Rapid and sensitive detection of Staphylococcus aureus (S. aureus) is critical for food safety and public health. Herein, this work developed an ABC (aptamer-B-PER-CRISPR) cascade detection platform, where B-PER stands for bidirectional primer exchange reaction, that integrates aptamer-mediated target recognition, B-PER isothermal amplification, and CRISPR/Cas12a trans-cleavage for highly sensitive and specific detection of S. aureus. Upon target recognition, the trigger strand T is released from the aptamer-T duplex, initiating B-PER to generate abundant products containing sequences recognized by the guide RNA (gRNA), which activate Cas12a to cleave the reporter probe, generating a detectable fluorescence signal. Under optimized conditions, the platform exhibited a linear range of 102 to 1010 CFU per mL with a regression equation of F520 nm = 575.36 log C + 3246.47 (R2 = 0.9992) and a limit of detection of 13 CFU per mL. The assay showed excellent specificity for S. aureus, with negligible cross-reactivity toward Vibrio parahaemolyticus and Salmonella. Recovery tests in spiked milk samples yielded average recoveries of 98.9-100.7%. Overall, the ABC platform offers high sensitivity, excellent specificity, robust anti-interference capability, and operational simplicity, demonstrating great potential for rapid and reliable detection of foodborne pathogens in complex matrices.
