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

Abstract

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.

Insights into the Formation of DNA–Magnetic Nanoparticle Hybrid Structures: Correlations between Morphological Characterization and Output from Magnetic Biosensor Measurements

“…Besides, unlike other well-established tools, for example, scanning electron microscopy (SEM), TEM, and conventional optical microscopy, AFM does not require coating or labeling of the sample, which is an advantage for understanding the underlying binding mechanisms for the formation of DNA–MNP hybrid structures. In particular, AFM has been used to characterize the morphology of Escherichia coli DNA-coils formed by rolling circle amplification and also to investigate the effect of an inert electrolyte in the interaction between calf thymus double-stranded DNA (dsDNA) and nonfunctionalized gold nanoparticles . Additionally, this technique was used to demonstrate that the decrease in the spin–spin relaxation time of water protons observed in NMR measurements can be ascribed to the oligomerization of iron oxide nanoparticles upon hybridization with the target DNA sequence .…”

Biomedical Applications of Microfluidic Devices: A Review

“…In isothermal amplification, the thermocycler, the fundamental part of PCR, is replaced by incubator or water bath-integrated microfluidics devices. Several types of microfluidic-based isothermal amplifications developed, including rolling circle amplification (RCA) [ 469 ], recombinase polymerase amplification (RPA) [ 470 ], and loop-mediated isothermal amplification (LAMP) [ 471 ], which were remarkably efficient and accurate. Ramachandran et al designed a microfluidics-based CRISPR where the CRISPR–Cas12 enzyme and a guide RNA were introduced to the device to bind to the selected target DNA and cleave it.…”

An Aggregation-Induced Emission Material Labeling Antigen-Based Lateral Flow Immunoassay Strip for Rapid Detection of Escherichia coli O157:H7

“…It is not suitable for the detection of a large number of samples and cannot monitor the epidemic in real time. 5 Methods based on molecular biology include polymerase chain reaction (PCR), 6 real-time PCR, 7 multiplex PCR, 8 loop-mediated isothermal amplification (LAMP), 9 rolling circle amplification (RCA), 10 and recombinase polymerase amplification (RPA). 11 Although molecular biology methods have high sensitivity, they require specialized equipment and skilled technicians to carry out complex operation procedures.…”