Ann Dermatol.  2014 Jun;26(3):338-342.

Identification of Dermatophytes by Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Analysis of Metalloproteinase-1

Affiliations
  • 1Department of Dermatology, Konkuk University School of Medicine, Seoul, Korea. kjahn@kuh.ac.kr
  • 2Research Institute of Medical Science, Konkuk University, Seoul, Korea.

Abstract

BACKGROUND
Transgenic 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.
OBJECTIVE
To 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).
METHODS
From 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.
RESULTS
The 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.
CONCLUSION
To 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.

Keyword

Metalloproteinases; Microsporum; Polymerase chain reaction; Restriction fragment length polymorphism; Trichophyton

MeSH Terms

Arthrodermataceae*
Base Sequence
Clinical Coding
Databases, Nucleic Acid
Digestion
DNA
Electrophoresis
Epidemiology
Metalloproteases
Microsporum
Molecular Biology
Plants
Polymerase Chain Reaction
Polymorphism, Restriction Fragment Length
Trichophyton
DNA
Metalloproteases

Figure

  • Fig. 1 Nucleotide sequence alignment of metalloproteinase-1 genes of various dermatophytes. Complements of the sequences highlighted in the yellow boxes were used as primers for the amplification of metalloproteinase-1.

  • Fig. 2 Restriction fragment length polymorphism patterns obtained upon DNA digestion with BsrDI. M: 100-base pair (bp) marker, 1: Microsporumcanis (IFM45829), 2: M. gypseum (IFM-5292), 3: Trichophyton mentagrophytes var. interdigitale (IFM-48155), 4: T. mentagrophytes var. mentagrophytes (CBS113880), 5: T. rubrum (ATCC28188), 6: T. tonsurans (CBS109036).


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