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, Ph.D, 1Masoomeh Shams-Ghahfarokhi, Ph.D, 1,*Mehdi Razzaghi-Abyaneh, Ph.D, 2
Department of Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Department of Mycology, Pasteur Institute of Iran, Tehran, Iran
Department of Mycology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
Department of Mycology, Pasteur Institute of Iran, Tehran, Iran
*Corresponding Address: P.O.Box: 14115-331 Department of Mycology Faculty of Medical Sciences Tarbiat Modares University Tehran Iran Email:shamsm@modares.ac.ir
The Cell Journal (Yakhteh) is an open access journal which means the articles are freely available online for any individual author to download and use the providing address. The journal is licensed under a Creative Commons Attribution-Non Commercial 3.0 Unported License which allows the author(s) to hold the copyright without restrictions that is permitting unrestricted use, distribution, and reproduction in any medium provided the original work is properly cited. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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 closely- related 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.