PCR and PCR–RFLP techniques targeting the DNA topoisomerase II gene for rapid clinical diagnosis of the etiologic agent of dermatophytosis

Abstract

Background : We have focused on the DNA topoisomerase II genes of several pathogenic fungi, and developed polymerase chain reaction (PCR) and PCR–restriction fragment length polymorphism (RFLP) methods targeting this gene for identification of dermatophytes. Objective : To assess the availability of the PCR-based identification for an etiologic study of dermatophytosis, by testing these PCR and PCR–RFLP methods for stability and reproducibility. Methods : Three hundred and fifty-six dermatophyte strains were isolated from 305 patients with tinea, and their genomic DNAs were used as templates for the PCR using primer mixes (PsT, PsME, dPsD1 or dPsD2) composed of gene-specific primers for identification of dermatophytes to the species level. The genomic DNAs of Trichophyton rubrum were further subjected to subrepeat element analysis of the nontranscribed spacer (NTS) of ribosomal DNA (rDNA). Results : In this study, six dermatophyte species ( T. rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Microsporum canis, Microsporum gypseum, and Epidermophyton floccosum) were obtained. In all cases, the identifications obtained from the PCR and PCR–RFLP targeting the DNA topoisomerase II gene coincided with those from the conventional morphological features-based identification technique. The sensitivity of the PCR-based identification was found to be a colony of approximately 3 mm in diameter. Furthermore, T. rubrum was divided into three groups (17 types) on the basis of the sizes and numbers of the products generated from the TRS-1 region, and three types from the TRS-2 region. Conclusion : The PCR and PCR–RFLP targeting the DNA topoisomerase II gene were rapid, stable, and reproducible for species identification of dermatophytes, and thus are convenient tools for an etiologic study of dermatophytosis.