Rapid and Specific Detection of Mycoplasma pneumoniae Using Pyrococcus furiosus Argonaute Combined with Recombinase Polymerase Amplification

Mycoplasma pneumonia is a common pathogen of human respiratory infections and one of the leading causes of community-acquired pneumonia. Early and rapid diagnosis of its infection is crucial for clinical treatment decisions. In this study, we innovatively combined recombinase polymerase amplification (RPA) with Pyrococcus furiosus Argonaute protein (PfAgo) to establish a novel molecular diagnostic method for Mycoplasma pneumonia. This assay demonstrated high specificity with no cross-reactivity with other common respiratory pathogens. The limit of detection was 2×104 copies μl⁻¹. Furthermore, evaluation using 37 clinical samples showed 100% specificity and 86.36% sensitivity compared to quantitative real-time PCR (qPCR). The entire workflow, including sample preparation, can be completed within 2.5 hours and requires only basic instrumentation. This method holds great potential for application in primary healthcare settings and resource-limited regions.