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

• Main Authors: H Mirzahoseini (Author), E Omidinia (Author), M Shams-Ghahfarokhi (Author), G Sadeghi (Author), M Razzaghi-Abyaneh (Author)
• Format: Book
• Published: Tehran University of Medical Sciences, 2009-03-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 2251-6085; 2251-6093
• Keywords: Dermatophytosis; PCR-RFLP; ITS region; Identification; Dermatophyte species; Public aspects of medicine; RA1-1270
• Source: Iranian Journal of Public Health, Vol 38, Iss 1 (2009)

Background: In the present study, a PCR-RFLP based molecular technique was designed to rapid identification of der­matophytes 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 Pas­teur 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 MvaI, HinfI and HaeIII. Results: Among 160 clinical samples examined, 6 dermatophyte species including Trichophyton mentagrophytes, T. ru­brum, 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 MvaI, HinfI and HaeIII en­zymes 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 al­lows identification of major pathogenic dermatophytes isolated in this study at species level in young 5-10 day-old colonies.