A point-of-care testing molecular diagnostic device integrating loop-mediated isothermal amplification and retroreflective optical analysis modules

Abstract

In this study, we developed a molecular diagnostic platform using portable equipment for point-of-care testing (POCT) to address the l imitations of traditional methods of Salmonella detection that are often labor-intensive, t ime-consuming, and require complex equipment. To miniaturize the process of DNA amplif ication, we employed loop-mediated isothermal amplif ication (LAMP) to amplify target genes at a constant temperature, replacing large thermocyclers with compact thermoelectric Peltier elements. We also introduced a non- spectroscopic analysis method , retroreflective, to overcome the drawbacks of f luorescence analysis, which involves complex and expensive equipment. Through this method, we converted the traditional f luorescence spectrometer into a simple system util izing an LED light source and a camera . Retroreflective Janus particles (RJPs) were introduced as optical probes, and specially designed LAMP primers were used to generate double-stranded molecular beacons with specific recognition sequences at each 5 ’ end. When combined with DNA-immobilized substrate and the avidin-immobilized RJPs, these amplicons formed a sandwich-type complex. Under non-spectroscopic conditions, the RJPs produced a bright optical signal that correlated with the concentration of DNA amplicons. We successfully demonstrated quantitative analysis of the foodborne pathogen Salmonella , achieving a limit of detection of 10 CFU by util izing the developed POCT platform.