出血性大肠埃希菌O157:H7可视化和实时荧光RPA检测方法的比较

Visualization and real-time fluorescence RPA detection methods for enterohemorrhagic Escherichia coli O157:H7

目的以检测出血性大肠埃希菌(EHEC)O157:H7为目的,基于重组酶聚合酶扩增(RPA)技术,分别建立可视化RPA检测方法和实时荧光RPA检测方法。方法根据出血性大肠埃希菌O157:H7的rfbE基因设计引物和探针,进行特异性、灵敏度检测,建立实时荧光RPA检测方法。结合SYBR Green I在核酸反应中的颜色变化特征,设计可视化RPA检测方法。结果建立的实时荧光RPA检测方法和可视化RPA检测方法,分别在35℃时反应不超过25 min即可检测EHEC O157:H7。设计的引物和探针仅对EHEC O157:H7出现特异性结果。两种检测方法的检测限低于10-5 ng/μL。结论EHEC O157:H7的可视化RPA检测方法和实时荧光RPA检测方法操作简便,且特异性强、敏感性高,可为EHEC O157:H7的现场快速检测和实验室检测提供新方法。

Objective To detect enterohemorrhagic Escherichia coli(EHEC)O157:H7,visualization recombinant enzyme polymerase amplification(RPA)detection and real-time fluorescence RPA detection methods were established based on RPA technology.Methods Primers and probes were designed based on the rfbE gene of EHEC O157:H7,and specificity and sensitivity were determined.A real-time fluorescent RPA method was established.Combined with the color change characteristics of SYBR Green I in nucleic acid reactions,a visualization RPA method for the detection of EHEC O157:H7 was established.Results The established real-time fluorescence RPA detection and visual RPA detection methods both detected EHEC O157:H7 in 25 min at a reaction temperature of 35℃.The designed primers and probes showed specific results only for EHEC O157:H7.The detection limits of the two methods were lower than 10-5 ng/μL.Conclusion The visual RPA detection and real-time fluorescence RPA detection methods of EHEC O157:H7 were easy to operate with strong specificity and high sensitivity.This study provides novel methods for the rapid detection of EHEC O157:H7 in the field and laboratory.