Identification and characterization of dermatophyte species and strains with PCR amplification

Abstract

The aim of the present study was to use two polymerase chain reaction (PCR) methods, with (GACA)₄ and non-transcribed spacer (NTS) as primers, to identify and characterize dermatophyte isolates from dogs and cats to a species and strain level. A total of 45 isolates from nine dermatophyte species were collected from pet dogs and cats and subjected to PCR amplification with the microsatellite primer (GACA)₄. Dermatophyte strains of three of the same species collected from four cities were subjected to PCR amplification with the NTS primer set. These two PCR methods were applied to identify and characterize the dermatophyte isolates to a species and strain level. Regional differences among the strain specificities were also examined. The results from PCR with (GACA)₄ demonstrated that strains from the same species produced similar PCR product band patterns. In addition, these patterns differed among species, indicating that (GACA)₄ primer-based PCR was able to distinguish between the various dermatophyte species. By contrast, dermatophyte isolates and/or strains within the same species revealed various band patterns with NTS-based PCR. In addition, the results indicated that regional differences contributed to the variations in PCR product band patterns. Therefore, the results of the present study indicate that the NTS-based PCR method is efficient in distinguishing dermatophytes to the strain level, while a combination of (GACA)₄ and NTS primer-based PCR methods is able to clarify dermatophyte isolates to a species and strain level. The present study provides information concerning the identification of pathogenic fungi and the epidemiological characteristics of fungal skin diseases.

Keywords: dermatophyte; microsatellite primer; non-transcribed spacer; polymerase chain reaction.

Figure 1

Identification of dermatophyte species using (GACA)₄ primer-based PCR. PCR products of various dermatophyte isolates are shown. Lanes: M, molecular weight marker; (A) 1–3, T. rubrum; 4–6, T. mentagrophytes; 7–9, M. canis; (B) 1–3, M. canis; 4–6, M. gypseum; 7–9, E. floccosum; (C) 1 and 2, C. albicans; 3 and 4, C. glabrata; 5 and 6, C. tropicalis; 7 and 8, C. parapsilosis; (D) 1, T. rubrum; 2, T. mentagrophytes; 3, M. canis; 4, M. gypseum; 5, E. floccosum; 6, C. albicans; 7, C. parapsilosis; 8, C. tropicalis; 9, C. glabrata. PCR, polymerase chain reaction; T. rubrum, Trichophyton rubrum; T. mentagrophytes, Trichophyton mentagrophytes; M. canis, Microsporum canis; M. gypseum, Microsporum gypseum; E. floccosum, Epidermophyton floccosum; C. albicans, Candida albicans; C. glabrata, Candida glabrata; C. tropicalis; Candida tropicalis; C. parapsilosis; Candida parapsilosis.

Figure 2

Characterization of 54 T. rubrum strains using PCR with the NTS-1 primer sets. Patterns were designated arbitrarily as A, B, C, D and E. Lanes: M, molecular weight marker; 1–54, 54 T. rubrum strains collected from four cities. PCR, polymerase chain reaction; NTS, non-transcribed spacer; T. rubrum, Trichophyton rubrum.

Figure 3

Characterization of 54 T. rubrum strains using PCR with NTS-2 primer sets. Patterns were designated arbitrarily as I and II. Lanes: M, molecular weight marker; 1–54, 54 T. rubrum strains collected from four cities. PCR, polymerase chain reaction; NTS, non-transcribed spacer; T. rubrum, Trichophyton rubrum.

Figure 4

Characterization of 26 T. mentagrophytes strains using NTS-based PCR. PCR products with (A) NTS-1 primer sets (patterns were designated arbitrarily as A, B, C and D) and (B) NTS-2 primer sets (patterns were designated arbitrarily as I and II). Lanes: M, molecular weight marker; 1–26, 26 T. mentagrophytes strains collected from four cities. PCR, polymerase chain reaction; NTS, non-transcribed spacer; T. mentagrophytes, Trichophyton mentagrophytes.

Figure 5

Characterization of 32 M. canis strains using NTS-based PCR. PCR products with (A) NTS-1 primer sets (patterns were designated arbitrarily as A, B, C and D) and (B) NTS-2 primer sets. Lanes: M, molecular weight marker; 1–32, 32 M. canis strains collected from four cities. PCR, polymerase chain reaction; NTS, non-transcribed spacer; M. canis, Microsporum canis.