Ultrasensitive Detection of Multiple Foodborne Pathogens Using CRISPR–Cas12a on a Finger-Actuated Microfluidic Device Integrated with a Modular Pressurizing Pump

Foodborne pathogens pose a serious threat to global health and the economy, causing gastrointestinal illnesses and potentially leading to fatalities. Here, we present a recombinase polymerase amplification (RPA)-CRISPR-Cas12a-based method for the detection of foodborne pathogens using target-specific CRISPR RNAs (crRNAs) on a reusable, reconfigurable finger-actuated microfluidic device. Unlike previous finger-actuated pushbutton-based microfluidic devices, the device incorporates a modular pressurizing pump (MoPP), a standalone, reconfigurable actuation module that not only enhances reusability and reduces cross-contamination risks but also provides a flexible interface that allows user-defined fluidic routing and multiplexed assay workflows. Using a MoPP-integrated finger-actuated microfluidic device, the RPA-CRISPR-Cas12a-based detection of three foodborne pathogens was validated with an optimized crRNA and RPA primer sequence. Genomic DNA (gDNA) extracted from pathogen-spiked milk samples further demonstrated real-world applicability, achieving a limit of detection (LOD) of 1.62, 1.84, and 1.01 CFU/mL for <i>Escherichia coli</i> O157:H7, <i>Salmonella</i> spp., and <i>Listeria monocytogenes</i>, respectively. The developed microfluidic RPA-CRISPR-Cas12a-based detection platform is expected to be a reconfigurable, user-friendly, and highly sensitive point-of-care testing system for monitoring foodborne pathogens throughout the food supply chain.