Ribosomal DNA Gene Analysis of Dematiaceous Fungi Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism / 대한피부과학회지

ABSTRACT

BACKGROUND: There are three kinds of diseases caused by dematiaceous fungi chromoblastomycosis, phaeohyphomycosis, and eumycotic mycetoma. The dematiaceous fungi have been identified and classified by morphological, biochemical and physiological tests. Recently molecular analysis has been introduced to the field of medical mycology. OBJECTIVE: Ribosomal DNA gene analysis of dematiaceous fungi using polymerase chain reaction(PCR)-restriction fragment length polymorphism(RFLP) is investigated. METHODS: The dematiaceous fungal strains studied were eight clinical isolates of chromoblastomycosis and phaeohyphomycosis agents(3 strains of Fonsecaea pedrosoi, 2 strains of Exophiala dermatitidis, 1 strain of Exophiala jeanselmei, 1 strain of Phialophora verrucosa, 1 strain of Rhinocladiella aquaspersa) and 4 standard strains(F. pedrosoi IFM 4889, E. dermatitidis IFM 4828, P. verrucosa IFM 4928, R. aquaspersa IFM 4930). Total twelve strains of dematiaceous fungi were cultured on Sabouraud dextrose broth and their DNA was extracted by bead-beating method. PCR-RFLP of ribosomal gene small subunit(SSU rDNA) and internal transcribed spacer(ITS)1-ITS2 ribosomal regions using the primer pairs NS1-NS2 and ITS1-ITS4, respectively were done. RESULTS: The PCR product obtained from the SSU rDNA amplification was of approximately 603 bp and restriction profile was clustered into two genetically heterogenous groups, the first one formed by F. pedrosoi and the second one formed by other dematiaceous fungi. In contrast, the amplified PCR products of ITS regions ranged in sized from 580 to 620 bp and restriction profile was clustered into five genetically heterogenous groups and it can be possible to differentiate dematiaceous fungi. But one clinical isolate of F. pedrosoi showed intra-specific variability. CONCLUSION: The PCR-RFLP analysis of SSU rDNA and ITS regions provided useful information for identification and classification of dematiaceous fungi.