Polymerase chain reaction-based detection of dermatophyte DNA with a fungus-specific primer system

Abstract

There is significant clinical interest in primers which are specific for fungi and do not hybridize to DNA of other eukaryotes or prokaryotes. Such primers would allow specific amplification of fungal DNA from human tissue samples containing fungi. Fungal identification to the species level could follow by direct sequencing or restriction analysis. Several previously described primer systems cross-react with DNA of plants and animals. We have designed a primer system that amplifies a fragment of the gene coding for the small ribosomal subunit 18S rRNA. Database searches and sequence analyses were performed using the HUSAR (Heidelberg Unix Sequence Analysis Resources) computer system at the German Cancer Research Centre, Heidelberg, Germany. Primers TR1 (5'-GTTTCTAGGACCGCCGTA) and TR2 (5'-CTCAAACTTCCATCGACTTG) bind to sequences which are homologous within the fungi, but differ from corresponding DNA fragments of plants and animals. The amplified fragment is 581 base pairs in length and contains variable, and therefore species-specific, regions. The DNA of Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton verrucosum, Trichophyton terrestre, Microsporum canis, Microsporum gypseum and Epidermophyton floccosum and of several yeast species was amplified by the primers, but not the DNA from 42 normal human skin samples. Furthermore, other DNA preparations from plants and animals, including those from radish, cabbage, wheat and mouse, did not show amplification reactions.