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

Abstract

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: <i>Microsporum canis</i>, <i>Nannizzia gypsea</i>, and <i>Trichophyton mentagrophytes</i>. Three qPCR platforms targeting the chitin synthase 1 (<i>CHS1</i>) 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 (<i>M. canis</i> ~56.1 °C, <i>N. gypsea</i> ~53.0 °C, <i>T. mentagrophytes</i> ~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 <i>Nocardia</i>, <i>Aspergillus</i>, <i>Fusarium</i>, <i>Sporothrix</i>, and <i>Candida</i>. 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.