1.
Progress in the development of field-based tools for
detec7on and characteriza7on of FMD Virus in situ
Dr. Veronica Fowler
Applied Diagnostics Research Coordinator, The Pirbright Institute

2.
LABORATORY
DIAGNOSIS
(Local or NRL)
DIFFERENTIAL
DIAGNOSIS
OF LOCAL
CLINICAL
OBSERVATION
=
CHALLENGING
Delays impact upon the potenDal size
and cost of an epidemic
“modern diagnosDc methods including pen-side tests – need to be developed that can
shiN the burden of diagnosis to veterinarians on the farm (2002)”.
FMD or VS?
(Vesicular lesions on dental pad-caUle)
FMD or SVD?
(Vesicular lesions on snout-pig)
The Problem

3.
Possible solu7ons:
5 portable real Dme RT-
PCR (rRT-PCR) and
17 isothermal publicaDons
No standardisa7on between methods

4.
Aims
Diagnos7c rRT-PCR Portable rRT-PCR
• SuperScript™ III PlaDnum™ One-Step
qRT-PCR Kit;
• Callahan et al (2001) primers and probe;
• Shaw et al (2007) cycling parameters;
• Performed on Stratagene Mx3005p or
endemic country equipment.
Portable rRPA
• T-COR 8 lyophilised reagents;
• Tetracore primers and probe;
• Modified Shaw et al (2007) cycling
parameters to include reduced RT and
extension Dme;
• Performed on T-COR 8 placorm;
• OpDgene ISO-001 lyophilised reagents;
• Dukes et al (2006) primers;
• Analysed on Genie II and LFD;
• 65oC for 30 mins.
Portable rRT-LAMP
• Undertake a standardised laboratory evaluaDon of three field-based tests, benchmarked against
the diagnosDc rRT-PCR;
• Evaluate two of the most promising field-based tests within FMD endemic sehngs.
• TwistDx lyophilised reagents;
• Abd El Wahed (2013) primers;
• Performed on Genie II.
• 42oC for 20 mins.
• The conserved 3D region was the target for all diagnosDc assay comparisons;
• Assays were evaluated with (automated and manual) and without extracDon methods.

5.
Analy7cal sensi7vity
Copy number of RNA standard
106 105 104 103 102 101 100 Nega7ve
Diagnos7c rRT-PCR + + + + + + +/- -
Portable T-COR 8 rRT-PCR + + + + + + +/- -
Portable rRT-RPA + + + + + - - -
Portable rRT-LAMP + + + + + + - -
RT-LAMP-LFD
• Using a diluDon series of FMDV 3D RNA standard (in carrier RNA):
• AnalyDcal sensiDvity was comparable to diagnosDc rRT-PCR for the portable T-COR 8
rRT-PCR and the rRT-LAMP/RT-LAMP-LFD.
• AnalyDcal sensiDvity was one log10 less for rRPA when compared to the diagnosDc
rRT-PCR.

6.
Diagnos7c sensi7vity
n = 43
• T-COR 8 had 100% concordance
detecDng to CT of 45;
• rRT-LAMP had 88% concordance
detecDng to CT of 30;
• RT-LAMP-LFD had 86% concordance
detecDng to CT of 30;
• rRPA had 77% concordance detecDng
to CT of 27.

7.
Performance of the assays with and without extrac7on
Epithelium
10-1 10-2 10-3 10-4 10-5 10-6 10-7

8.
Serum
Performance of the assays with and without extrac7on
10-1 10-2 10-3 10-4 10-5 10-6 10-7
+/-
+/-
+/-

9.
OP fluid
Performance of the assays with and without extrac7on
10-1 10-2 10-3 10-4 10-5 10-6 10-7

10.
Field evalua7on: rRT-LAMP
1:5
1:10 1:10
RT-LAMP-Tp
RT-LAMP-Tp
RT-LAMP-Tp

11.
Field evalua7on: rRT-LAMP concordance with rRT-PCR

12.
Field evalua7on: T-COR 8 rRT-PCR

13.
Field evalua7on: T-COR 8 rRT-PCR concordance with
laboratory rRT-PCR

14.
Ini7al field evalua7on: Serotyping with the T-COR 8
rRT-PCR

15.
Future direc7ons: Innova7ve, community-led disease
surveillance systems
• Building on previous work
(Fowler et al 2014);
• Recently demonstrated that
it is possible to:
– Serotype viruses using
portable rRT-PCR from LFD
eluDon wash;
– Sequence (Sanger and NGS)
viruses from LFD eluDon
wash;
– Infer geneDc relaDonships
between farms.
– All from LFDs two years post
field test.
• Highlights the potenDal of
this new approach to
improve pracDcal tools for
disease surveillance.
(● SAT1; ● A)

16.
Summary and future direc7on
• The analy7cal sensi7vity of portable rRT-PCR and RT-LAMP are equivalent to
diagnosDc rRT-PCR (10-1 RNA copies);
• AnalyDcal sensiDvity of rRPA is one log10 less than rRT-PCR;
• Excellent concordance between portable rRT-PCR, RT-LAMP and the diagnosDc rRT-
PCR;
• Portable rRT-PCR and RT-LAMP can be performed in the absence of RNA extrac7on
on blood, epithelium, swabs and OP fluid;
• It is possible to serotype in situ using the portable rRT-PCR using East African
primers and probe;
• Bringing this all together our future vision is to develop a community-led
molecular epidemiological approach using portable diagnos7cs. BBSRC GCRF
applicaDon submiUed (Veronica Fowler, Tiziana Lembo, Richard Reeve, Mark
Bronsvoort, Don King, Kasia Bankowska, Satya Parida).

17.
Acknowledgements
Emma Howson (Pirbright)
Jemma Wadsworth (Pirbright)
Kasia Bankowska (Pirbright)
Tiziana Lembo (Glasgow)
Richard Reeve (Glasgow)
Paulo F. Raphael (SUA)
Graham Friemans (Pirbright)
David King (Pirbright)
Sarah Cleaveland (Glasgow)
Nick Lyons (Pirbright)
Donald King (Pirbright)
Valerie Mioulet (Pirbright)
Bryony Armson (Pirbright)
Rolf Rauh (Tetracore)
Bill Nelson (Tetracore)
Eunice Chepkwony (KVS)
Christopher Kasanga (SUA)
Sengiumva Kandusi (SUA)
WRLFMD reference laboratory staff
Duncan Clarke (OpDgene Ltd)
Daniel Guizaw
EuFMD
Livestock owners