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Application of PCR-RFLP to Rapid Identification of the Main Pathogenic Dermatophytes from Clinical Specimens

Application of PCR-RFLP to Rapid Identification of the Main Pathogenic Dermatophytes from Clinical Specimens,H Mirzahoseini,E Omidinia,M Shams-Ghahfar

Application of PCR-RFLP to Rapid Identification of the Main Pathogenic Dermatophytes from Clinical Specimens  
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Background: In the present study, a PCR-RFLP based molecular technique was designed to rapid identification of der- matophytes in clinical specimens. Skin scrapings obtained from human cases suspected to dermatophytosis were studied in order to identify involved etiological fungi. Methods: In this experimental study, the specimens (skin scrapings) of patients referred to Mycology Department of Pas- teur Institute of Iran were inoculated on Petri dishes contained selective agar for pathogenic fungi (SAPF) and incubated at 25º C until visible growth of fungal colonies. The colonies were examined for standard morphological characteristics after visible growth on the agar medium. A small portion of each fungal colony was further studied by restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified internal transcribed spacer (ITS) region of ribosomal DNA (rDNA). PCR amplicons were electrophoresed on 2% agarose gel after digesting by different restriction enzymes including Mva I, Hinf I and Hae III. Results: Among 160 clinical samples examined, 6 dermatophyte species including Trichophyton mentagrophytes, T. ru- brum, T. verrucosum, T. tonsurans, Microsporum canis and Epidermophyton floccosum were finally identified based on the colony morphology and microscopic criteria. Specific PCR products and RFLP patterns for Mva I, Hinf I and Hae III en- zymes allowed the rapid identification and reliable differentiation of isolated dermatophytes at the genus or species level for 5-10 day-old colonies. Conclusions: The results showed that PCR-RFLP analysis of the ITS region of rDNA is a rapid and reliable tool which al- lows identification of major pathogenic dermatophytes isolated in this study at species level in young 5-10 day-old colonies.
Published in 2009.
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