"In field diagnostics- the Genie and the LAMP"
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LAMP is a DNA amplification technology which enables rapid and sensitive detection and when run on the Genie III platform enables detection of pests and diseases outside of the laboratory at any point in the agri-food chain where decisions are being made. The target audience are researchers, agri-business and forestry experts, farmers and foresters and any other interested in being introduced to this portable molecular diagnostic tools on plant diseases. Do not hesitate to contact EMPHASIS project through Facebook, Twitter, email (emphasisproject@gmail.com), Youtube or through our website (http://www.emphasisproject.eu/) if you want to be updated on webinars dates and content and book a ticket. To watch on Youtube: https://youtu.be/Dg-lAjuCYuQ
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"In field diagnostics- the Genie and the LAMP"
- 1. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 634179. EMPHASIS Effective Management of Pests and Harmful Alien Species - Integrated Solutions Ed Haynes Fera Science Ltd, UK In field testing – the Genie and the LAMP
- 2. Why LAMP? • Short reaction time (approximately 30 minutes) • Sensitivity and specificity are similar to lab-based methods (real-time PCR) • No thermal cycling = simple equipment • Tolerance of inhibitors = crude extraction methods • Simplified extraction methods reduce the chance of errors • Highly efficient amplification simplifies interpretation of results (clear -/+)
- 3. Surveillance and monitoring • Detection at the point of decision making • Inspectors at ports of entry • Agronomists confirming pathogen identity • Packers and processors for latent infection • Seed testing and certification
- 4. Results in 10-15 mins How it works
- 5. LAMP: Loop-mediated isothermal AMPlification • 3 pairs of primers (internal, external, loop) • Bst DNA polymerase with strand-displacing activity • Internal primers generate product containing single-stranded loops F3c B2cF3 B2 3’ 5’ 5’ 3’ F1c F2 B2 B1c F1c F1 B1 B1c FL FLc BLc BL Internal primer FIP Internal primer BIP B3 B3c F2c F2 Loop primer F-loopExternal primer F3 Loop primer B-loop External primer B3 Notomi et al. 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research 28:e63.
- 6. Amplification of DNA 3’ 5’ 5’ 3’ double-stranded DNA target 3’ 5’ primer binding to one strand of target DNA polymerase extending primer from its 3′ end3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ some DNA polymerases (eg Taq) exhibit 5′-3′activity 5’ 3’
- 7. Amplification of DNA 3’ 5’ 5’ 3’ double-stranded DNA target 3’ 5’ primer binding to one strand of target DNA polymerase extending primer from its 3′ end3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ some DNA polymerases (eg Bst) exhibit strand displacing activity 5’ 3’
- 8. Loop-mediated isothermal AMPlification Internal primers - consist of 2 regions Internal primer BIP Internal primer FIP F3c F2c F1c B1c B2c B3cF3 F2 F1 B1 B2 B3 3’ 5’ 5’ 3’ F1c F2 B2 B1c 5′ 3′ 5′3′
- 9. Loop-mediated isothermal AMPlification Internal primers - consist of 2 regions Internal primer BIP Internal primer FIP F3c F2c F1c B1c B2c B3cF3 F2 F1 B1 B2 B3 3’ 5’ 5’ 3’ F1c F2 B2 B1c 5′ 3′ 5′3′
- 10. Loop-mediated isothermal AMPlification External primers – initiate strand displacement F3c F2c F1c B1c B2c B3cF3 F2 F1 B1 B2 B3 3’ 5’ 5’ 3’ F1c F2 B2 B1c External primer F3 External primer B3 5′3′ 5′ 3′
- 11. Loop-mediated isothermal AMPlification Loop formation in the displaced strand 5′3′ 5′ 3′ 5′ 3′ single-stranded loop
- 12. Loop-mediated isothermal AMPlification Loop formation B1c B2c B3cF1c F2 F1 5’ 3’ B1c B2c B3cF1 F2 F1c Extension of internal primer BIP etc B1c B2c B3cF1 F2 F1c B2 B1c B3 F1c F2c F1 B2 B1c B1 Dumbbell-like structure 5’3’ http://loopamp.eiken.co.jp/e/lamp/anim.html
- 13. Loop-mediated isothermal AMPlification • LAMP product consists of various sized structures consisting of alternately inverted repeats of the target sequence - +
- 14. Real-time LAMP Real-time detection using a fluorescent intercalating dye Before amplification, little double-stranded DNA, low fluorescence After amplification, lots of double-stranded DNA, high fluorescence
