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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
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 -/+)
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
Results in
10-15 mins
How it works
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.
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’
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’
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′
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′
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′
Loop-mediated isothermal AMPlification
Loop formation in the displaced strand
5′3′
5′ 3′
5′
3′
single-stranded
loop
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
Loop-mediated isothermal AMPlification
• LAMP product consists of various sized structures consisting of
alternately inverted repeats of the target sequence
- +
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
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
Crops of interest
Potato (Solanum tuberosum) Oilseed Rape (Brassica napus)
Crops of interest
Potato Olives Citrus
Crops of interest
Rice
Fruits
Horticultural
Pines
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
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
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
• 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
LM=Leptosphaeria maculans
Assay Development
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)
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
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
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

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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
  • 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