DNA and ATP synergistically triggered Argonaute-mediated sensor for the ultrasensitive detection of viable Salmonella without DNA extraction and amplification

Foodborne pathogens present a significant threat to human health. In this study, a phage-assisted DNA and ATP synergistically triggered Argonaute-mediated fluorescent (PDAAF) biosensor was developed for the ultrasensitive detection of viable Salmonella with DNA extraction-free and amplification-free. The phage can lyse viable Salmonella to release DNA and ATP, which can reflect the amount of bacteria present. First, the two-step cleavage ability of Pyrococcus furiosus Argonaute (PfAgo) was used to identify the target DNA and cleave fluorescent probes. Second, the bivalent aptamer recognized the ATP and released guide DNA that combined with PfAgo to cleave fluorescent probes. The DNA and ATP synergistically triggered double cleavage in a single tube, greatly enhancing the detection sensitivity for Salmonella (limit of detection was 20 CFU/mL). The spiked recoveries (87.72%-113.97%) and real sample analysis demonstrated that the PDAAF biosensor possesses good accuracy and applicability, effectively enabling the DNA extraction- and amplification-free detection of foodborne pathogens.