Open AccessRestriction Analysis of β‐Tubulin Gene for Differentiation of the Common Pathogenic Dermatophytes
Author(s) -
Abastabar Mahdi,
Mirhendi Hossein,
RezaeiMatehkolaei Ali,
Shidfar Mohammad Reza,
Kordbacheh Parivash,
Makimura Koichi
Publication year - 2014
Publication title -
journal of clinical laboratory analysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 50
eISSN - 1098-2825
pISSN - 0887-8013
DOI - 10.1002/jcla.21649
Subject(s) - restriction enzyme , dermatophyte , biology , restriction fragment length polymorphism , genetics , genbank , agarose gel electrophoresis , restriction digest , restriction fragment , terminal restriction fragment length polymorphism , gene , restriction site , microbiology and biotechnology , polymerase chain reaction
Background Identification of dermatophytes at the species level, relying on macro‐ and microscopic properties of the colonies is time‐consuming, questioned in many circumstances, and requires considerable expertise. In this study, we examined the potency of a new genetic marker, β‐tubulin (BT2) gene, for differentiation of dermatophytes in an in silico and experimental restriction fragment length polymorphism (RFLP) profile. Methods The BT2 sequences of dermatophyte species were retrieved from GenBank and analyzed using bioinformatics softwares to choose suitable restriction enzyme(s). Forty reference culture collections and 100 clinical isolates were PCR‐amplified using the primers T1 and Bt2b and consequently subjected to virtual RFLP analysis. The dermatophytes were identified according to specific lengths of bands in agarose gel electrophoresis. Results After digestion of partially amplified β‐tubulin gene with the restriction enzyme Fat I, three dermatophyte species, that is, Microsporum gypseum , M. canis, and Trichophyton verrucosum yielded unique restriction maps while the remaining species including T. interdigitale, T. rubrum, T. tonsurans, T. schoenleinii , and T. violaceum , were identified by further restriction digestion by Alw 21I, Mwo I, and Hpy CH4V endonucleases. The length of RFLP products was same as of those expected by computer analysis. Conclusion The two‐step BT2 restriction mapping used in this study is an effective tool for reliable differentiation of the clinically relevant species of dermatophytes.