Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis
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...
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Frontiers Media S.A.
2023-03-01
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| Series: | Frontiers in Veterinary Science |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fvets.2023.1101502/full |
| _version_ | 1797856294927859712 |
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| author | Duan S. Loy Renata Spuri Gomes Enakshy Dutta Bruce W. Brodersen John Dustin Loy |
| author_facet | Duan S. Loy Renata Spuri Gomes Enakshy Dutta Bruce W. Brodersen John Dustin Loy |
| author_sort | Duan S. Loy |
| collection | DOAJ |
| description | 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 <10 parasites/extraction at −20°C for 14 days, which should be considered for long-term storage. In summary, direct RT-qPCR was found to be equivalent or superior to qPCR and PBS was not significantly different from TF transport media. The findings of the current study allows for more flexibility during sample collection and transport and ultimately enhancement of TF surveillance programs. |
| first_indexed | 2024-04-09T20:38:08Z |
| format | Article |
| id | doaj.art-97c58af2ed454cc3a27093c2bef95ed0 |
| institution | Directory Open Access Journal |
| issn | 2297-1769 |
| language | English |
| last_indexed | 2024-04-09T20:38:08Z |
| publishDate | 2023-03-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Veterinary Science |
| spelling | doaj.art-97c58af2ed454cc3a27093c2bef95ed02023-03-30T07:01:12ZengFrontiers Media S.A.Frontiers in Veterinary Science2297-17692023-03-011010.3389/fvets.2023.11015021101502Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysisDuan S. Loy0Renata Spuri Gomes1Enakshy Dutta2Bruce W. Brodersen3John Dustin Loy4School of Veterinary Medicine and Biomedical Sciences, Nebraska Veterinary Diagnostic Center, University of Nebraska-Lincoln, Lincoln, NE, United StatesSchool of Veterinary Medicine and Biomedical Sciences, Nebraska Veterinary Diagnostic Center, University of Nebraska-Lincoln, Lincoln, NE, United StatesDepartment of Statistics, University of Nebraska-Lincoln, Lincoln, NE, United StatesSchool of Veterinary Medicine and Biomedical Sciences, Nebraska Veterinary Diagnostic Center, University of Nebraska-Lincoln, Lincoln, NE, United StatesSchool of Veterinary Medicine and Biomedical Sciences, Nebraska Veterinary Diagnostic Center, University of Nebraska-Lincoln, Lincoln, NE, United StatesTritrichomonas 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 <10 parasites/extraction at −20°C for 14 days, which should be considered for long-term storage. In summary, direct RT-qPCR was found to be equivalent or superior to qPCR and PBS was not significantly different from TF transport media. The findings of the current study allows for more flexibility during sample collection and transport and ultimately enhancement of TF surveillance programs.https://www.frontiersin.org/articles/10.3389/fvets.2023.1101502/fulltimetemperatureRNA stabilityPBSTritrichomonas foetusdirect reverse transcription quantitative real-time PCR |
| spellingShingle | Duan S. Loy Renata Spuri Gomes Enakshy Dutta Bruce W. Brodersen John Dustin Loy Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis Frontiers in Veterinary Science time temperature RNA stability PBS Tritrichomonas foetus direct reverse transcription quantitative real-time PCR |
| title | Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis |
| title_full | Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis |
| title_fullStr | Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis |
| title_full_unstemmed | Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis |
| title_short | Time and temperature stability of Tritrichomonas foetus in phosphate-buffered saline as evaluated by a reverse transcription real-time PCR assay and field analysis |
| title_sort | time and temperature stability of tritrichomonas foetus in phosphate buffered saline as evaluated by a reverse transcription real time pcr assay and field analysis |
| topic | time temperature RNA stability PBS Tritrichomonas foetus direct reverse transcription quantitative real-time PCR |
| url | https://www.frontiersin.org/articles/10.3389/fvets.2023.1101502/full |
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