR反应扩增出目的基因,把志贺菌,副溶血性弧菌、大肠埃希菌O157、肠道致病性大肠埃希菌,产肠毒素性大肠埃希菌,金黄色葡萄球菌,单核细胞增生李斯特菌、蜡样芽胞杆菌8种菌混在一起提取核酸进行PCR扩增,结果表明此PCR方法具有良好的特异性。结论本研究建立的多重PCR检测方法能快速检测志贺菌,副溶血性弧菌和大肠埃希菌O157,具有快速,灵敏,特异的特点,能广泛应用于食源性疾病检测,食物中毒检测等领域。

Objective To establish a multiplex PCR method for simultaneous detection of Shigella,Escherichia coli O157 and Vibrio parahaemolyticus. Methods Shigella,Escherichia coli O157 and Vibrio parahaemolyticus were cultured by LB medium.Three pairs of primers had been designed according to IpaH gene of Shigella,t1 gene of Vibrio parahaemolyticus and eaeA gene of Escherichia coli O157. The target genes of these pathogens were amplified by multiplex PCR and the reaction efficiency and specificity of the reaction were improved by optimizing the reaction system. Results The multiplex PCR method established in this experiment had high specificity while eight kinds of bacteria DNA were mixed in one PCR reaction tube,and the detection limit of the method was 5 cfu/ml for Shigella,Escherichia coli O157 and Vibrio parahaemolyticus. Conclusion The multiplex PCR assay established in this study can rapidly detect Shigella,Vibrio parahaemolyticus and Escherichia coli O157. It has rapid,sensitive and specific characteristics and can be widely used in food sanitation detection,foodborne illness detection.