A Relay CRISPR/Cas12a System for Extraction-Free and Ultrasensitive Detection of <i>Staphylococcus aureus</i> in Complex Food Samples

<i>Staphylococcus aureus</i> (SA) is a predominant foodborne pathogen, frequently associated with nutrient-rich matrices. However, rapid and ultrasensitive detection of SA in complex food samples remains a formidable challenge. In this work, we developed a relay biosensing system for detecting SA-16S rDNA that combines a magnetic bead-anchored APE1 DNA walker with an asymmetric CRISPR/Cas12a system. The magnetic bead-anchored DNA walker enables efficient separation of SA-targets from complex food matrices, while generating abundant activators through APE1-mediated catalytic cleavage. Employing spatially decoupled reactions with differential crRNAs, the asymmetric Cas12a system achieves programmable cascade amplification. Dual-mode fluorescence/electrochemical readout supports both laboratory analysis and on-site detection. The system demonstrates exceptional sensitivity, capable of detecting trace amounts of SA-16S rDNA (7.5 aM by fluorescence, 1.0 fM by electrochemistry) and SA (4 CFU/mL by fluorescence, 34 CFU/mL by electrochemistry). Overall, this work provides a modular and programmable biosensing strategy for bacterial identification, thereby facilitating advanced nucleic acid analysis within complex biological matrices.