Species-identification of dermatophytes Trichophyton, Microsporum and Epidermophyton by PCR and PCR-RFLP targeting of the DNA topoisomerase II genes

Background: We have focused on the DNA topoisomerase 11 genes of pathogenic fungi and have previously applied polymerase chain reaction (PCR)-based identification of several species including the some of the major dermatophyte species. Objective: To identify the dermatophytes (18 species) to a species level by PCR and PCR-restriction fragment Length polymorphism (RFLP) techniques, without determining the nucleotide sequence. Methods: The genomic DNAs of the dermatophytes (ten species of Trichophyton, seven species of Microsporum, and Epidermaphytbn floccosum) were amplified by PCR using a common primer set (dPsD1) for the dermatophytes, followed by nested PCR using other primer sets (dPsD2, PsT and PsME) that contained primers specific for the DNA topoisomerase 11 genes of the dermatophytes. PCRs using PsT and PsME were used for the species-identification of Trichophyton, Microsporum and E. floccosum. The PCR products generated by dPsD2 were digested with restriction enzymes (Hinc 11, Hinf, Afl 11 and PflM 1), and the restriction profiles were analyzed. Results: Of the eighteen species of dermatophytes, five species (T rubrum, T violaceum, M. canis, M. gypseum and E. floccosum) were specifically identified by the PCR using PsT and PsME to the species level, and the remaining species were identified by the unique restriction profiles for each species in the PCR-RFLP analysis, except that the restriction profile of T mentagrophytes var. interdigitale was identical to that of T mentagrophytes var. quinckeanum. Conclusion: PCR and PCR-RFLP techniques targeting the DNA topoisomerase 11 gene are simple and rapid, and quite useful as tools for the identification of dermatophytes to the species level. (C) 2003 Elsevier Ireland Ltd. on behalf of Japanese Society for Investigative Dermatology. All rights reserved.