沙门菌、志贺菌和大肠杆菌O157∶H7的多重PCR快速检测体系的初步探讨

目的建立能在12小时内同时快速检测沙门菌、志贺菌和大肠杆菌O157∶H7的多重PCR体系。方法碱性蛋白胨水(BPW)非选择性增菌6h;100℃10min制备DNA模板;根据大肠杆菌O157∶H7的uidA基因、志贺菌的ipaH基因及沙门菌的invA基因序列设计各菌引物,进行PCR扩增及电泳检测。同时优化反应体系,测定体系灵敏度和特异性。结果该多重PCR体系能在12h内同时检测3种目的菌;灵敏度为10～30cfuml;通过对23株目的菌和15株非目的菌检测,提示该体系特异性高。结论初步探讨出能在12h内快速、灵敏、特异地同时测定沙门菌、志贺菌和大肠杆菌O157∶H7的多重PCR体系。

Primarily study on a multiplex PCR-based system for the rapid detection of Salmonella spp., Shigella spp. and Escherichia coli O157∶H7

ObjectiveTo establish a multiplex PCR-based system for the simultaneous detection of Salmonella spp., Shigella spp. and Escherichia coli O157∶H7 in 12 hours.Methods After 6 h nonselective enrichment in BPW, DNA template were prepared at 100℃ for 10min. Three sets of primers were designed to amplify the gene segments of invA of Salmonella spp, ipaH of Shigella spp, and uidA of E. coli O157∶H7, and the products were analyzed by electrophoresis. At the same time, this system was optimized,and the specificity and sensitivity of this system were evaluated.Results Three target bacteria were detected in 12 h by using this multiplex PCR-based system. The sensitivity of it was up to 10-30cfu/ml, and the high specificity was demonstrated by detecting 23 target stains and 15 non-target stains.Conclusion A rapid,specific, and sensitive multiplex PCR-based system for the simultaneous detection of Salmonella spp., Shigella spp. and E. coli O157∶H7 in 12 h has been studied primarily.