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Diagnostics for Pests and Diseases
What can it do for you?
James Woodhall
Detection and Surveillance
Technologies Team
Fera
Introduction
• Exciting time to be in diagnostics
• Many new technologies emerging
• New DNA sequence data on P&Ds
• Engage with the industry early
• Feedback & new ideas
• Develop diagnostic solutions for P&D
problems
Overview of talk
• Key diagnostic methods
PCR
Field testing - LAMP
Arrays
NGS
• Strength & weakness
• Examples of where they are applied
Identifying, detecting and
diagnosing P&D at Fera
Polymerase chain reaction (PCR)
Polymerase chain reaction (PCR)
DNA Extraction
20 minutes-
4 h
90 minutes 60-90 minutes
Real-time PCR chemistries -
TaqMam
DNA Extraction
• Planting Material
• Seeds
• Soil & Compost
• Water
• Air
TaqMan is our tool of trade
used in soft fruit:
• Blackcurrant reversion virus, Strawberry
crinkle virus, Strawberry mottle virus,
Strawberry mild yellow edge virus,
Strawberry vein banding virus, Strawberry
latent ringspot virus, Arabis mosaic virus,
Rasberry ringspot virus, Tomato black ring
virus.
• Colletotrichum acutatum, Phytophthora
cactorum, Phytophthora fragariae/rubi
24/7 operation during outbreaks
National survey within a few days!
Standard method v PCR v
Real-time PCR
Polyscytalum pustulans
Fera
DNA extraction
& real-time PCR
Cold Store
20 weeks storage and
visual assessment
Relationship between DNA and
observation of disease
R2 = 0.65
Woodhall et al.,
2010
Real-time PCR for Sclerotium
cepivorum directly in soil
Woodhall et al.,
2012
Detection of Verticillium dahliae
in soil
• EU Project on olives with WUR
• HDC Project with ADAS for Strawberries
Molecular tool box
• Sensitive Verticillium species complex assay
• Verticillium dahliae & Verticillium longisporum
• Aggressive olive strain assay “d-type”
• VCG Specific assays (differences in
pathogenicity)
• Resistance Races
Detection of Verticillium in soil
Harris test v PCR (pg DNA g soil)
Harris testing (wet sieving) v
TaqMan direct PCR from soil
TaqMan Harris testing
0.62 1
1.07 3.5
6.49 1
27.91 2.1
6.37 5.9
1.20 1.4
2.88 3.1
0.42 0
0.50 0.7
6.47 9
2.71 4.9
PCR
Robust
Easy to use
Rapid
Cost
Portability
Sensitivity
Specificity
Development time
lab
(day)
(DNA Extraction)
The move to on-site testing
• Speed and cost
• rapidly implement management options
• Import inspections consignments can be
released more quickly
• Demonstration purposes – confidence
• Research tool – field work, screening
samples
Development of on-site testing
• Lateral flow devices (LFD)
Lateral flow devices
Robust
Easy to use
Rapid
Cost
Portability
Sensitivity
Specificity
Development time
?
?
X
Cepheid SmartCycler
Smiths Detection
BioSeeq
Portable platforms for PCR-
based detection
Portable real-time PCR
Robust
Easy to use
Rapid
Portability
Cost
Sensitivity
Specificity
Development time
?
X
?
