Multi-enzyme isothermal rapid amplification assay for the detection of Escherichia coli O157∶H7

Abstract

ObjectiveA rapid enrichment and detection method for Escherichia coli O157∶H7 was developed by using multienzyme isothermal rapid amplification （MIRA） fluorescence method combined with metal organic frameworks immunomagnetic beads.MethodsUsing rfbE gene as the target， the primers， probes and reaction system were screened， and the specificity， sensitivity and practical application of this method were investigated.ResultsThe detection limit of Escherichia coli O157∶H7 was 1.18×105 CFU‧mL-1， and the detection limit of DNA concentration was 9 pg‧μL-1. The detection process was completed in 20 minutes. The test results of 47 strains （24 target strains and 23 non-target strains） were consistent with real-time PCR （RT-PCR）.ConclusionA method based on metal-organic framework immunomagnetic beads enrichment combined with MIRA assay is developed in this study. The method is simple， rapid and suitable for rapid enrichment and detection of Escherichia coli O157∶H7 in food.

Keywords

• multienzyme isothermal rapid amplification
• metal-organic framework immunomagnetic bead
• rfbe gene
• escherichia coli o157∶h7