PfAgo-DNAzyme cascade amplification improves Salmonella typhimurium detection

A DNA preamplification-free method was developed for sensitive detection of Salmonella typhimurium ( S. typhimurium ) by combining Pyrococcus furiosus Argonaute ( Pf Ago), a programmable small DNA-guided endonuclease and label-free DNAzyme, a functional nucleic acid (FNA). The effects of 5′-phosphorylated/hydroxylated single-strand DNA (5′P/OH ssDNA), including chimeric ssDNA-RNA and ssDNA-RNA-DNA (DNAzyme) on Pf Ago-mediated cleavage activity, were systematically investigated. Two detection strategies were developed: (1) A 63 nt 5′P DNAzyme was used as a guide DNA (gDNA) to mediate Pf Ago cleavage of probe MB1, resulting in a fluorescence signal inversely proportional to S. typhimurium concentration. RNase H2 from S. typhimurium (STH2) cleaved the DNAzyme, thereby reducing the signal. The limit of detection (LOD) was 4.5 × 10 CFU mL −1 , with a linear range from 8.0 × 10 to 6.4 × 10 4  CFU mL −1 (2) A 64 nt 5′OH DNAzyme was cleaved by STH2 to generate a 50 nt 5′P ssDNA, triggering Pf Ago-mediated MB2 cleavage. The fluorescence signal positively correlated with the concentration of S. typhimurium , with the LOD reduced to 2.0 × 10 CFU mL −1 and a detection range from 3.2 × 10 to 8.0 × 10 4  CFU mL −1 . Compared with the previous LOD of 3.2 × 10 3  CFU mL −1 achieved using the same DNAzyme labeled with a fluorescence probe and quencher, but without Pf Ago, the sensitivity of these methods was significantly improved, with increases of 71- and 160-folds, respectively. This method offers advantages of simplicity, specificity, rapidity, and sensitivity, making it suitable for point-of-care testing and holding promising potential for environmental monitoring, food safety, and clinical diagnosis.