Target-Amplification-Free Detection of Pathogenic Bacteria Via a Cascaded Nuclease Reaction

The translation of nucleic acid testing to point-of-care settings is hindered by the reliance on target amplification, which introduces complexity and contamination risks. Herein, we report a target-amplification-free assay for the direct detection of Staphylococcus aureus 16S rRNA, utilizing a cascaded DNAzyme and Nicking endonuclease reaction (DNECR). This system integrates a panel of target-specific multicomponent DNAzyme (MNAzyme) for primary recognition and signal amplification with a sterically blocked bipedal DNA walker (BDW) for cascade signal amplification. Upon target binding, the activated MNAzyme cleaves the blocker to initiate the BDW, which then traverses a spherical nucleic acid track via nicking endonuclease activity, generating amplified fluorescent signals. This cascaded design achieved a detection limit of 102 CFU/mL for cultured S. aureus with high specificity. Clinical validation using 12 patient sputum samples demonstrated 100% diagnostic sensitivity and specificity, confirming the potential of DNECR as a robust, amplification-free platform for rapid pathogen detection at the point of care.