OS16 - 4.P4.c Tailed Primers Enhance Real-Time RT-PCR Detection of FMD Virus - D. Lefebvre
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This document summarizes a presentation on enhancing the specificity of real-time RT-PCR detection of foot-and-mouth disease virus through the use of tailed primers. The presentation evaluated adding tails to the primers for two different gene targets, 5'-UTR and 3D, and found that tailed primers improved detection of FMDV, with tailed 3D primers showing a consistent Cq value shift across multiple virus strains. Tailed primers enhance the robustness and performance of RT-PCR assays for FMDV detection and can help authorities demonstrate that a sample does not contain the virus by improving specificity and reducing risks of contamination, cross-reactivity, or detecting small amounts of viral RNA.
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OS16 - 4.P4.c Tailed Primers Enhance Real-Time RT-PCR Detection of FMD Virus - D. Lefebvre
- 1. Open Session of the EuFMD - Cascais –Portugal 26-28 October 2016 TAILED PRIMERS ENHANCE REAL- TIME RT-PCR DETECTION OF FOOT AND MOUTH DISEASE VIRUS F. Vandenbussche, E. Mathijs, D. Lefebvre, K. De Clercq and S. Van Borm
- 2. Use of a portable real-time reverse transcriptase-polymerase chain reaction assay for rapid detection of foot-and-mouth disease virus. Callahan JD, Brown F, Osorio FA, Sur JH, Kramer E, Long GW, Lubroth J, Ellis SJ, Shoulars KS, Gaffney KL, Rock DL, Nelson WM. J Am Vet Med Assoc. 2002 Jun 1;220(11):1636-42. 3D 5’-UTR 3D and 5’-UTR Reference methods
- 3. But what about specificity? When your authorities demand you to demonstrate that a sample does NOT contain FMDV…
- 4. But what about specificity? When your authorities demand you to demonstrate that a sample does NOT contain FMDV… • Contamination ? • Cross-reactivity ? • PCR artefact ? • … • Small amount of FMD viral RNA ?
- 5. But what about specificity? When your authorities demand you to demonstrate that a sample does NOT contain FMDV… • Contamination ? • Cross-reactivity ? • PCR artefact ? • … • Small amount of FMD viral RNA ? To improve the robustness and overall performance of the assays ?!
- 7. 5’-UTR: dilution series with tailed and non-tailed primers
- 8. 5’-UTR: effect of tailing of forward and/or reverse primers
- 9. 3D: 5 / 50 FMDV strains showed Cq value shift (3.43 ± 0.11)
- 10. 3D: 5 / 50 FMDV strains showed Cq value shift (3.43 ± 0.11)
- 12. Thank you for your attention!