Molecular probes for diagnosis of fungal infections

TLDR

The authors designed 21 species‑specific nucleic acid probes targeting the 28S rRNA gene of 50 fungal pathogens and standardized a universal PCR‑based detection workflow involving alkaline guanidine‑phenol extraction, universal primer amplification, and probe hybridization or sequencing for all fungi. Testing on fungal isolates and clinical specimens demonstrated high specificity of the probe‑based detection system.

Abstract

We have developed 21 specific nucleic acid probes which target the large subunit rRNA genes from Aspergillus flavus, Aspergillus fumigatus, Aspergillus glaucus, Aspergillus niger, Aspergillus terreus, Blastomyces dermatitidis, Candida albicans, Candida (Torulopsis) glabrata, Candida guilliermondii, Candida kefyr, Candida krusei, Candida lusitaniae, Candida parapsilosis, Candida tropicalis, Coccidioides immitis, Cryptococcus neoformans var. gattii, Cryptococcus neoformans var. neoformans, Filobasidiella neoformans var. bacillispora, Filobasidiella neoformans var. neoformans, Histoplasma capsulatum, Pseudallescheria boydii, and Sporothrix schenckii. A section of the 28S rRNA gene from approximately 100 fungi, representing about 50 species of pathogens and commonly encountered saprophytes, was sequenced to develop universal PCR primers and species-specific oligonucleotide probes. Each step in the process of detection and identification was standardized to a common set of conditions applicable without modification to all fungi of interest and all types of clinical specimens. These steps consist of DNA extraction by boiling specimens in an alkaline guanidine-phenol-Tris reagent, amplification of a variable region of the 28S rRNA gene with universal primers, and amplicon identification by probe hybridization or DNA sequencing performed under conditions identical for all fungi. The results obtained by testing a panel of fungal isolates and a variety of clinical specimens indicate a high level of specificity.