可视化-环介导等温扩增技术检测肠出血性大肠杆菌O157:H

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

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)0157:H7的分析方法。方法以EHEC 0157: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EHEC0157: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 0157:H7,与试验对照菌株EHEC 026、0145、045、0121,及其他主要大肠杆菌血清型025、078、0103、O111、0127、0138、0139、0141,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 0157: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。

Objective To establish a method for determination of enterohemorrhagic Escherichia coli(EHEC)0157: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 0157: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 0157:H7,and no cross-reaction was observed with any of the other test bacteria,including the control strains EHEC 026,0145,045,0121,other major Escherichia coli serotypes 025,078,0103,O111,0127,0138,0139,0141,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 0157:H7 culture.Conclusion The established LAMP-LED detection method has good specificity and sensitivity,and the results of operation detection are visible.