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Vandenbussche Frank, Lefebvre David , De Leeuw Ilse, De Clercq Kris

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. Introduction: rRT-PCR for FMDV.

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Vandenbussche Frank, Lefebvre David , De Leeuw Ilse, De Clercq Kris

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  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. Introduction: rRT-PCR for FMDV • 5’-UTR rRT-PCR

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

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

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

  6. Results for the diagnostic specificity using field samples • Negative sheep serum (N=382) • Negative cattle serum (N=372) • Negative pig serum (N=372) • 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. • No false-positive test result was observed in the 5’-UTR/ IC/EC rRT-PCR. • No doubtful test results (Cp 40-50) were observed in neither of the two rRT-PCRs.

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