水体污染中常见致病菌的多重PCR分子检测研究

目的]建立同时检测沙门氏菌、志贺氏菌、绿脓杆菌、肠出血型大肠杆菌和副溶血弧菌等5种水体常见致病菌的多重PCR检测技术,为这些致病菌感染的快速诊断提供实验依据。方法]筛选设计5对特异引物,建立优化的多重PCR扩增体系和条件。结果]对5种致病菌的检测,多重PCR灵敏度分别为：肠出血型大肠杆菌O157102 cfu、志贺氏菌102 cfu、副溶血弧菌102 cfu、绿脓杆菌101 cfu、沙门氏菌102 cfu。应用于人工污染水样及天然水样的检测,均有清晰、特异的预期条带产生,并与传统检测结果一致,每个样品所需时间为6～8 h,相对于传统检测方法,极大地缩短了检测时间,提高了检测灵敏度。结论]该方法适用于大通量样品的检测研究,可推广应用于食品检测、环境监测等领域。

STUDY ON POL YMERASE CHAIN REACTION MOLECULAR DETECTION OF FIVE KINDS OF COMOM PATHOGENIC BACTERIA IN POLLUTED WATER

Objective]To develop a multiplex PCR assay for simultaneously detecting Salmonella,Shigella,Pseudomonas aeruginosa,enterohemorrhagic Escherichia coli(EHEC)and Vibrio parahaemolyticus in polluted water,to provide experimental evidence for rapid diagnosis of these pathogenic bacterium infection.Methods]Five pairs of specific primers were screened and designed to compose optimized system and conditions of multiplex PCR.Results]The detection sensitivity of multiplex PCR were 102cfu,102cfu,102cfu,101cfu and 102cfu for EHEC,Shigella,Vibrio parahaemolyticus,Pseudomonas aeruginosa and Salmonella,respectively.The detection results of polluted water and natural water by multiplex PCR were consistent with that of traditional assay,and expected straps appeared to be clear and specific.Besides,it took multiplex PCR 6-8 hours to detect a sample.Compared with traditional assay,the detection time was shortened greatly,and the sensitivity improved obviously.So multiplex PCR method was more sensitive,specific and efficient,and the processing was rapid and simple.Conclusions]The multiplex PCR method is suitable for detection of large sample,and it can be applied in food detection and environmental monitoring.