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

Abstract

ObjectiveTo 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.MethodsPrimers 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.ResultsThe 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.ConclusionThe 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.

Keywords

• recombinant enzyme polymerase amplification technology
• enterohemorrhagic escherichia coli o157：h7
• visualization rpa
• chromogenic reaction
• real-time fluorescence rpa
• detection method
• foodborne pathogenic