Identification of dermatophytes by polymerase chain reaction-restriction fragment length polymorphism analysis of metalloproteinase-1.

Abstract

BackgroundTransgenic research on metalloproteinase-1 is an emerging field in the area of plant molecular biology. The new method reported here can similarly be applied in fungal molecular biology to identify different dermatophytes. Our method is more accurate than traditional methods such as molecular analyses.ObjectiveTo identify Trichophyton rubrum, T. mentagrophytes var. mentagrophytes, T. tonsurans, T. mentagrophytes var. interdigitale, Microsporum canis and M. gypseum, by using the restriction fragment length polymorphism (RFLP) analysis and polymerase chain reaction (PCR) to detect polymorphisms in the metalloproteinase-1 gene (MEP1).MethodsFrom each fungal strain, we isolated genomic DNA and performed PCR to amplify the region coding for metalloproteinase-1. Primers for the metalloproteinase-1 gene were designed based on the sequence in NCBI GenBank. Subsequently, we purified the amplified PCR product and performed RFLP analysis. After restriction enzyme digestion, BsrDI (NEB, England), the samples were subjected to electrophoresis. Four different patterns of DNA fragments were observed among 6 fungal species.ResultsThe DNA fragments for T. mentagrophytes var. mentagrophytes, T. mentagrophytes var. interdigitale and T. tonsurans showed similar patterns on electrophoresis and were not distinguishable, whereas T. rubrum, M. canis, and M. gypseum showed different patterns.ConclusionTo our knowledge, it is the first study to introduce the analysis of the nucleotide sequence of metalloproteinase-1 enzyme to study differentiation in dermatophytes. Based on our results, more accurate differentiation and subtyping of T. rubrum and T. mentagrophytes var. interdigitale might be possible. This might contribute to better understanding of the epidemiology and pathogenesis of dermatophyte.