1.
Open Session of the EuFMD - Cascais –Portugal 26-28 October 2016
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
1. O/ME-SA/Egy-72
2. A/Africa/G-VII (2006 & 2009)
3. O/ME-SA/PanAsia 2
4. A/Asia/Iran-05 Bar-08 (2010)
5. SAT2/VII/Ghb-12 (2012)
6. SAT2/VII/Alx-12 (2012)
7. O/EA-3 (2012)
8. A/Africa/G-IV (ISM-12) (2012)
Donald King (FMD Global and Regional update, 2014)

4.
OS16
L 2A 3CPrimary cleavages
Poly(C)
L 1A 1B 1C 1D 2A 2B 2C 3A 3B 3C 3D
Secondary cleavages 1B/RNA? 3C 3C 3C 3C 3C 3C
Kilobases
0 1 2 3 4 5 6 7 8
FMDV RT-PCR target
Protease
Carboxy-terminal
self-cleaving
ProteaseCapsid NTP binding* Polymerase
Genome-linked
(VPg)
* putative functions
Membrane-binding
VP4 VP2 VP3 VP1
VPG
5’UTR
AAA (n)
3’UTR
variable VP1 region
-sequences used for isolate characterisation
conserved IRES and 3D regions
targets for pan-serotype reactive assays

5.
One Step Multiplex RT-PCR Strategy
2B2AVP1
A 750 bp
O 283 bp
SAT2 666 bp

6.
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
500 bp
200 bp 283 bp
666 bp
750 bp

9.
A
SAT2 O
A SAT2 O NTC M

10.
SO Sharqia-72
O
EA-3
2015
mixed A&O
O
200 bp
500 bp
A
O panasia2
SAT2
283 bp
750 bp
666 bp
O/EA-3
2014
A /Iran-05 NTCMM

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

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

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

14.
Serotype SAT2
666 bp
M 106 105 104 103 102 10 NTC
500 bp

15.
Serotype A
750 bp
M 106 105 104 103 102 10 NTC
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.
Acknowledgement
• 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
Special Thanks
Kees van Maanen
(EuFMD)