A programmable aptamer-triggered B-PER/CRISPR cascade amplification platform for ultrasensitive detection of Staphylococcus aureus.

Abstract

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.