An Efficient Workflow for Fungal Nucleic Acid Detection in Sputum.

Respiratory fungal infections are challenging to diagnose due to non-specific symptoms and limitations of conventional methods. This study established an integrated, rapid workflow for detecting three common pathogenic fungi, combining an optimized nucleic acid extraction method with a TaqMan-based multiplex real-time qPCR system. The high-salt lysis method yielded high-purity nucleic acid extracts, with performance comparable to or better than commercial kits. The multiplex qPCR assay was optimized for high specificity, sensitivity, and reproducibility, with detection limits as low as 10 CFU/mL for some fungi. Clinical validation using 14 sputum samples demonstrated high agreement with conventional culture, and the assay detected a low-load infection missed by culture. This integrated approach shows great potential for rapid, sensitive, and specific clinical detection of respiratory fungal infections.