Gel-Free Detection of Tritrichomonas foetus in Cats: Preliminary Analytical Evaluation and Method Agreement Assessment of a One-Tube Nested Real-Time PCR-HRM Assay

Feline tritrichomonosis caused by Tritrichomonas foetus is an important cause of chronic or intermittent large-bowel diarrhea in cats. Nested PCR is useful for detecting low parasite burdens in fecal samples, but gel-based endpoint interpretation requires post-amplification handling and may increase the risk of amplicon contamination. This study describes a one-tube nested real-time PCR-HRM assay for qualitative detection of T. foetus DNA in feline fecal extracts. The assay was adapted from a previously described single-tube nested PCR workflow by integrating real-time fluorescence monitoring and high-resolution melting analysis as a closed-tube endpoint. Analytical evaluation included post-extraction matrix-matched sensitivity testing, an exclusivity panel, matrix blank assessment, a checkerboard carryover experiment, and within-platform repeatability assessment of HRM temperatures. Method agreement was assessed using 147 fecal samples from diarrheic cats and compared with gel-based single-tube nested PCR and outer-primer PCR. Both nested methods detected 16/147 samples, whereas outer-primer PCR detected 5/147 samples. All 16 nested-positive amplicons were confirmed by Sanger sequencing and showed 99.51–100.00% identity to T. foetus reference sequences. Across 95 target-associated dual-peak HRM reactions, the low-temperature melting domain showed a mean Tm of 76.18 ± 0.24 °C, and the high-temperature melting domain showed a mean Tm of 81.57 ± 0.42 °C. However, because no independent reference standard was available, diagnostic sensitivity and specificity were not determined. The assay should therefore be interpreted as a preliminary analytical evaluation and method-agreement study of a closed-tube alternative to gel-based nested PCR.