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