1

INRA PMDV / MDO

GMO detectionat the INRA stationof Versailles, France

Cécile Collonnier INRA PMDV / MDO

INRA PMDV / MDO

Activities performed in :

• Lab. of GMO Detection Methodologies (MDO)

• Unit of Phytopathology and Detection Methodologies of Versailles (PMDV)

• Station of Versailles – Grignon

• National Institute for Agronomical Research (INRA)directly related to the French Ministry of Agriculture

INRA PMDV / MDO

Context of creation

• Important public concern about GMO in the EU since 1996

• No available methods for the application of GMO regulations emitted since 1997

• France less involved in GMO detection than other countries

INRA PMDV / MDO

MDO history

• created ex nihilo in January 1999.

• one year to prepare and equipe the laboperational in 2000.

• New lab built in 2003installation planned for March 2004

INRA PMDV / MDO

MDO Scientific team:10 people with various specialties

Permanent people :• Dr Yves Bertheau – senior scientist (team leader) • Dr André Kobilinsky – senior scientist (statistics)• Georges Berthier – engineer (QRT-PCR, QA)• Carole Couture – engineer • Marie-Noëlle Duplan – research assistant (QRT-PCR)• Marcel Romaniuk – research assistant (DNA extraction)• Colette Audeon – technician (plasmid collection, QA)

People presently under contract :• Dr Cécile Collonnier – junior scientist (QRT-PCR)• Dr Sophie Fernandez – junior scientist (microarrays)• Francine Boyer – research assistant (plasmid collection, microarrays)

INRA PMDV / MDO

MDO general objective

Become a pole of expertise in qualitative and quantitative GMO detection methods

2

INRA PMDV / MDO

MDO missionsTo develop and/or adapt GMO detection methods,

To participate to the national and international efforts of methods standardization (AFNOR, CEN, ISO…),

To collaborate to proficiency testing and to methods validation throughring trials,

To participate and/or coordinate French and European networks,

To participate and/or coordinate French and European research programs,

To be an expertise lab for the French Ministry of Agriculture.

INRA PMDV / MDO

MDO = coordinator of theFrench network of GMO detection labs

• 4 labs: MDO + GMO detection units..- ..of the Fraud repression services- ..of the Plan Protection Department of the Ministry of

Agriculture- ..of Geves (seeds and cultivars protection and control)

• roles: homogenise technical and regulatory knowledge, supply standards and methods …

INRA PMDV / MDO

List of Labs in the French Network

• Lab. National de la Protection des Végétaux, unité de détection des OGM (Orléans, France)

• Lab. de la Direction Générale de la Concurrence de la Consommation et de la Répression des Fraudes (Strasbourg, France)

• Lab. BioGEVES du Groupe d’Etude et de Contrôle des Variétés et des Semences (Magneraud, France).

• Lab. Méthodologies de la détection des OGM (MDO) (Versailles, France).

INRA PMDV / MDO

MDO = a member of the European Network of GMO Laboratories (ENGL)

Responsabilities:• methods developments• validation and proficiency

studies• technology transfer• reference materials (IRMM)• sampling strategies and

procedures• databases and bio-informatics• training

INRA PMDV / MDO

ENGL Structure:

JRC

Steering Commitee

PlenaryObserversMembers

co-ordination, organization,contact to EC and stakeholders

proposals for tasks, work programme and projects

discussion of SC proposals, transfer of information,participation in break out groups and projects

comm

un

ication

MDO

MDO

MDO = also a member of ENGL steering commitee

INRA PMDV / MDO

• = European network safety assessment of genetically modified food crops.

• subsidised by the EC in the 5th frameworkprogramme

• MDO participates to the 4th working group « Traceability and Quality Assurance »through 2 programs: QPCR-GMOFOOD

GMO-CHIPS

MDO contributes to ENTRANSFOOD

3

INRA PMDV / MDO

MDO participates to normalisation groups

• AFNOR: Y. Bertheau = Former chairman of the AFNOR commission for standardisation of GMO detection methods

• CEN: Y. Bertheau = Task leader of the European quantitative PCR ad-hoc group of the CEN/TC 275/WG 11

• ISO: Y. Bertheau = Coordinator of the CEN/ISO French delegation INRA PMDV / MDO

To provide tools to comply with the Regulations

1829/03/EC and 1830/03/EC...

Traceability along the food and feed supply chains,

Mandatory labelling independently of the presence of recombinant DNA or protein,

Methods and control samples to be provided by the companies and validated by JRC + ENGL.

