A PfAgo-assisted artificial nucleic acid circuit for extraction-free and ultrasensitive detection of Staphylococcus aureus in dairy products.

Staphylococcus aureus is a major foodborne pathogen associated with dairy products; however, rapid and reliable detection remains challenging due to complex food matrices. Herein, we developed a one-step isothermal biosensing strategy based on an artificial nucleic acid circuit-assisted Argonaute system (PfAgo-ANCA) for rapid and sensitive detection of S. aureus. By targeting the species-specific nuc gene, the platform utilizes the high-temperature endonuclease activity of Pyrococcus furiosus Argonaute (PfAgo). The strategy integrates programmable PfAgo-mediated target recognition with downstream artificial nucleic acid circuit-based signal amplification, converting target cleavage into an amplified fluorescence signal without conventional nucleic acid extraction, purification, or amplification. Under optimized conditions, the PfAgo-ANCA platform achieved a detection limit of 100 CFU/mL for pure S. aureus cultures and exhibited excellent specificity against S. aureus. In artificially contaminated milk and dairy beverage samples, the assay maintained a detection limit of 100 CFU/mL. Owing to the high-temperature reaction characteristics of PfAgo, the platform integrates bacterial thermal lysis and enzymatic signal amplification within a sealed single-tube format, thereby eliminating the need for conventional DNA extraction and purification procedures. Furthermore, the modularity of the PfAgo-ANCA platform enabled adaptation to different genetic targets through guide DNA redesign, supporting dual-gene detection of S. aureus and enterotoxin-associated genes. Overall, PfAgo-ANCA provides a distinct and modular strategy that combines programmable target recognition, circuit-based signal amplification, and simplified sample processing, showing considerable potential for rapid food safety monitoring.