1. LABORATORY VALIDATION OF TWO REAL TIME RT-PCR METHODS FOR FOOT-AND-MOUTH DISEASE VIRUS WITH AN INCREASED DIAGNOSTIC SPECIFICITY Vandenbussche Frank, Lefebvre David, De Leeuw Ilse, De Clercq Kris 22 October 2015 GFRA Scientific Meeting, Hanoi, Vietnam

2. o 5’-UTR rRT-PCR Introduction: rRT-PCR for FMDV

3. o 3D rRT-PCR 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. o Evaluation of the 5’-UTR and 3D rRT-PCRs Introduction: rRT-PCR for FMDV

4. o Excellent results for each assay for: Analytical sensitivity and specificity Repeatability and reproducibility Linearity of the PCR Diagnostic sensitivity and specificity using reference samples o Field samples negative for FMDV: regularly Cp 38-45 (doubtful result) Validation of the 5’-UTR and 3D rRT-PCRs at CODA-CERVA (ISO 17025 accreditation)

6. 5’-ATATATATATATACTGGGTTTTACAAACCTGTGA …..............TGACCCAAAATGTTTGGACACT… ATATATATATATACTGGGTTTTACAAACCTGTGA………………….. TATATATATATATGACCCAAAATGTTTGGACACT… ATATATATATATACTGGGTTTTACAAACCTGTGA………………….. TATATATATATATGACCCAAAATGTTTGGACACT… ATATATATATATACTGGGTTTTACAAACCTGTGA … General principle demonstrated for the FMDV 3D forward primer

7. o Negative sheep serum (N=382) o Negative cattle serum (N=372) o Negative pig serum (N=372) o 1 of the 1126 negative samples yielded a false-positive test result in the 3D/IC/EC rRT-PCR (Cp 39.78) with the cut-off value for positive set at Cp 40. o No false-positive test result was observed in the 5’-UTR/ IC/EC rRT-PCR. o No doubtful test results (Cp 40-50) were observed in neither of the two rRT-PCRs. Results for the diagnostic specificity using field samples