An Integrated Hydroxyl Magnetic Bead–Microfluidic RPA Platform for Simultaneous Detection of Seven Foodborne Pathogens

The contamination and outbreaks caused by foodborne pathogens highlight the need for developing a rapid, reliable, and field-deployable detection strategy suitable for food safety monitoring and environmental surveillance. However, traditional detection methods usually require time-consuming procedures, specialized instruments, trained personnel, or separate reactions for different pathogens, making them unsuitable for rapid on-site multiplex screening. To address these limitations, we developed a rapid multiplex detection strategy combining hydroxyl magnetic bead-based DNA preparation with recombinase polymerase amplification (RPA) on a high-throughput centrifugal microfluidic chip. In this workflow, bacterial DNA was first extracted and purified from complex samples using hydroxyl magnetic beads, and then introduced into the microfluidic chip for parallel RPA amplification and real-time fluorescent signal detection in a closed system. With this detection platform, seven common foodborne pathogens, including E.coli O157:H7, Salmonella, S. anreus, V.Parahaemotyticus, L.monocytogenes, Enterobacter sakazakii, and Brucella, were simultaneously detected within 85 min, with limits of detection at the 10² CFU/mL level. The system showed excellent specificity, good reproducibility with coefficients of variation below 5%, and reliable performance in artificially contaminated beef and wastewater samples. This hydroxyl magnetic bead-assisted microfluidic RPA strategy provides a rapid, sensitive, high-throughput, and user-friendly method for on-site screening of multiple foodborne pathogens, showing great potential for food safety surveillance, outbreak response, and routine quality control, especially in resource-limited settings.