1.
Open Session of the EuFMD - Cascais –Portugal 26-28 October 2016
ESTABLISHMENT AND VALIDATION OF TWO
DUPLEX ONE-STEP REAL-TIME RT-PCR ASSAYS
FOR DIAGNOSIS OF FOOT-AND-MOUTH DISEASE
Kamila Gorna, Anthony Relmy, Aurore Romey, Eve Laloy, Stephan Zientara,
Sandra Blaise-Boisseau, Labib Bakkali-Kassimi
Université Paris-Est, Anses, Laboratoire de Santé Animale, Laboratoire OIE de référence FMD,
UMR1161 Virologie (Anses-Inra-Enva), 14 Rue Pierre et Marie Curie
94700 Maisons-Alfort, France
Contact : labib.bakkali-kassimi@anses.fr

2.
OS16

3.
OS16
3DL VP4 VP2 VP3 2A 2B 2C 3A 3B 3CVP1
VPg
5’ 3’
IRES
3B 3B
Conserved region within all
FMDV serotypes
1- Mix IRES/B-actin 2- Mix 3D/B-actin Conserved region within all
FMDV serotypesMastermix
Internally controlled one-step rtRT-PCR
RNA
5 µl
20 µl
 Master mix with all reagents
 Can be aliquoted
 Stable for at least one year

4.
ONE-STEP DUPLEX
rtRT-PCR
 Ensuring adequate efficiency of RNA extraction
 Confirming the absence of PCR-inhibitors in each
sample,
 Less handling means less cross-contaminations
 Tests can be produced in advance as a ready-to-
use master mix, which can be controlled before
use and stored at−20◦C as an aliquot, making
diagnostic results more reproducible and accurate.
 Can be easily implemented in routine analysis
 Automated

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OS16
Validated according to OIE and ISO/IEC 17025 standards
Accredited under ISO/IEC 17025
 Analytical specificity (ASp): Seven FMDV serotypes are detected. None amplification for SVDV and VSV strains
 Analytical sensitivity (Asn)/ Limit of detection (LoD): LoD PCR = 7 TCID50/ml et LoD METHODE = 50 copies RNA
 Diagnostic sensitivity (DSn): (positives n=40) 100% (3D); 97% (IRES)
 Diagnostic specificity (DSp): (negatives n=79) 100% (3D and IRES)
 Repeatability intra et inter assays: (CV max intra = 2,96; CV max inter =6,73)
Master mix was provided to some laboratories
Method available : Gorna K. et al. 2016, J Virol Methods. doi: 10.1016/j.jviromet.2016.03.020

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OS16
Would like to
know more ?
Look at the poster

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OS16
Sandra
Blaise Boisseau
Anthony
Relmy
Aurore
Romey
Eve
Laloy
Kamila
Gorna
Thank you for your attention