Development of a multiplex RT-LAMP for the discrimination of FMD from other vesicular diseases
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1 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 1
2 The Problem FMD or VS? (Vesicular lesions on dental pad-cattle) FMD OR VS? DIFFERENTIAL DIAGNOSIS OF LOCAL CLINICAL OBSERVATION = CHALLENGING FMD or SVD? (Vesicular lesions on snout-pig) LABORATORY DIAGNOSIS (Local or NRL) 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). 2
3 Solutions? FMDV antigen LFDs - + DIFFERENTIAL DIAGNOSIS OF LOCAL CLINICAL OBSERVATION = POSSIBLE Mobile rrt-pcr RT-LAMP 3
4 FMDV antigen detection: Lateral-flow devices 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 (Boehringer Ingelheim) - + Ferris et al., 2009: J. Virol. Methods 4 4
5 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 5
6 FMDV---- VSV or SVD---- Control---- FMDV---- VSV or SVD---- Control---- The Solution FMD or VS? DIFFERENTIAL DIAGNOSIS OF LOCAL CLINICAL OBSERVATION = POSSIBLE FMD or SVD? Multiplex RT-Loop Mediated Isothermal Amplification (RT-LAMP) assay Molecular lateral flow devices (LFD) VSV/SVD + FMDV + = Improved decision algorithm 6 6
7 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 7
8 Multiplex RT-LAMP: Rapid with simple detection FMDV VSV/SVD Control FMDV, VSV and SVD can be detected in less than ten minutes at 10 8 copies Flc Biotin FMDV + Labelled beads (anti-flc antibody linked to gold beads) TEST Anti-Flc antibody CONTROL Anti-Ig antibody DIG Flc VSV/SVD + Labelled beads (anti-flc antibody linked to gold beads) TEST Anti-DIG antibody CONTROL Anti-Ig antibody 8
9 Multiplex RT-LAMP: Analytical sensitivity comparable to rrt-pcr FMDV --- VSV/SVD --- Control --- Multiplex RT-LAMP (RNA standard) RT-LAMP FMDV RT-LAMP-LFD rrt-pcr -/ RT-LAMP SVD RT-LAMP-LFD rrt-pcr RT-LAMP VSV RT-LAMP-LFD rrt-pcr Cannot be assayed as different target Multiplex RT-LAMP (RNA dilution) VSV RT-LAMP RT-LAMP-LFD rrt-pcr - - +/- +/
10 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 NJ NJ NJ29336 NJ29344 NJ 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
11 Summary and impacts 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 11
12 Co-authors Emma Howson (Pirbright) Donald King (Pirbright) Valerie Mioulet (Pirbright) Bryony Armson (Pirbright) Miki Madi (Pirbright) Acknowledgements 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 12
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