Molecular probes for the detection of pathogenic fungi in the presence of human tissue.

Four primer systems, amplifying fragments of the gene coding for the small ribosomal subunit (18S rRNA) were characterised with pure cultures of 65 medically relevant fungal species plus two mushrooms. A primer cocktail (TR1/CA1-TR2/AF2) amplified 59 of 67 fungal species; the universal fungal primer 1 (UF1) in combination with the eukaryotic primers S3 or EU1 amplified 64 and 65 of 67 fungal species, respectively. The design of an additional primer (RZY1) enabled the amplification of the missing members of the zygomycetes. The primer systems amplified all the medically relevant fungi tested. These included eight Candida spp. and seven other yeast species, 13 dermatophytes, 32 moulds (including six zygomycetes and five dimorphic fungi) and two mushrooms. Eleven controls including DNA from Schistosoma mansoni, Escherichia coli, Mycobacterium tuberculosis and man were not amplified. The oligonucleotide CA hybridised with C. albicans, C. tropicalis and C. parapsilosis; the oligonucleotide TR hybridised with the 13 dermatophytes; the oligonucleotide AF hybridised with Aspergillus fumigatus, A. flavus, A. terreus, A. nidulans, A. versicolor, A. tamarii, A. clavatus, A. fischeri, but not with A. niger or A. versicolor; and the oligonucleotide HC hybridised with three varieties of Histoplasma capsulatum. These oligonucleotides did not hybridise with the other fungi nor the controls. The specificity of the newly designed primer systems was confirmed by selective amplification of fungal DNA from human lung tissue spiked with fungal biomass and from vitrectomy fluid of a patient with candida endophthalmitis.