PfAgo-guided SERS signal conversion for amplification-free and semi-quantitative detection of Salmonella typhimurium in food and physiological samples

Rapid detection of foodborne pathogens is critical for food safety. Conventional qPCR offers high sensitivity but relies on nucleic acid amplification, increasing operational complexity and false-positive risks. Here, we developed a biosensor platform named PAF-SERS ( P f Ago-mediated A mplification- F ree S urface- E nhanced R aman S cattering Biosensor) for detecting Salmonella typhimurium ( S. typhimurium ) without pre-amplification. In this platform, Pf Ago specifically cleaves target DNA guided by a single-stranded DNA guide, generating a linker sequence that enables quantitative detection through SERS signal conversion. The assay completes within 55   min, with a detection limit as low as 10 CFU/mL and a linear range of 10^ 1 to 10^ 7 CFU/mL, exhibiting high specificity and reproducibility. We further applied a support vector machine (SVM) algorithm to accurately classify low-concentration samples. The method was successfully validated in milk, eggs, chicken, and feces and blood from infected mice, demonstrating its potential for rapid, on-site detection without bulky instruments.