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ISTA Seminar for Specified Trait Testing, 16 June 2008, Bologna, Italy INTERNATIONAL SEED TESTING ASSOCIATION (ISTA)

Latest Developments in the Area

of Detection Methods

Enrico Noli

LaRAS, University of Bologna


Global status of Commercialized Biotech Crop: 2007

Source: (ISAAA)


Who needs GMO detection methodsThose who want GM varieties: Trait Purity

Biotech producers, seed companies, farmersTo assist ”variety conversion”To track different productsTo assure compliance of biotech varieties to commercial purity standards

Those who don’t: Adventitious PresenceSeed companies, farmers, food & feed industry,

enforcement authorities, testing laboratoriesTo monitor products after commercial release To assure segregation of products To check compliance with legislation (authorized vsnon-autorized, labelling thresholds)


The view of ISTA on GMO testing (Position Paper 2001)

“ISTA seed testing laboratories are competence centres for varietal puritytesting of GM varieties as well as for the detection, identification and quantificationof GM seeds in conventional seed lots and provide uniform test results all over the world”


Methods for GMO detection

• Analysis of the phenotype – Biossays– Protein-based methods

•ELISA•lateral flow stick

• Analysis of the genotype– DNA-based methods

•end-point PCR•real-time PCR


Trait Purity testing

• Biossays for HT• Soak test• Spray test

• Simple• Reasonable cost (20-30 US$)

• Take time• Difficult interpretation

Usually 200-400 individual seeds/seedlings


Trait Purity testing

• Protein-based methods •ELISA• Immunochromatography(lateral flow strips)

• Relatively simple• Results in hours

•Cost 50-100 US$

Usually 100-200 individual seeds


AP testing

•Quantification- Does the lot comply with thresholds?

• Detection- Does the lot contain GMOs?

• Identification- If lot is positive, is/are the GMO(s) authorised?- Necessary for correct quantification


AP testing - Methods utilised

• Protein-based•ELISA•Immunostrips

• DNA-based (PCR)

Test conducted on bulk samples


DNA-based methods end-point PCR Amplification

Electrophoresis

Detection

ResultResult: : presencepresence//absenceabsence


DNA-based methods real time PCR

Amplification

Reference geneTransgene

Real time monitoring

Data analysis

ResultResult: qualitative/quantitative: qualitative/quantitative


• Rapidity (no electrophoresis)• Specificity (based on probe hybridisation)• Sensitivity (automated optical detection)• Low risk of contamination (no post-PCR manipulation)

• and, of course, possibility of quantification

Advantages of real time PCR

In the EU, all the assays validated by the Community Reference Laboratory are based on real time PCR


GM targets for PCR:Different levels of specificity

Gene specific targets

Construct specific targets

Event specific targets

LOW

HIGH

spec

ifici

ty

genepromoter promoter terminator plantplant gene

Screening targets


AP testing

Detection problems• Availability of common elements

(based on BIOTRADESTATUS and AGBIOS databases)

Number of events containing a given element


AP testing

Identification problems• Increasing number of transgenic events on the

market• Assays informativity

How many assays are necessary How many assays are necessary for unambiguous identification?for unambiguous identification?


AP testing

• Dichotomic keys

GM cornGM cornT-nos

13 EVENTS

9 EVENTS

bar4

EVENTS5

EVENTS

cry3Bb1MON88017 NK603

P-ractGA21 MIR604

cry1Fa2TC1507 DAS-59122-7

+ -

+

T-PINII2

EVENTS2

EVENTS

+

+ +

-

bla

-

T14 T25

2 EVENTS

+ pat676676680

MON810

+ --

blaDBT418 TC6275

T-35SBT176 B116

+ + -- T-35S3

EVENTS2

EVENTS

+ -

-

-

-

+

T-35SCBH-351 MS3,

MS6

+ -

bar3

EVENTS10

EVENTS

+ -

CP4-epsps6

EVENTS4

EVENTS

+ -

patBt11 MON863

+ -

P-ract2

EVENTSMON809

P-ractMON832 MON801

MON802

-- ++

neo3

EVENTS3

EVENTS

+ -

P-35S2

EVENTS2

EVENTS

-+


AP testing

• Strategies (Challenges) for improvement

• Simplicity

• Speed of testing

IncreaseIncrease

throughputthroughput


AP testingIncrease throughput → PCR - MULTIPLEXING

Hernandez et al., 2005J. Agric. Food Chem., 53 (9), 3333 -3337, 2005

MON810

Bt11

T25

GA21


AP testingProblems with PCR-multiplexing

Hernandez et al., 2005J. Agric. Food Chem., 53 (9), 3333 -3337, 2005

0.05% each event

0.1 0.5 0.9 non GMMW


High throughput

PCR with labelled primers(multiplexing and/or

multipooling)