• About 90 minutes due
to thermal cycling
X
Field based extraction
& inhibition
LAMP: Loop-mediated isothermal AMPlification
• Notomi et al. 2000
• 3 pairs of primers (internal, external, loop)
• Bst (or similar) DNA polymerase with strand-displacing activity
• Internal primers generate product containing single-stranded loops
• typical reaction conditions: 30 minutes @ 65°C
F3c
B2c F3
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-loop External primer F3
Loop primer B-loop External primer B3
F3c F2c F1c B1 B2 B3 3’ 5’
F1c F2
B1c B2c B3c F3 F2 F1
F3c F2c F1c B1 B2 B3 3’
5’
5’
3’ B1c B2c B3c F1c F2 F1 5’ 3’
F1 B1c B2c B3c
F3c F2c F1c B1 B2 B3 3’ 5’
F1c F2 3’
B1c B2c B3c F1
F2
F1c
B1c B2c B3c F1
F2
F1c B2 B1c
B3
F1c
F2c
F1
B2
B1c
B1
Loop primers not shown
F3
• Non-denatured target • Isothermal reaction at 65°C • 10-60 minute reaction time
Detection of LAMP products
- +
0
250
500
0 5 10 15 20 25 30 35 40
flu
ore
sc
ence
minutes
-
+
- +
- + - +
Genie I, II & III
• touchscreen
• Intuitive user interface
• Can connect to computer for additional analysis, archiving of results
• Battery-powered
Genie Output
Time to positive
(Tp)
Annealing
temperature
DNA extraction
Dilute 1:10 to
1:40
DNA extraction
• Crude extraction – preferably single step, no equipment
• LAMP reagents set up in advance
• Samples added to reactions using disposable inoculating loops (no pipettes)
Sensitivity with assay to rDNA ITS
y = -1.205ln(x) + 19.564 R² = 0.9044
5
10
15
20
25
30
0.001 0.01 0.1 1 10 100 1000 10000
Tim
e t
io p
osit
ive (
min
ute
s)
DNA Concentration (pg/µl)
y = -1.306ln(x) + 30.528 R² = 0.9961
15
20
25
30
35
40
0.001 0.01 0.1 1 10 100 1000 10000
Cycle
th
resh
old
(C
t)
DNA Concentration (pg/µl)
Burning the midnight LAMP
LAMP
Robust
Easy to use
Rapid
Portability
Cost
Sensitivity
Specificity
Development time
?
LAMP for detection of Chalara fraxinea
150 samples tested by LAMP 20th - 23rd November: 34% infected, 66% C. fraxinea-negative
Primer design and initial test 17th -26th October
Development of field extraction method for ash
31st October – 16th November
Improved assay design 9th -16th November
Finalised protocol for validation ready 19th November
-10000
0
10000
20000
30000
40000
50000
60000
0 5 10 15 20 25 30
Flu
ore
scen
ce
Time (minutes)
NTC H. albidus
H. pseudoalbidus C.f-infected ash
Citizen Science
LAMP assays in development Chalara fraxinea ash dieback
Plant control (COX) Liriomyza huidobrensis
leaf miners Liriomyza control assay L. sativae L. trifolii P. kernoviae Guignardia citricarpa citrus black spot
CMS potato ring rot
Dothistroma septosporum red band needle blight
Phytophthora ramorum P. lateralis P. austrocedrae Cryphonectria parasitica sweet chestnut blight
Ceratitis capitata medfly
Ceratocystis platani plane canker/plane wilt
D. pini red band needle blight
Lecanosticta acicola brown spot needle blight
Giberella circinata pitch canker of pine
Agrilus planipennis emerald ash borer
Trichoferus campestris velvet longhorn beetle
P. pseudosyringae Ceratocystis fagacearum oak wilt
P. infestans
OptiGene kits – due January ‘15
Further development, more validation – next batch of kits
What about for testing for more
than one target?
• Microarrays
• Developed for expression studies
• Could be used for screening for pathogens ?
Generic detection
• Glass slide microarray
• 8729 capture probes
• 1132 different viruses from 40 viral families
• Animals, fish, plants, insects includes
zoonotics
Diagnostic platforms: Clondiag
• Labelling with biotin / colorimetric detection
• Inexpensive
• Array reader approx 6000 USD
• Array (196 features) approx 20 USD
Virus detection
• TSWV • PepMV
Sensitivity
Mycorray – Grapevines
EU consortium
Disease Pathogen
Botryosphaeria
canker and
dieback (Black
Dead Arm)
Neofusicoccum parvum (Botryosphaeria parva)
Neofusicoccum luteum (Botryosphaeria lutea)
Diplodia seriata (Botryosphaeria obtusa)
Botryosphaeria dothidea
Lasiodiplodia theobromae (Botryosphaeria rhodina)
Esca disease Phaeomoniella chlamydospora
Phaeoacremonium aleophilum
Fomitiporia mediterranea
Dead arm Eutypa lata
Phomopsis viticola
Black foot disease Ilyonectria liriodendra (Cylindrocarpon liriodendri)
Ilyonectria macrodidyma (Cylindrocarpon macrodidymum)
Mycorray
‘Black goo’
Even more targets or
unknowns- DNA sequencing
technology Capillary based DNA
sequencing
High throughput
pyrosequencing
High throughput short read
sequencing
3130XL GS-FLX-Titanium +
(also GSjunior)
HiSeq 2000
(also MiSeq /HiSeq2500)
Applied Biosystem Roche-454 Illumina
(Hostile take over from Roche!)
