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

Summary

This research developed a new rapid test called FRET-PCR that can identify three common fungal infections in dogs within 2.5 hours, compared to 2-4 weeks for traditional culture methods. The test works by detecting specific genetic markers in fungal DNA and measuring temperature changes that differ for each fungal species. When compared to two other molecular tests (SYBR Green and EvaGreen), the FRET-PCR method was more accurate and sensitive, even able to detect a single copy of the target gene. This faster, more accurate identification will help veterinarians choose the right treatment more quickly and prevent spread of fungal infections.

Background

Conventional diagnosis of dermatophytosis relies on fungal culture and microscopic examination, which are time-consuming and lack sensitivity. Microsporum canis, Nannizzia gypsea, and Trichophyton mentagrophytes are the most common dermatophytes causing superficial fungal infections in canines. Molecular diagnostic methods, particularly real-time PCR, offer technical advantages in speed, sensitivity, and specificity over conventional methods.

Objective

This study aimed to develop and compare real-time PCR assays for the simultaneous detection and differentiation of three major dermatophytes in dogs using SYBR Green, EvaGreen, and dual-emission fluorescence resonance energy transfer (FRET) platforms targeting the chitin synthase 1 gene.

Results

The FRET assay demonstrated superior performance with a detection limit of a single gene copy and produced distinct melting profiles (M. canis ~56.1°C, N. gypsea ~53.0°C, T. mentagrophytes ~51.8°C). SYBR Green and EvaGreen showed reduced sensitivity and cross-reactivity with non-target fungi. FRET-qPCR achieved 100% specificity across all tested strains with no false positives in non-dermatophyte controls.

Conclusion

The FRET-based qPCR assay provides a rapid, reliable tool for accurate species-level identification of the three most prevalent canine dermatophytes with results within 2.5 hours. Its superior analytical performance, high specificity, and tolerance for low DNA inputs make it well-suited for clinical implementation and molecular surveillance of dermatophytosis in companion animals.
  • Published in:Journal of Fungi (Basel),
  • Study Type:Comparative Laboratory Study,
  • Source: PMID: 41149898, DOI: 10.3390/jof11100708
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