Species identification and strain differentiation of dermatophyte fungi byanalysis of ribosomal-DNA intergenic spacer regions

Restriction fragment length polymorphisms (RFLPs) identified in the ribosom al-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplif ied from the small-subunit (18S) rDNA and adjacent internal transcribed spa cer (TTS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short re petitive sequences in the intergenic spacers of other fungi. Fourteen indiv idual RFLP patterns (DNA types A to N) were recognized among 50 random clin ical isolates of T. rubrum, A majority of strains (19 of 50 [38%]) were cha racterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA t ypes E to N) were represented by one or two isolates only. A rapid and simp le method was also developed for molecular species identification of dermat ophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclea se MvaI produced unique and easily identifiable fragment patterns for a maj ority of species. However, some closely related taxon pairs, such as T. rub rum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguish ed. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differen tiation of T. rubrum and for species identification of common dermatophyte fungi.