三种食源性致病菌多重PCR检测方法的建立

舒畅; 姜琛璐; 钟慈平; 李林

doi:10.13386/j.issn1002-0306.2014.12.001

三种食源性致病菌多重PCR检测方法的建立

Establishment of a multiplex PCR detection method for three foodborne pathogens

摘要

摘要: 目的:建立同时快速检测沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7的多重PCR方法。方法:根据沙门氏菌的invA基因、志贺氏菌的ipaH基因及肠出血性大肠杆菌O157∶H7的uidA基因设计3对引物,通过单因素实验、L9(34)正交实验优化反应体系,并对多重PCR扩增的敏感性进行分析。结果:3对引物能特异性扩增出495、620、252bp的目的片段;在最优多重PCR反应体系下,多重PCR检测3种致病菌的灵敏度达104CFU/mL;将该法应用于人工污染实验,可在5h内得到准确、稳定的检测结果。结论:该方法操作简单、检测特异性和灵敏度较高,能够实现对沙门氏菌、志贺氏菌和肠出血性大肠杆菌O157∶H7 3种食源性致病菌的快速监控和诊断。

Abstract: Objective:To establish a rapid multiplex polymerase chain reaction (PCR) method for the simultaneous detection of Salmonella spp., Shigella spp. and Enterohemorrhagic Escherichia coli O157∶H7. Methods:Three pairs of primers had been designed based on the invA gene in Salmonella spp., ipaH gene in Shigella spp.and uidA gene in Enterohemorrhagic Escherichia coli O157∶H7, single-factor experiment and L9 (34) orthogonal experiment were used to optimize multiplex PCR amplification system, and the sensitivity of multiplex PCR was also analyzed. Results:Fragments of 495, 620, 252 bp were obtained respectively. Under the optimized condition, the detection sensitivity of multiplex PCR for three pathogens was 104CFU/mL. This method had been applied to artificial experiment, the result was accurate and steady, which could be obtained within 5h.Conclusion:A simple, specific and sensitive multiplex PCR method had been established and it was valuable for rapid monitoring and diagnosis for Salmonella spp., Shigella spp. and Enterohemorrhagic Escherichia coli O157∶H7.