DNA extraction

Sample

Hybridization

Detection

MICROARRAYgeneral concept

Capture probes spotted on slide

(plant species-, genes-, or event-specific sequences)


Amplified targets

Sample DNA MICROARRAYgeneral concept

Multiplex PCRwith primers

Hybridization and detection

Sample 1

Sample 2

P35SP35S

lectinlectin

TT--nosnos

invertaseinvertase

P35SP35S lectinlectin

TT--nosnosinvertaseinvertase


PCR(with biothynilated primers)

PCR1: Tnos, P35SPCR2: Pnos/nptII, CaMVPCR3: Pat, Cry1Ab, EPSPSPCR4: maize, soybean,

rapeseed, plant generic

ARRAYGMO target elements

- P35S- Tnos- Pat- Cry1Ab(Bt176), Cry1Ab(Bt11),

Cry1Ab(Mon810) - CP4-EPSPS, m-EPSPS- Pnos/nptII

Species specific targets- Invertase (maize)- Cruciferin (rapeseed)- Lectin (Soybean)- rBCL (plant universal)

Control target- CaMV

Hybridization

Detection

DNA extraction

Sample

GMO-Dual Chip®


GMO-Dual Chip®

1. Specific binding of biothynilatedproducts

2. Binding of anti-biotin conjugated gold

3. Silver precipitation catalysed by gold


•False positive rate very low•False negative rate <5% down to 0.1% level for all GM targets, down to 0,045% for 7 of them

Rep.1 Rep.2 Rep.3

P35S, TnosPCR controlepsps

cruciferinrbcL, lectin

Orientation markers

Detection control

invertase

GMO-Dual Chip®

Feed sample containing RR soybean, rapeseed and maize


Quantification of AP presence

• Real-time PCR is the most precise method forgene quantification

• However quantification is strongly affected bysample properties

•DNA quantity

•DNA quality (extraction methods and matrices)

•Purity and integrity

•Amplification efficiency (DNA quality and PCR assay


Effect of DNA quality on amplificationefficiency and quantification

22

24

26

28

30

32

34

-1.2 -0.7 -0.2 0.3 0.8 1.3 1.8 2.3

log of target amount (ng)

Ct 1.15

1.101.051.000.950.900.85

Sample efficiency(transgene and reference)

1.11.05

10.950.9

GMO% estimate

6.095.545.004.463.94

GMO% estimate(transgene efficiency=1)

1.832.965.008.9116.91

Standard curve efficiency =1

True GMO content = 5%


How to check amplification efficiency

• Test efficiency on dilution series of each sample DNA both for GM and reference target

• Compare with efficiency of standard samples

• Accept quantitative estimate only if efficiencies are similar

384-well plate

96-well plate

More More controlscontrols neededneeded


Chemistries for real time quantification

•TaqMan®

•MGM (minor groove binding)

•LNA (locked nucleic acid)

•Molecular Beacon

•Scorpion™

•D-LUX™ (light upon extension)

•Plexor™

•CPT® (cycling probe technology) Hyd

roly

sis

•SYBR® GreenIntercalating dye

Pro

bes

Prim

ersFRET-basedquenching


TaqMan®

3’ 5’

5’ 3’

reporter quencer

3’ 5’Taq

5’ 3’

3’ 5’Taq

5’ 3’


LNA (locked nucleic acid)

HH

• Increased thermal stability of duplexes• Shorter probes (8-mer vs ≅25-mer TaqMan) →

shorter amplicons (50-150 bp)

• Facilitated assay design, especially for construct- or event-specific assays

• Easily adopted • Costs comparable to ordinary TaqMan


Amplification methods alternative to PCR

•Real multiplexing possibilities

•Linear amplification

•Isothermal conditions

•Low sensitivity to inhibitors

•Amplification on microarray

LDR (ligase detection reaction)NASBA (Nucleic Acid Sequence Based Amplification)RCA (Rolling Cycle Amplification)

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