- 15. LAMP Instrumentation Genie II • Two sample blocks – 16 tubes • 7” touchscreen interface • Wired USB communications • Single-colour fluorescence • Internal battery • Forced-air cooling • Available since 2011 Genie III • Single sample block – 8 tubes • 4.3” touchscreen interface • Wired USB communications • Bluetooth and WiFi • Two-colour fluorescence • Internal battery • Active (Peltier) cooling • Real-time PCR capability • Available since 2013 Genie HT • 12 sample blocks – 96 tubes • 10.1” touchscreen interface • Wired USB communications • Bluetooth and WiFi • Two-colour fluorescence • Mains power only • Forced-air cooling • Integrated barcode reader • Supports instruction videos • Available 2017
- 16. Crops of interest Potato (Solanum tuberosum) Oilseed Rape (Brassica napus)
- 17. Crops of interest Potato Olives Citrus
- 19. Heterobasidion irregulare Garbel. & Otrosina • Heterobasidion irregulare is a major fungal pathogen of pines in North America accidentally introduced into Central Italy during World War II. • Currently distributed in pine stands along 103 km of coastline of central Italy, including Rome. • The disease can result in root rot, butt rot, reduced growth, and mortality of host trees. • Recently, the pathogen has been included in the A2 EPPO list of organisms recommended for regulation all over Europe. (Garbelotto and Gonthier, 2013) (Gonthier et al., 2014) Heterobasidion irregulare
- 20. Produces brown rot of peach and other stone fruits is one of the most serious diseases of stone fruits worldwide. Quarantine pathogen – EPPO List A2 (Pellegrino et al. 2009) The fungus attacks both flowers and mature fruit. Infected fruits eventually dry up into desiccated mummies, which serve as overwintering reservoirs of the fungus Monilinia fructicola
- 21. WORKFLOW Identification of a specific gene and primers design Optimization of LAMP reaction conditions Validation of the specificity and sensitivity of the assay Design of an internal control assay (e.g. cytochrome oxidase) Test on field samples Development of assays
- 22. • Using available data from whole genome comparative analysis between Heterobasidium irregulare and its closely related sympatric species H. annosum s.s. • LAMP assays were designed to different genes • Target species: H. irregulare • Non target species: H. annosum s.s. • The most promising results: intergenic region scaffold_2 assay and Calmodulin gene assay • Next activities: Optimisation of the assay, specificity (by testing on full panel of species within genus) and sensitivity 45 min at 65°C H. irregulare H. annosum (NON-TARGET) Region on Scaffold_2 H. irregulare H. annosum (NON-TARGET) Calmodulin Unito-Forestry Assay Design
- 24. Assay Validation Tp (min:sec) Anneal (°C) Target ‘Ca. L. asiaticus’ 08:15 86.5 Other species in Genus ‘Ca. L. africanus’ - - ‘Ca. L. solanacearum’ - - Related bacterial species Burkholderia sp. - - Enterobacter sp. - - Ochrobactrum anthropi - - Pseudomonas marginalis pv. marginalis - - Xylella fastidiosa - - Pantoea agglomerans - - Pseudomonas syringae pv. persicae - - Other citrus plant pathogens Guignardia citricarpa on C. macroptera - - Guignardia citricarpa on Pomelo - - Elsinoe fawcetti on C. aurantifolia - - Guignardia mangiferae - - Xanthomonas axonopodis pv. citri on C. aurantifolia - - Xanthomonas axonopodis pv. citri on C. aurantifolia - - Vector species Diaphorina citri - - Dilution LAMP Real-time PCR Tp No. positive CT No. positive Neat 8:00 - 8:15 3 / 3 26.0 3 / 3 10 -1 11:45 - 26:15 2 / 3 29.5 3 / 3 10 -2 - 0 / 3 32.9 3 / 3 10 -3 - 0 / 3 39.3 (2 / 3 ) 10 -4 - 0 / 3 - 0 / 3 ‘Candidatus Liberibacter asiaticus’ (CLas)
- 25. Pathogen Target Assay designed Tested on DNA Tested on infected sample Assay validated A. alternata Alt a 1 allergene P P P P L. biglobosa beta-tubulin P P L. maculans nitrate reductase P P P P Pyrenophora tritici repensis GPDH P P V. longisporum elongation factor P P P P Pyrenopeziza brassicacea beta-tubulin P P Peronspora belbahrii ITS P P P Heterobasidium irregulare Intergenic region scaffold-2 P P Fusarium fujikuroi Elongation factor P P Fusarium oxysporum f.sp. lactucae RAPD-SCAR marker P P Pythium ultimum Beta-tubulin P P ‘Candidatus Liberibacter asiaticus’ secA P P P ‘Candidatus Liberibacter africanus’ secA P P P ‘Candidatus Liberibacter solanacearum’ secA P P P LAMP assays update
- 26. Conclusions • LAMP – sensitive, specific DNA amplification method • Genie – easy to use platform for performing LAMP tests • LAMP kits for plant pathogens are being developed within the project • LAMP kits will be validated in line with EPPO standard 7/98 and available from OptiGene
- 27. Acknowledgments • Fera – Jennifer Hodgetts, Jenny Tomlinson, Sioban Ostoja-Starzewska & Neil Boonham • NIAB – Jane Thomas, Tom Wood & Rosa Caiazzo • Optisense – Michael Andreou • Agroinnova – Davide Spadaro & Sara Franco Ortega for more information, visit www.emphasisproject.eu or email Neil.Boonham@fera.co.uk