Development of a TaqMan probe-based multiplex real-time PCR for the simultaneous detection of four clinically important filamentous fungi

Filamentous fungi present significant health hazards to immunocompromised individuals globally; however, the prompt and precise identification of them during infection remains challenging. In this study, a TaqMan probe-based multiplex real-time PCR (M-qPCR) assay was developed to detect simultaneously the target genes of four important pathogenic filamentous fungi: ANXC4 gene of Aspergillus fumigatus, EF1-alpha gene of Fusarium spp., mitochondrial rnl gene of Mucorales, and hcp100 gene of Histoplasma capsulatum. In this M-qPCR assay, the limit of detection (LoD) to all four kinds of fungi was 100 copies and the correlation coefficients (R-2) were above 0.99. The specificity of this assay is 100%, and the minimum detection limit is 100 copies/reaction. In conclusion, an M-qPCR detection assay was well established with high specificity and sensitivity for rapid and simultaneous detection on four important filamentous fungi in the clinic.