6类致泻大肠埃希氏菌多重PCR检测体系的建立

目的构建食源性致泻大肠埃希氏菌高效、快速、特异性强的检验方法。方法根据6类致泻大肠埃希菌11种毒力基因引物,采用煮沸法制备细菌基因组DNA作为模板构建致泻大肠埃希氏菌单菌多重PCR检测体系;采用试剂盒法制备细菌基因组DNA作为模板构建6类菌十重PCR检测体系,对2种检测体系进行优化并对其进行特异性、重复性验证。结果构建了6类致泻大肠埃希氏菌多重PCR检测体系,包括单菌多重PCR、6类菌十重PCR检测体系。仅6类致泻大肠埃希氏菌阳性菌株有特异性条带产生;不同类型致泻大肠埃希氏菌菌株均检出其特异性阳性条带,该多重PCR检测方法重复性好,稳定性强。结论该方法可高效快速地确定待测样是否为大肠埃希氏菌及其致病型,为食品中致泻大肠埃希氏菌的监控、预警及爆发引起食物中毒后溯源等提供参考。

Establishment of multiplex PCR detection method for 6 strains of diarrheagenic Escherichia coli

Objective To establish an effective, rapid and specific detection method for foodborne diarrheagenic Escherichia coli. Methods Based on 11 virulence gene primers of diarrheagenic E. coli, multiple PCR detection system of single type strain and decuple PCR detection system of 6 strains were constructed by using bacterial genome DNA prepared by the boiling method and kit method respectively as template. The 2 detection systems were optimized, then their specificity and repeatability were verified. Results Multiplex PCR detection systems for 6 strains of diarrheagenic E. coli were established, including multiple PCR detection system for single type strain and decuple PCR detection system for 6 strains. The specific bands were amplified only in the 6 strains of diarrheagenic E. coli. Different kinds of diarrheagenic E. coli were detected the same specific positive bands, indicating that the repeatability and stability of the multiplex PCR detection systems were good. Conclusion This method can effectively and rapidly determine whether the test samples are E.coli and their pathotype, which can provide references for the monitoring, warning and tracing of food poisoning outbreaks caused by diarrheagenic E. coli.