变性高效液相色谱检测食品中致泻性大肠杆菌

[目的]应用多聚酶链式反应(polymerase chain reaction,PCR)结合变性高效液相色谱(denaturing high-performance liquid chromatography,DHPLC)技术建立食品中致泻性大肠杆菌的快速检测方法.[方法]分别根据4种致泻性大肠杆菌的特异性毒力因子基因序列设计引物,PCR扩增产物经变性高效液相色谱进行快速检测.以肠产毒性大肠杆菌等32株试验菌株做特异性检测;4种致泻性大肠杆菌标准菌株稀释成不同梯度,做灵敏度检测.[结果]试验结果表明该方法有很好的特异性,且灵敏度高,检测限可达到:肠产毒性大肠杆菌27 CFU/mL、肠致病性大肠杆菌33 CFU/mL、肠出血性大肠杆菌25 CFU/mL、肠侵袭性大肠杆菌42 CFU/mL.[结论]该方法可以快速、准确地检测食品中的致泻性大肠杆菌,是食品中病原菌检测的新技术和新方法.

Denaturing high-performance liquid chromatography for identifying four categories of diarrheagenic Escherichia coli

Abstract: [Objective] We report a new molecular technique for the analysis of four categories of diarrheagenic Es-cherichia coli based on the separation of PCR-amplified target fragments by denaturing high-performance liquid chroma-tography (DHPLC). [Methods] Enteropathogenic E.coli, enterotoxigenic E.coli, enterohemorrhagic E.coli and enteroin-vasive E.coli were identified by analyzing four specific virulence genes. [Results] A total of 32 bacterial strains were tested, giving no false-positive or false negative results. The detection limits were as low as: ETEC 27 CFU/mL, EPEC 33 CFU/mL, EHEC 25 CFU/mL and EIEC 42 CFU/mL. [Conclusion] The data suggest that PCR-DHPLC will be useful for diarrheagenic Escherichia coli detection and identification.