Rolling circle amplification and its application in microfluidic systems for Escherichia coli O157:H7 detections

Rolling circle amplification (RCA) has been widely used to enhance detection signals as its long single-stranded RCA products can provide multiple binding sites for signal probes for sensitive detections. In the current study, we employ atomic force microscopy (AFM) to monitor the RCA products during the course of the RCA process over time. Subsequently, the results of the RCA obtained from the AFM study are combined with those from the conventional electrophoresis method to optimize RCA reactions for rapid and sensitive detection. We show that there appears to be an inhomogeneous RCA initiation phase in early to mid-stage of the RCA reaction where some chains grow faster while others grow slower or remain dormant, an observation that has not been reported in the literature. Furthermore, we demonstrate that the RCA can significantly enhance detection signals by up to 100-fold. We also show that the Escherichia coli O157:H7 detection with the RCA can be carried out in different food matrices with excellent detection sensitivities and specificities. In conclusion, these results suggest that a microfluidic device in combination with RCA signal enhancement is a simple and robust approach to sensitive whole-cell detection in food samples. Practical applications The current research has exciting potentials for applications in sensitive detections of food samples for food safety inspections.