Internal Transcribed Spacer rDNA and TEF-1α Gene Sequencing of Pathogenic Dermatophyte Species and Differentiation of Closely Related Species Using PCR-RFLP of The Topoisomerase II

Zahra Salehi, Masoomeh Shams-Ghahfarokhi, Mehdi Razzaghi-Abyaneh, Zahra Salehi, Masoomeh Shams-Ghahfarokhi, Mehdi Razzaghi-Abyaneh

Abstract

Objective: Precise identification of dermatophyte species significantly improves treatment and controls measures of dermatophytosis in human and animals. This study was designed to evaluate molecular tools effectiveness of the gene sequencing and DNA-based fragment polymorphism analysis for accurate identification and differentiation of closelyrelated dermatophyte species isolated from clinical cases of dermatophytosis and their antifungal susceptibility to the current antifungal agents.

Materials and methods: In this experimental study, a total of 95 skin samples were inoculated into mycobiotic agar for two weeks at 28˚C. Morphological characteristics of the isolated dermatophytes were evaluated. DNA was extracted from the fungal culture for amplification of topoisomerase II gene fragments and polymerase chain reaction (PCR) products were digested by Hinf I enzyme. Internal transcribed spacer (ITS) rDNA and TEF-1α regions of the all isolates were amplified using the primers of ITS1/4 and EF-DermF/EF-DermR, respectively.

Results: Based on the morphological criteria, 24, 24, 24 and 23 isolates were identified as T. rubrum , T. interdigitale, T. tonsurans and E. floccosum, respectively. PCR-restriction fragment length polymorphism (RFLP) results provided identification pattern of the isolates for T. rubrum (19 isolates), T. tonsurans (28 isolates), T. interdigitale (26 isolates) and E. floccosum (22 isolates). Concatenated dataset results were similar in PCR-RFLP, except six T. interdigitale isolates belonging to T. mentagrophytes.

Conclusion: Our results clearly indicated that conventional morphology and PCR-RFLP were not able to precisely identify all dermatophyte species and differentiation of closely related species like T. interdigitale and T. mentagrophytes, while ITS rDNA and TEF-1α gene sequence analyses provided accurate identification of all isolates at the genus and species level.

Trial registration: ClinicalTrials.gov NCT01115634.

Keywords: Dermatophytes; Gene Sequencing; Polymerase Chain Reaction-Restriction Fragment Length Polymorphism; Topoisomerase II.

Conflict of interest statement

There is no conflict of interest in this study.

Copyright© by Royan Institute. All rights reserved.

Figures

Fig.1
Fig.1
Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) electrophoretic patterns of dermatophytes species by amplification of topoisomerase II gene and digestion of the Hinf I enzyme. Lane M; 100 bp DNA ladder, Lane 1; T. tonsurans, Lane 2; T. tonsurans (CBS 130924), Lane 3;T. interdigitale, Lane 4; T. mentagrophytes, Lane 5; T. rubrum, Lane 6; T. rubrum (PFCC 51431), Lane 7; E. floccosum, and Lane 8; E. floccosum (CBS 767.73).
Fig.2
Fig.2
Bayesian tree based on the combined dataset. Phylogenetic analysis of the combined dataset with TIM2+G model of the 95 clinical isolates, four standard strains and Fusarium, as the out-group. Posterior probabilities more than 60% are given for the appropriate clades.

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