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Development of a multiplex RT-LAMP for the discrimination of FMD from other vesicular diseases

Dr. Veronica Fowler Applied Diagnostics Research Coordinator, The Pirbright Institute

2

The Problem

LABORATORY DIAGNOSIS

(Local or NRL)

DIFFERENTIAL DIAGNOSIS OF LOCAL CLINICAL

OBSERVATION =

CHALLENGING

FMD OR VS?

Delays impact upon the potential size and cost of an epidemic

“modern diagnostic methods including pen-side tests – need to be developed that can shift the burden of diagnosis to veterinarians on the farm (2002)”.

FMD or VS? (Vesicular lesions on dental pad-cattle)

FMD or SVD? (Vesicular lesions on snout-pig)

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Solutions?

DIFFERENTIAL DIAGNOSIS OF LOCAL CLINICAL

OBSERVATION =

POSSIBLE

• FMDV antigen LFDs

• Mobile rRT-PCR

• RT-LAMP

- +

4

FMDV antigen detection: Lateral-flow devices

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• Developed collaboration with international partners

• Quick and simple to perform

• Used in the UK (during 2007)

• Rapid (<10 mins) confirmation of FMD in the field

• Also useful in the Lab for triage of samples

• Recognises all seven FMDV serotypes

• Similar assay performance to lab-based Ag-ELISA

• LFD marketed by

Ferris et al., 2009: J. Virol. Methods

- +

(Boehringer Ingelheim)

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FMDV detection by mobile real-time RT-PCR

• Non-specialist user

1. Nucleic acid extraction

2. PCR set-up

3. Analysis

• Location on/or near farms

• Sample to report < 60 mins

• Powered by car auxiliary/battery

• Platform for other diseases

• Uses mature and established technologies

• Equivalent to lab-based methods

• Expensive • Can only run one sample at a time

Madi et al., 2012: The Vet Journal

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The Solution

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DIFFERENTIAL DIAGNOSIS OF LOCAL CLINICAL

OBSERVATION =

POSSIBLE

Multiplex RT-Loop Mediated Isothermal Amplification (RT-LAMP) assay

Molecular lateral flow devices (LFD)

Improved decision algorithm

=

FMDV + VSV/SVD +

Co

ntr

ol-

---

VSV

or

SVD

----

FMD

V--

--

Co

ntr

ol-

---

VSV

or

SVD

----

FMD

V--

--

FMD or VS? FMD or SVD?

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Loop mediated isothermal amplification (RT-LAMP)

• Isothermal autocyling strand-displacement DNA synthesis technique

• Utilises six primers (IP’s, EP’s and Loops)

• Formation of loop structures enables explosive polymerase-based enzymatic amplification

• Generates double-stranded, multi-sized amplicons

• Sensitivity equivalent to rRT-PCR

• Rapid detection of nucleic acid

• Accommodate reverse transcription (RT-LAMP)

• Detection of multiple pathogens (multiplexing)

• Test can be performed using simple heat source

• End point visualisation using simple molecular LFD’s

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FMDV, VSV and SVD can be detected in less than ten minutes at 108 copies

TEST Anti-Flc antibody

CONTROL Anti-Ig antibody

Labelled beads

(anti-flc antibody linked to gold beads)

Biotin

Flc

DIG

Flc

TEST Anti-DIG antibody

CONTROL Anti-Ig antibody

(anti-flc antibody linked to gold beads)

Labelled beads

FMDV +

VSV/SVD +

Multiplex RT-LAMP: Rapid with simple detection

Co

ntr

ol

VSV

/SV

D

FMD

V

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Multiplex RT-LAMP: Analytical sensitivity comparable to rRT-PCR

Multiplex RT-LAMP (RNA standard) 100 101 102 103 104 105 106

FMDV

RT-LAMP - + + + + + +

RT-LAMP-LFD - + + + + + +

rRT-PCR -/+ + + + + + +

SVD

RT-LAMP - - - - - + +

RT-LAMP-LFD - - - - + + +

rRT-PCR - - - - - + +

VSV

RT-LAMP - - - + + + +

RT-LAMP-LFD - - - + + + +

rRT-PCR Cannot be assayed as different target

FMD

V -

--

VSV

/SV

D -

--

Co

ntr

ol -

--

Multiplex RT-LAMP (RNA dilution) 10-7 10-6 10-5 10-4 10-3 10-2 10-1

VSV

RT-LAMP - - + + + + +

RT-LAMP-LFD - - + + + + +

rRT-PCR - - +/- +/- + + +

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Multiplex RT-LAMP: Simple sample preparation

• RT-LAMP can be performed directly on clinical (epithelium) samples (1:10 dilution)

Direct Multiplex RT-LAMP A A A Asia 1 Asia 1 Sat 1 Sat 2 Sat 2

FMDV clinical samples

RT-LAMP + + + + + + + +

rRT-PCR + + + + + + + +

NJ 27405 NJ 27324 NJ 27946 NJ29336 NJ29344 NJ 27775

VSV clinical samples

RT-LAMP + + + +(36) + +

rRT-PCR + + + - + -

UKG 24/72 UKG 50/22 UKG 51/72 UKG 63/73

SVD clinical samples RT-LAMP

+ + + +

rRT-PCR + + + +

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• Test provides an improved decision algorithm via:

Rapid pathogen detection within 10 minutes.

Performance at penside on ‘raw’ clinical samples.

• Test confidence via:

Comparable limit of detection to rRT-PCR.

• Test desirability via:

Disposability.

Assay can be performed using non technical heating systems.

Easily adaptability for use as a field kit (e.g. by use of dry down reagents and closed system)….see E Howson talk

Summary and impacts

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Co-authors Emma Howson (Pirbright)

Donald King (Pirbright)

Valerie Mioulet (Pirbright)

Bryony Armson (Pirbright)

Miki Madi (Pirbright)

WRLFMD reference laboratory staff

Luis Rodriguez(USDA)

Steve Pauszek (USDA)

Mike McIntosh (APHIS)

Fernando Torres (APHIS)

Tammy Beckham (IIAD)

Melissa Berquist (IIAD)

Rapidia-field

Optigene Ltd

Acknowledgements