可视化-环介导等温扩增技术检测肠出血性大肠杆菌O157:H7

目的 建立可视化的环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)联合横向流动试纸条(lateral flow dipstick, LFD)方法检测肠出血性大肠杆菌(enterohemorrhagic Escherichia coli, EHEC)O157:H7的分析方法。方法 以EHEC O157:H7的遗传标志性基因z3276基因序列为检测靶标设计6条LAMP特异性引物, 其中2条环引物分别标记异硫氰酸荧光素和地高辛, 用LFD检测扩增产物, 建立EHEC O157:H7 LAMP-LFD检测方法。结果 所建立的检测方法能够特异性的检测EHEC O157:H7, 与试验对照菌株EHEC O26、O145、O45、O121, 及其他主要大肠杆菌血清型O25、O78、O103、O111、O127、O138、O139、O141, 及禽致病性大肠杆菌(avian pathogenic Escherichia coli, APEC)E058、肠致病性大肠杆菌(enteropathogenic Escherichia coli, EPEC)SEC627、EPEC SEC689、K12-MG1655、BL21, 和其他种属的受试菌, 不存在交叉反应; 对EHEC O157:H7纯培养物的最低检测限为1.3 CFU/mL。结论 本研究所建立的LAMP-LFD检测方法特异性和敏感性良好, 操作简单, 结果可视。

Determination of enterohemorrhagic Escherichia coli O157: H7 by visualization-loop mediated isothermal amplification

Objective To establish a method for determination of enterohemorrhagic Escherichia coli (EHEC) O157: H7 by visualization-loop mediated isothermal amplification (LAMP)-lateral flow dipstick(LFD). Methods Totally 6 LAMP-specific primers were designed targeting the genetic marker gene of z3276 from EHEC O157:H7, and 2 loop primers were respectively labeled with fluorescein isothiocyanate and digoxin. The amplification products were detected by LFD, then the LAMP-LFD detection method was established. Results This assay could specifically detect EHEC O157:H7, and no cross-reaction was observed with any of the other test bacteria, including the control strains EHEC O26, O145, O45, O121, other major Escherichia coli serotypes O25, O78, O103, O111, O127, O138, O139, O141, and avian pathogenic Escherichia coli (APEC) E058, enteropathogenic Escherichia coli (EPEC) SEC627, EPEC SEC689, K12-MG1655, BL21, and other species. The minimum detection limit of this assay was 1.3 CFU/mL for EHEC O157:H7 culture. Conclusion The established LAMP-LED detection method has good specificity and sensitivity, and the results of operation detection are visible.