Identification ofSporothix schenckiiof various mtDNA types by Nested PCR Assay

We employed a nested PCR assay to detect Sporothrix schenckii DNA of 38 strains (including all the 24 mtDNA types) collected from different areas of the world, in tissue of eight mice infected with ATCC10268 strain of the fungus, and the skin biopsies of nine patients with sporotrichosis. In addition, the same procedures were used with two strains of Ceratocystis minor and isolates of 10 species of other pathogenic fungi. The outer primers SS(1) and SS(2) and inner primers SS(3) and SS(4) of the 18S rRNA gene of S. schenckii were employed. A 152 bp fragment was detected in all 38 strains of S. schenckii, eight animal specimens and nine human skin biopsies, but not samples of C. minor and the other fungal species. The detection limit of 50 fg of S. schenckii DNA extract was determined with ethidium bromide staining. In summary, we demonstrated that nested PCR assay could identify S. schenckii of all the mtDNA types and in isolates recovered from different areas of the world. The nested PCR assay seems to be highly sensitive and specific and provides a rapid method for diagnosis of sporotrichosis under conditions of contamination avoidance.