700bp x 96 750bp x 1 million 150bp x 3 billion
(HiSeq2500 x2)
Specific targets
Barcoding,
microsatellites
Genome sequencing,
transcriptomes,
metagenomics, amplicon
sequencing
Re-sequencing, transcriptomes,
genome sequencing
Identifying agents of disease
suppression
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
Olpidium
brassicae
Apodus deciduus
Botryotinia fuckeliana
Davidiella sp. B23B
Gaeum
annomyces gram
inis var. avenae
Geom
yces sp. BC9
Geom
yces sp. LC-03-010
Lecanora carpinea
Lewia sp. B19A
Phaeoacremonium
sp. JD-115.1
Phoma herbarum
Preussia fleischhakii
Tetracladium m
axilliforme
Woollsia m
ycorrhizal fungus VII
uncultured ectomycorrhiza (Scabropezia)
Calocera viscosa
Cryptococcus aerius
Helicobasidium
longisporum I
Lepiota sp. E.C. Vellinga 3327
Phanerochaete rimosa
Trichosporon gracile
uncultured basidiomycete
uncultured ectomycorrhiza (Sistotrem
a)
Glom
us geosporum
Polymyxa gram
inis
unassigned
Control
Suppressive
Next generation sequencing &
Maize lethal necrosis in Kenya
From Autumn 2011: a mystery disease
spreading through maize crops across
country - Major losses
Feb-April 2012: No pathogen found in
samples using traditional diagnostics
May 2012: Use of Pyrosequencer
= non-targeted approach
June 2012: Rapid design of
real-time PCR
> Screening & Future control Two viruses identified =
Maize lethal necrosis disease
Maize chlorotic mottle virus
Sugarcane mosaic virus
Getting to the root of the
issue...
• Problem known >10 years
• Growers losing up to 10%
yield
• Unable to ‘grade out’ internal
problems leading to whole
crop rejection
• Previous work (HDC FV382)
suggested a possible loose
association with virus but only
looked at symptomatic
individuals
Deep Sequencing
• other possible viruses?
• 12 necrotic carrots & 12 healthy
• Mix of virus and symptomatic status
• 2 MiSeq Runs : 16 million reads
Relative abundance of
sequence reads
Symptomatic
Asymptomatic
Summary of NGS
Expected:
• Carrot mottle virus
• Carrot red leaf virus
• Carrot red leaf associated RNA
Unexpected:
• Beet western yellows associated RNA
• Carrot yellow leaf virus
Novel
• a novel virus in the same genus as CYLV (Carrot closterovirus 1)
• 2 novel Betaflexiviruses (genus tentatively named ‘chordovirus’)
• a novel carrot Torradovirus (CTV-1)
New virus discovery: the impact of
new technology
0
2
4
6
8
10
12
14
16
1980 1985 1990 1995 2000 2005 2010
Mean = 1.3 new viruses per year
Routine Molecular
diagnostics
NGS comes on
line
Sequencing costs and ease
GS-FLX-Titanium
1pence per/kbp
Oxford nanopore
0.1 pence per/kbp
Mi-SEQ
0.01 pence per/kbp
All these technologies linked in
some way to:
Remote sensing
‘Big Data’ and GIS
Piggy-back onto
existing monitoring
& surveillance
systems
Researchers Diagnosticians Inspectors Industry &
general public Remote & real-time monitoring
The future of diagnostics?
Conclusions
• More technologies than ever
• Greater sequence data – specific diagnostics
Species=> strains => fungicide resistance
• Dialogue between scientists and industry
Its now not what diagnostics can do for
you…….
But what can you do for diagnostics…
Acknowledgements
• Neil Boonham, Jenny Tomlinson, Sioban Ostoja-
Starzewska, Catherine Harrison, Melanie Sapp,
John Elphinstone, Adrian Fox, Ian Adams, Toby
Hodges, Anna Skelton, Kate Perkins, Jen
Hodgetts, Roy Macarthur