INRA PMDV / MDO

...MDO develops DNA-basedGMO detection methods

Development, Determination of the performance criteria using CRM or internal standards, Validations

INRA PMDV / MDO

Performance criteria of detection methods

• Sensitivity (limit of detection, limit of quantification)

• Specificity• Accuracy• Ruggedness• Precision (trueness)• Applicability• Practicability• Fit for purpose

INRA PMDV / MDO

but MDO also works at all the levels of the general detection procedure

• Sampling / sub sampling• Grinding / homogenization • Analyte extraction• Analyses (standards, controls)

• Results interpretation (measure uncertainty) and expression

INRA PMDV / MDO

• Qualitative PCR

• Quantitative Real-time PCR

• Microarrays

Techniques chosen at MDO

4

INRA PMDV / MDO

GMO identification

Negative

Positive

Authorized?No Yes

Illegal or temporarily

tolerated at 0.5%

Assay individual

ingredients

Less than 0.9%No need for labeling

Labeling required More than 0.9%

GMO quantification

GMO detection

GMO screening

Fort

uito

us

pres

ence

INRA PMDV / MDO

Gene

Plant genome

1 12 23 3

Promoter Terminator

Insert (simplified)Plant genome

1 : Screening (P35S / Tnos / nptII…)

PCR target for the GMO and plant species

2: Construct specific3: GMO identification

Housekeeping genesChoice criteria:• specificity• copy number• sequence conservation

GMO

INRA PMDV / MDO

ABI-PRISM 7700

Real Time PCR at MDO

INRA PMDV / MDO

QRT- PCR Taqman® chemistry

• Polymerization

5’

5’

3’ 5’

3’5’

forwardprimer

R

R = Rapporter

Q = Quencher

Denaturating Hybridization

QQ

INRA PMDV / MDO

Quantitative PCR (QRT-PCR)TaqmanProbe

Plant genome Insert

Forward Primer Reverse Primer

PCR product

Ct : Cycle threshold

Standard curveCt = f (Log concentration)

INRA PMDV / MDO

Micro-arrays

Diagnostics : presence of sequences specific of GMO, plants, controls…

DNA extractionAmplification and labeling

Surface activated with specific capture probes

Ampliconshybridization

SamplingGrinding, homogeneization

5

INRA PMDV / MDO

Arrayer

• More than 3000 points/ h.

• Important choice of size

INRA PMDV / MDO

Detector integrated into a computer

INRA PMDV / MDO

Example of a DNAchip for GMOApplication• GMO detection and identification

Interest • Limited number of PCR• Easy and cost-effective method• Multi-analytical

Users• Enforcement and private labs• Agro-food Companies

Fixation ctlPos Hyb ctl

P-FMVP-masCryIAb

PatNeg Hyb ctl.

bar P-nosbarnase T-tmlbarstar T-nos

hsp70Int. IVS 6FS gene

Fixation ctl

Fixation ctl

EPSPSIVS2nptII

Soja sp Tomato spCorn sp Canola sp

gox Lac Z

Other targets: standards, controls...

INRA PMDV / MDO

MDO statistical expertise

• Identify the sources of measure uncertainty and optimize the protocols

• Decrease both Buyer’s and Seller’s Risk(see fig)

Means:Increase Precision of Analytical MethodsImprove sampling and testing schemes

INRA PMDV / MDO

0

20

40

60

80

100

0 0,5 1 1,5 2

GM Seed (%)

Lots

Acc

epte

d (%

)

1000 seed

Acc/Rej = 1%

3000 seed6000 seed

(Seller’s Risk)

(Buyer’s Risk)

INRA PMDV / MDO

2%1000

(20 GM)

1000(0 GM)

1x106 seed

(10,000 GM)

1%

1%1000

(10 GM)

0%

Multiple control plans: sampling

6

INRA PMDV / MDO

MDO participates to various programs

• ACTA « filières sans OGM » = First European study on the feasibility of a non GM food chain (financed by 37 Europeanorganisations) (1999-2000)

• CNIEL (2003)• QPCR-GMOFOOD (2000-2003)• GMO-CHIPS (2001-2004)

To come ...• SIGMEA• proposal in the frame of the FP 6 INRA PMDV / MDO

CNIEL(AQS R99/16)

« Recherche de séquences nucléiques et de protéines d’OGM végétaux dans les produits animaux »

• 3 French partners: INRA Jouy, INRA Versailles, AFSSA

• supported by the French Ministry of Agriculture

• devoted to the detection of GM DNA and proteins in the blood and milk of cows fed with GM Bt176 maize event.

INRA PMDV / MDO

Artificial contamination of bloodand milk with Bt176 products

ELISA Mass

Spectrometry

CryIAb proteinsgenomic DNA Bt176

QRT-PCR

P35S (cf3/cR4)

Adh (F3/R4)

CryIAb (cry03/04)

CNIEL Strategy

PCR-ELISA

P35S (cf3/cR4)

Adh (F3/OGM-14)

CryIAb (cry03/04)

INRA PMDV / MDO

Results

Extraction protocol for blood set up.

