Simultaneous detection of multiple foodborne pathogens using a CRISPR/Cas12a-based pump-free microfluidic chip

The development of microfluidic chips for nucleic acid detection provides efficient technical support for monitoring food safety. With the increasing maturity of CRISPR technology, it has the advantages of high specificity and high sensitivity in the detection of single or multiple nucleic acids. In this study, a microfluidic biosensor based on the CRISPR/Cas12a system was constructed using a pump-free microfluidic chip as the carrier, with a focus on the rapid, simultaneous detection of Listeria monocytogenes, Staphylococcus aureus, Escherichia coli O157:H7, and Cronobacter sakazakii. For each target pathogenic bacterium, two replicate channels for each pathogen were established, along with corresponding negative and positive controls, which effectively ensured the reliability and repeatability of the detected results and successfully achieved the simultaneous high-sensitivity, high-specificity, and high-accuracy detection of multiple foodborne pathogens. The detection sensitivity of the sensor for S. aureus, E. coli O157:H7, L. monocytogenes, and C. sakazakii was as low as 10³ CFU/mL, 10³ CFU/mL, 10² CFU/mL, and 10³ CFU/mL, respectively. This integrated CRISPR/Cas12a sensor chip has the advantages of the simultaneous efficient detection of multiple pathogens, parallel verification, and control settings, and the detection results can be visualized by fluorescence, indicating broad application prospects in the field of on-site rapid nucleic acid analysis.