Dual-Emission FRET-PCR Outperforms SYBR Green and EvaGreen for Accurate Discrimination of Primary Canine Dermatophytes: Microsporum canis, Nannizzia gypsea, and Trichophyton mentagrophytes

Conventional diagnosis of dermatophytosis relies on fungal culture and microscopic examination, methods that are often time-consuming and lack sensitivity. This study aimed to develop and compare real-time PCR assays for the simultaneous detection and differentiation of three major dermatophytes in dogs: Microsporum canis, Nannizzia gypsea, and Trichophyton mentagrophytes. Three qPCR platforms targeting the chitin synthase 1 (CHS1) gene—SYBR Green, EvaGreen, and dual-emission fluorescence resonance energy transfer (FRET)—were evaluated. The FRET assay demonstrated the highest performance, achieving a detection limit of a single gene copy per reaction and producing distinct melting profiles that enabled accurate species discrimination (M. canis ~56.1 °C, N. gypsea ~53.0 °C, T. mentagrophytes ~51.8 °C). In contrast, SYBR Green and EvaGreen assays showed reduced sensitivity and cross-reactivity with non-target fungi. All assays were validated using three ATCC reference strains, ten clinical isolates of the target dermatophytes, and nine additional fungal species, including Nocardia, Aspergillus, Fusarium, Sporothrix, and Candida. Overall, FRET-qPCR exhibited a 100% specificity and a detection limit of one copy of target gene per reaction, offering a rapid, reliable tool for accurate diagnosis and molecular surveillance of dermatophytosis in companion animals.