肠出血性大肠杆菌O157∶H7的快速可视化检测

摘要

目的 应用环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术和羟基萘酚蓝(Hydroxynaphtholblue,HNB)指示剂建立肠出血性大肠杆菌(EHEC) O157∶H7快速可视化检测方法.方法 针对EHEC O157∶H7脂多糖编码基因rfbE保守区设计LAMP引物及环引物,反应体系加入染料HNB作为LAMP扩增的指示剂,结合LAMP浊度仪优化扩增条件,根据HNB的颜色变化进行结果判定,并评价检测方法的特异性和灵敏度.结果 本方法最低检出限约为100拷贝/反应管,高于普通PCR;检测特异性高,仅EHEC O157∶H7反应管HNB颜色由紫罗兰变成天蓝色,而肠产毒性大肠杆菌(ETEC)、肠致病性大肠杆菌(EPEC)、肠侵袭性大肠杆菌(EIEC)、肠聚集性大肠杆菌(EAEC)、宋内志贺菌、福氏志贺菌均不发生变色反应；体系的扩增效率高于普通PCR,40 min内出结果.结论 建立的基于颜色判定的EHEC O157∶H7 LAMP检测方法具有特异、灵敏、设备要求简单等特点,适用于EHEC O157∶H7现场快速检测.%Objective A loop-mediated isothermal amplification (LAMP) assay was developed for rapid visual detection of Enterohemorrhagic Escherichia co1li O157∶ H7.Methods A set of six specific primers specific to eight regions of rfbE gene were designed.The reaction was performed in a single tube at 63 ℃ with the addition of hydroxynaphthol blue (HNB) dye prior to amplification.Use Loopamp Realtime Turbidimeter to grope for amplification conditions.The result was determined by the color change of HNB,and the sensitivity and specificty of LAMP method were evaluated.Results The results showed the detection limit of the LAMP assay was 100 copies per tube which was higher than conventional PCR method.The specificity of the LAMP assay was high.Only Enterohemorrhagic Escherichia coli O157∶H7 tube changed color.Conclusion These results suggested that the LAMP assay with HNB dye provided a useful tool for the rapid detection of Enterohemorrhagic Escherichia coli O157∶ H7.