变性高效液相色谱检测食品中致泻性大肠杆菌

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

摘要:

摘要：【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。

Abstract:

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.