Development and mechanistic exploration of a rapid visual fluorescent MIRA-PfAgo assay for the detection of Staphylococcus aureus.

Staphylococcus aureus (S. aureus) is a major pathogen responsible for both clinical and community-acquired infections, and its rapid and specific detection is essential for early infection management. To address the limitations of existing detection methods, such as laborious procedures and dependence on sophisticated instruments, this study coupled the efficient amplification capability of multienzyme isothermal rapid amplification (MIRA) with the sequence-specific DNA cleavage activity of Pyrococcus furiosus Argonaute (PfAgo) and constructed a dual-modal MIRA-PfAgo detection system that enables both real-time fluorescence monitoring and visual result interpretation. This system allows the detection of S. aureus within 1 h, with a limit of detection (LOD) as low as 1 copy/μL. In addition, evaluation using 267 clinical isolates demonstrated an accuracy of 99.63% and a specificity of 100%, highlighting its considerable potential as an efficient and reliable tool for clinical pathogen detection. Furthermore, molecular simulation analysis suggested that, in the Mn2+-mediated reaction system, target DNA binding enhanced the overall stability of the PfAgo complex and induced conformational rearrangement of the catalytic center, as well as remodeling of the bimetallic ion coordination environment, thereby enabling precise target DNA recognition and specific cleavage by PfAgo. These results further provide supplementary molecular-level structural support for understanding the molecular basis of efficient and specific PfAgo-based nucleic acid detection.