| Popis: |
Tritrichomonas foetus (TF) is a significant reproductive pathogen of cattle, and sample collection, handling, transport, and testing are significant hurdles to surveillance programs. Recent methods have been developed that allow for the direct detection of TF using a reverse transcription real-time PCR (direct RT-qPCR) approach. To evaluate these methods, a comparative analysis was conducted to assess the technical performance of this assay with a commercially available real-time PCR (qPCR) assay. In addition, the evaluation of two types of collection media (PBS and TF transport tube) was conducted that evaluated sample stability from 0 to 3 days when stored at 4°C or 25°C. Extended incubation times for PBS media were also evaluated (5, 7, and 14 days) at both refrigeration and frozen temperatures to evaluate the effect of extended transport time on samples. Limits of detection (LODs), dynamic range, and RNA stability were assessed using lab-cultured TF spiked into samples of normal bovine smegma collected in PBS or TF transport media, and performance was assessed on field samples collected in parallel. 100% agreement was found between direct RT-qPCR and qPCR at 10 parasites/extraction and a LOD of 1 parasite/extraction. Differences in detection were not observed in either collection media when incubated at either temperatures for up to 3 days of incubation. In addition, the extended incubation experiments indicate that samples containing 10 parasites/extraction can be detected at 4°C for 5 days with a mean Cq 26.34 (95% CI: 23.11–29.58) and detected at −20°C for 7 or 14 days, with a mean Cq 29.55 (95% CI: 27.73–31.37). A significant decrease in detectable RNA was observed in samples containing |