Good sensitivity for the 3 QRT-PCR systems tested :

LOD = 2 copies

INRA PMDV / MDO

QPCR-GMOFOOD(QLK1-1999-01301)

• devoted to the development of edge-fragment based QRT-PRC tests for GMO quantification

INRA PMDV / MDO

T25 maize- LibertylinkTM (Bayer CropScience)

after sequencing

public data

ADN maïsP35S* pUC18 P35S pat T35SpUC18

bla*

bla*

pUC18 P35S pat T35S

bla*

GMO targets : edge fragments

7

INRA PMDV / MDO

species specific targets

• Maize – Test Adh

• Rapeseed– Test PE3-PEPCase

• Tomatoe – β fructosidase

• Potatoe– β fructosidase

INRA PMDV / MDO

Development of multiplex-PCRs

INRA PMDV / MDO

Qualitative tetraplex

INRA PMDV / MDO

MAIZE GMO insert

Forward PrimerTaqman

probe Reverse Primer

GMO’ specific PCR productsAmplified fragment sizes have to be longer than 70 bp (for qualitative PCR) and to be shorter than 200 bp (for quantitative PCR).

Bt11 171 bp T25 151 bp ADH 134 bpBt176 104 bp Mon810 90 bp

Primer selection

INRA PMDV / MDO

Experimental design used to optimise the m-PCR reaction, with as few as possible combinations.

Studied parameters and values:• Copy Number of GMO: 100 and 500 copies of each GMO target.• With or without ballast DNA: 0 ng or 100 ng of non-GM maize.• Primers concentrations: 1 µM or 2 µM.• Primers ratio: ½ or ¼ (GMO/Maize)• With or without DMSO: 0 or 5 %. • MgCl2 Concentration: 1.5mM, 1.9mM, 2.2mM, 2.5mM.• Annealing temperature: 57°C, 58°C, 59°C, 60°C.

Qualitative PCR Optimization

INRA PMDV / MDO

after multiplex-PCR optimisation

Simplex PCRswith specific primers

Bt11

Bt176Adh

T25

Mon810

Multiplex before optimisation(2,000 and 200 copies each GMO)

Multiplex after optimisation(2,000 and 200 copies each GMO)

Bt 176AdhT25Mon 810

Bt 176Adh

ballast DNA amount

[primers]

primer concentration ratio

[MgCl2]

T°C

[DMSO]

DNA target amount

8

INRA PMDV / MDO

Duplex quantitative assays

( Bt176, T25 and Mon810 / Adh)

INRA PMDV / MDO

Duplex-Quantitative Real Time-PCR

OGM specific probes (VIC-labelled)

Adh probe (Fam-labelled)

MAIZE GM insert

Forward PrimerTaqman

probe Reverse Primer

GMO specific PCR product

INRA PMDV / MDO

GMO-CHIPS(G6RD-CT2000-00419)

• devoted to the development of(i) time- and cost-effective consensus

PCR tests and microarrays(ii) (ii) a matrix approach for detection of

unknown GMOs

INRA PMDV / MDO

PCR and LABELING

Consensus primer 1

Consensus primer 2

Hybridization on specificcapture probes

GMO constructions

PAT CryIA Bar nptII EPSPS

Specific capture probes

Hybridisation results analysis

INRA PMDV / MDO

Bt11

+ hy Ctl 5.86nM

Bt176 GA21 Mon810

T25,T45,Topas

RRS Starlink OXY-235

Pat gene

buffer

P-35S

T-nos test

nptII test

CaMV test (a)

CaMV test (b)

+ hy Ctl 11.72nM

+ hy Ctl 23.44nM

+ hy Ctl 46.875nM

+ hy Ctl 93.75nM

+ hy Ctl 187.5nM

+ hy Ctl 375nM + hy Ctl 750nM

+ hy Ctl 1.5µM + hy Ctl 3µM - hy Ctl - hy Ctl

Rapeseed Sugar beet

Tomato

EF Bt11

EF Bt176

EF GA21

EF T25

EF RRS EF Starlink

EF Mon810

Maize Soybean

Fix Ctl 5nM

Fix Ctl 80nM

buffer

Fix Ctl 20nM

Fix Ctl 2.5nM

Fix Ctl 40nM

Fix Ctl 10nMFix Ctl 160nM

buffer

GMOchips design(04/10/2003)

INRA PMDV / MDO

Specific GMOchips- Sensitivity test

Bt11 0.3% Bt11 0.1% Bt11 0.03% Bt11 0%

Bt11 1%Bt11 2%Bt11 100% Bt11 5%

9

INRA PMDV / MDO

Specific GMOchips- Sensitivity for GMOs in a Mixture

Bt11 Starlink RRS GA21MIX2-a 0,3% 33% 33% 33%

PCR OPP35S1/OPTnos2

Bt11 GA21CBH351RRS

Pat gene

P35S

PCR OPEPS1B/OPTnos2

INRA PMDV / MDO

Hybridization on chip:

