Rapid and Ultrasensitive Detection of <i>Staphylococcus aureus</i> by a One-Pot System Integrating <i>Pyrococcus furiosus</i> Argonaute with Loop-Mediated Isothermal Amplification

<i>Staphylococcus aureus</i> (<i>S. aureus</i>) is one of the most common pathogens associated with oral and maxillofacial space infections (OMSI), significantly impairing patients' quality of life and posing substantial public health risks. Traditional detection methods are usually time-consuming and operationally complex, which limits their applicability for rapid on-site detection. This study introduces a novel, ultrasensitive nucleic acid detection system combining loop-mediated isothermal amplification (LAMP) with <i>Pyrococcus furiosus</i> Argonaute (<i>Pf</i>Ago) in a one-pot strategy. The combination of LAMP and <i>Pf</i>Ago not only improves specificity and sensitivity but also effectively reduces the risk of aerosol contamination and false positives. The system detects the <i>nuc</i> gene of <i>S. aureus</i>, enabling rapid detection within 50 min with a limit of detection (LOD) of 10⁰ Colony Forming Units (CFU)/ml for bacterial solutions and 10^-5 ng/μl for plasmids. This system exhibited exceptional specificity by effectively differentiating <i>S. aureus</i> from other common OSMI bacteria, and was successfully applied to samples from animal OMSI infection models. This innovative one-pot system offers a rapid, reliable, and effective solution for <i>S. aureus</i> detection, with potential applications in human health and public safety.