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 2015GFRA Scientific Meeting, Hanoi, Vietnam

o 5’-UTR rRT-PCR

Introduction: rRT-PCR for FMDV

o 3D rRT-PCRUse 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

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)

5’-ATATATATATATACTGGGTTTTACAAACCTGTGA …

..............TGACCCAAAATGTTTGGACACT…

ATATATATATATACTGGGTTTTACAAACCTGTGA…………………..

TATATATATATATGACCCAAAATGTTTGGACACT…ATATATATATATACTGGGTTTTACAAACCTGTGA…………………..

TATATATATATATGACCCAAAATGTTTGGACACT…ATATATATATATACTGGGTTTTACAAACCTGTGA …

General principle demonstrated for the FMDV 3D forward primer

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