UNIVERSAL FUNGUS-SPECIFIC PRIMER SYSTEMS AND GROUP-SPECIFIC HYBRIDIZATION OLIGONUCLEOTIDES FOR 18S RDNA

We designed two primer systems that amplify a fragment of the gene cod ing for the small ribosomal subunit (18S rRNA). A broadly reactive, ye t fungus-specific, primer cocktail comprises two previously published primers, TR1 and TR2, which specifically amplify dermatophytes, and tw o newly designed primers, CA1 and AF2, which specifically amplify Cand ida and Aspergillus respectively. This primer cocktail amplifies a DNA fragment of approximately 578 basepairs (bp) in length (from position 838 to 1415), which contains variable, possibly species-specific regi ons (V5, partly V7). Another newly designed primer, UF1 (universal fun gal primer 1), along with the eukaryotic primer S3 amplifies a 926-bp fragment (from position 263 to 1188) that includes the variable region s V3, V4 and V5. Both primer systems amplified DNA from Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fum igatus, Penicillium marneffei, Fusarium oxysporum and Trichophyton men tagrophytes, but not the DNA from Prototheca zopfii, Escherichia coli or humans. The previously published oligonucleotides TR and HC, which are specific for dermatophytes and Histoplasma respectively, and the n ewly designed group-specific oligonucleotides, CA and AF, hybridized w ith T. mentagrophytes, Histoplasma capsulatum, C. albicans and, A. fum igatus respectively, but not with the other six fungi or with the thre e controls.