PCR Fluorescent Probe-based Detection of Aspergillus Spp., Cryptococcus neoformans, and Pneumocystis jirovecii

This protocol describes a standardized procedure for the qualitative detection of Aspergillus spp., Cryptococcus neoformans, and Pneumocystis jirovecii in clinical sputum samples using a PCR fluorescent probe-based nucleic acid detection kit. The procedure involves clinical sample collection, alkaline liquefaction pretreatment, automated nucleic acid extraction, and multiplex real‑time PCR detection. Aspergillus spp., C. neoformans, and P. jirovecii are detected using target‑specific fluorescent probes (FAM, VIC, and CY5 channels, respectively), with an internal control (ROX channel) integrated for quality assurance. Assay validity and sample result interpretation are based on defined cycle threshold (Ct) cut-off values. A positive control must exhibit S-shaped amplification curves with Ct ≤ 33.7 in all channels, while the negative control must show no amplification. For clinical samples, a Ct value ≤ 33.7 in the FAM channel indicates positivity for Aspergillus spp., and a Ct value ≤ 36 in the VIC or CY5 channel indicates positivity for C. neoformans or P. jirovecii, respectively.