1. Development of One step Multiplex RT-PCR for differentiation of FMDV serotypes A, O and SAT2 in Egypt
Amir A. Shehata

2. FMD distribution pattern

3. FMD situation in Egypt O/ME-SA/Egy-72 A/Africa/G-VII (2006 & 2009)
O/ME-SA/PanAsia 2
A/Asia/Iran-05 Bar-08 (2010)
SAT2/VII/Ghb-12 (2012)
SAT2/VII/Alx-12 (2012)
O/EA-3 (2012)
A/Africa/G-IV (ISM-12) (2012)
Donald King (FMD Global and Regional update, 2014)

4. FMDV RT-PCR target conserved IRES and 3D regions variable VP1 region
* putative functions
Membrane-binding
Genome-linked
(VPg)
5’UTR
Carboxy-terminal
self-cleaving
3’UTR
Protease
Capsid
NTP binding*
Protease
Polymerase
VPG L 1A 1B 1C 1D 2A 2B 2C 3A 3B 3C 3D
VP4
VP2
VP3
VP1
AAA (n)
Poly(C)
Primary cleavages L 2A 3C
Secondary cleavages
1B/RNA? 3C 3C 3C 3C 3C 3C
Brief update of recent improvements in FMDV diagnosis
Move on to discuss the development of pen-side assays for the detection of FMDV
conserved IRES and 3D regions
targets for pan-serotype reactive assays 1
variable VP1 region
-sequences used for isolate characterisation 2 3 4 5 6 7 8
Kilobases 4

5. One Step Multiplex RT-PCR Strategy
VP1 2A 2B
A bp
SAT bp
O bp

6. 2- Pan-serotypic detection of FMDV RNA (Reid et al.,2000)
Methods
1- RNA Extraction
2- Pan-serotypic detection of FMDV RNA (Reid et al.,2000)
3- Assay Validation

7. Assay Validation To check specificity and / or cross reactivity:
7 archival samples representing the strains recorded in Egypt except A/ Africa/G-VII.
BHK-21 cell culture and Epithelial tissue from FMD negative cattle and buffalo.
45 clinical and T/C samples collected from 2012 till 2016 were tested by Ag ELISA and the
developed assay.
- Confirmation by nucleotide sequencing of amplified products.
Determination of Limit of Detection (LOD)

8. Determination Of Specificity
O/ME-SA/Pan-Asia 2
O/ME-SA/Sharqia-72
SAT2/VII/Ghb-12
SAT2/VII/Alx-12
A/Asia/Iran-05
A/Africa/G-IV
Mixed sample
C -Ve
Marker
O/ EA-3
750 bp
666 bp
500 bp
200 bp
283 bp

9. A SAT O NTC M A
SAT2 O

10. S SAT2 EA-3 2015 2014 A O mixed A&O O/EA-3 O O panasia2
A /Iran-05 M
O Sharqia-72 M
O/EA-3
2014
NTC O
EA-3
2015
SAT2
750 bp
500 bp A
666 bp
283 bp
200 bp
mpcr for seminar O

11. M SAT2/ Alx-12 NTC G-IV O/EA-3 (2016) A /Iran-05 A/ Africa/ 500 bp

12. S
BHK-21
Buffalo
Cattle
A +ve control
500 bp
200 bp

13. Limit Of Detection Serotype O M 106 105 104 103 102 10 NTC 500 bp

14. Serotype SAT2
M NTC
666 bp
500 bp

15. Serotype A
M NTC
750 bp
500 bp

16. Conclusion & Recommendation
The developed Multiplex RT-PCR assay differentiate accurately between FMDV A, O and SAT2 serotypes in Egypt and will save cost, time and effort.
It is highly recommended to continuously monitor the circulating strains by nucleotide sequencing to be sure that the developed assay is fit for purpose and to detect any further changes in the circulating strains.

17. Kees van Maanen (EuFMD)
Acknowledgement
Special Thanks
Kees van Maanen (EuFMD)
Animal Health Research Institute:
Hanaa A. Ahmed
Faculty of veterinary medicine – Cairo University :
Mohamed A. Shalaby.
Ayman H. El-Deeb.
Swedish University of Agricultural Sciences :
- Jean-Francois Valarcher