GMO : Bt 176, Bt 11, Mon 810, GA 21, RRS, GT 73, OXY 235, Starlink,T 25,Topas 19/2,T 45

Sensitivity tests : 100, 5, 2, 1, 0.3, 0.1 et 0.03 %

Actual results

Reference genes : sucrose synthase (maize, soybean),

rubisco activase (rapeseed, tomato, sugar beet)

Small fragments : P35S (INRA),nptII and T-nos (CSIC)

Edge fragments : Bt 11, Bt 176, T25, RRS, GA21, Mon810, Starlink

Creation of a plasmid bank: with specific fragments of the11 GMO tested.

Prevalidation of the chip in the upcoming weeks

INRA PMDV / MDO

SIGMEA European program (STREP)

Cf Mr Messean’s presentation

•Study of the impact of GMO introduction in european agricultural landscapes

•MDO will contribute to the :

- Comparison of protein- and DNA- based methods for on-site monitoring of GMOs in soybean and maize;

- Development of an endogenous species specific reference gene detection system for sugar beet;

- Validation of the methods developedINRA PMDV / MDO

Strip test

• Obtain quick, easy & accurate results < 5 minutes

• Analysis in the field• Significant benefit to the

customer– Low cost– Highly reliable results

Scope extension to some processed products...

INRA PMDV / MDO

MDO participates to Proficiency testing

• BIPEA (EU)• AACC (USA)

INRA PMDV / MDO

MDO contributes to Methodvalidation (JRC)

• Mon 810• Bt11• Adh• T25

Soon:• GMO-CHIPS microarray prevalidation

10

INRA PMDV / MDO

MDO ACCREDITATION programme

• ISO 17025 (Quality assurance)• French Norme XP-V 03-020-1• Application domain :

– Matrix: seeds, plants– Maize and Soja

INRA PMDV / MDO

Trainings at MDO

• Proteus program: 1 PhD student on the detection of unknown GMO (K. Cankar, NIB)

• students under- and post-graduate from France and abroad.

• scientists from France and abroad

INRA PMDV / MDO

Main results or current developments• CaMV control for P35S screening• Adh reference gene for corn (JRC’s validation)• T25 quantitative identification (JRC’s validation)• Qualitative and Quantitative event-specific Multiplex• Generic fractional design plans for rapid and cost-

effective PCR optimization• Cost-effective consensus PCR and micro-arrays

methods to face the increasing number of approved GMO

• Methods to detect unknown GMO• Ways to improve precision and accuracy of the

Quantitative detection methodsINRA PMDV / MDO

Subjects of futur work• Control and sampling plans • Compatibility of the methods (proteins / nucleic acids;

control plans; grains vs. DNA copies, screening vs. identification …) and international rules and standards (ISTA vs. CEN / ISO)

• Identification of the critical points of doc-based traceability

• Technical challenges :– Procedures (apparatus, grinding, homogenization, analyte extraction, extraction

automation, DNA assay…) and measures biases (calibration, measure uncertainty…)

– Standards (availability, stability, continuity, type, analyte content of tissues)

INRA PMDV / MDO

Thank you !

INRA PMDV / MDO

11

INRA PMDV / MDO

GMO detection and standardization

INRA PMDV / MDO

Standardization• National bodies (AFNOR, DIN…)• CEN TC 233 and TC 275• ISO (CEN docs + vertical method e.g. Oleagineous)

• Other standardization bodies (ISTA, AACC, AOAC…)

Validation: • collaborative trials (ISO 5725, IUPAC harmonized

protocol…) • “Single Lab” and Performance criteria of Codex

Alimentarius

INRA PMDV / MDO

French standardization and accreditation

• Standard XP V 03-020-1 (published December 2000)

– DNA based methods– General guideline and requirements

• Accreditation program under way

INRA PMDV / MDO

Current CEN Projects

• TC 233, • Mandate of the EFTA• 54 standards on biotechnology

(laboratory requirements and organization, apparatus performances, sampling during GMO releases…)

INRA PMDV / MDO

CEN/TC 275: Food analysis - horizontal methods

WG 11: Genetically modified foodstuffs

Task:

Elaboration of standards for the detection of genetically modified organisms and derived products

Current CEN Projects

INRA PMDV / MDO

Work Programme of CEN/TC 275/WG 11:agenda item

• General requirements and definitions 7.5

• Sampling - 12/04* 7.6

• Nucleic acid extraction - 12/02* 7.3

• Qualitative nucleic acid based methods - 12/02* 7.2

• Quantitative nucleic acid based methods - 12/04 * 7.1

• Protein based methods - 09/00* 7.4* Document available at CS for enquiry

Current CEN Projects

12

INRA PMDV / MDO

Detection methods