Self-Propagating Rolling Circle Amplification Enabled by a Dual-Function Fluorogenic Primer for Ultrasensitive Detection of Salmonella

Abstract Sensitive detection of foodborne pathogenic bacteria is essential for food safety monitoring and public health protection. Herein, we report a self-propagating hyperbranched rolling circle amplification (SP-HRCA) biosensing strategy enabled by a dual-function fluorogenic primer (DFP) for ultrasensitive detection of Salmonella. In this strategy, recombinase polymerase amplification (RPA) combined with λ-exonuclease (λ-exo)-assisted strand-selective digestion was employed to convert double-stranded DNA amplicons into target-specific single-stranded DNA intermediates, enabling efficient coupling between RPA and rolling circle amplification (RCA). More importantly, a specially engineered DFP was introduced as the central amplification relay unit. Upon hybridization with RCA products, the DFP simultaneously generated fluorescence signals through hairpin opening and functioned as a newly activated primer to initiate subsequent amplification events. This design transformed conventional fluorescent reporters from passive signal outputs into active amplification propagators, thereby intrinsically coupling signal transduction with amplification propagation. Through recursive amplification propagation, hyperbranched RCA nanostructures were continuously generated, resulting in markedly enhanced fluorescence output. Under optimized conditions, the proposed strategy exhibited a linear response from 101 to 106 cfu/mL with a detection limit of 8 cfu/mL. In addition, excellent specificity and satisfactory performance in practical food samples were achieved. This work provides a versatile strategy for constructing self-propagating nucleic acid amplification systems for ultrasensitive biosensing applications.