Detection methods in support to

the EU legislation on GMO

www.jrc.ec.europa.eu

Principles of EU-GMO policy 1. Zero Tolerance for un-authorised GMOs

2. Authorisation for

a. cultivation,

b. and/or use in food

c. and/or use in feed

3. Authorisation requires

a. Risk assessment (EFSA: European Food Safety Authority)

b. EURL GMFF validated method for detection, identification and

quantification of the authorised event

c. CRMs

4. Authorisation triggers labelling of GM-food or feed products

a. containing GMO as ingredient

b. with unavoidable GMO-contamination >0.9% of total ingredient

No method – no authorisation

GM food and feed in the EU

59 GM event authorised for food/feed use

Methods for their identification and quantification (0.9%)

21 GM events pending authorisation

Methods to enforce the Minimum Required Performance

Limit (0.1%) in feed

1) European Union Reference Laboratory under Regulation (EC) No 882/2004 on official controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules

1) European Union Reference Laboratory under Regulation (EC) No 1829/2003 on GM food and feed

The European Union Reference Laboratory for GM Food & Feed: 2 legal mandates from 2 EU Regulations

EURL – NRLs: the instrument for internal market control

Official Control laboratories

NRLs (28 MS 36 National

Reference Laboratories)

5 3 November 2015

European Commission

EURL

EURL

EURL

EURL on GMOs

National Reference Laboratories implement the official controls and must: - Coordinate National Official Control Laboratories - Work in accordance with internationally approved performance standards and thus, - Use methods of analysis that have been validated.

EU Reference Laboratories:

- Make reference methods available; - Organise comparative testing; - Training & workshops

- Coordinate adoption of new technologies - Do not exercise controls but may intervene in cases of dispute.

Validation - Verification and confirmation that a method is fit for purpose - Purpose: Detection, identification, and quantification of GMO

in food and feed

- Validation against pre-defined method performance criteria - http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm

- 5 step validation process - Step 1&2: Completeness and scientific assessment of dossier - Step 3: In-house testing - Step 4: International validation ring trial (12 ENGL labs) - Step 5: Reporting to EFSA

EURL GMFF

mandate acc. to Reg. (EC) No 1829/2003 and implementing Regulations

Method acceptance and performance criteria

General items

• Applicability

• Practicability

Module ‘DNA extraction’

• Concentration

• Yield

• Structural integrity

• Purity

Analytical module

• Specificity

• Dynamic range

• Amplification efficiency

• R2

• Trueness

• LOQ and LOD

• Repeatability SD

• Reproducibility SD

• False positive rate

• False negative rate

• POD

Gene specific

Construct specific

Event-specific method (qPCR => probe)

Specific

ity

Low

High

Plant DNA Plant DNA Promoter Gene Terminator

Type of PCR methods

GMOMETHODS: The European Union Database of

Reference Methods for GMO Analysis

Available at: http://gmo-crl.jrc.ec.europa.eu/gmomethods/ More information: Bonfini et al. Journal of AOAC international (2012)

10

The GMOMETHODS database

• Criteria for inclusion of detection methods:

• Validated in a collaborative trial, according to the principles and

requirements of ISO 5725 and/or IUPAC protocol

• Verified by the EU-RL GMFF in the context of compliance with a EU

legislative act.

• Unique identifiers for each method, linked to the full information

of specific primer and probe sequences

• About 140 PCR methods (quali/quanti/EL/CN/EV/TX)

11

Producing GMO CRMs for implementing EU legislation

Certification of GMO CRMs

GMO content established by accurate weighing (taking purity and water content into account), verified by qPCR

Homogeneity studies ensuring the same GMO content in each unit

Stability studies and post certification monitoring ensuring the same GMO content over time *

* Including stock control to trigger timely reproduction

ERM-BF410 and ERM-BF410k (RUR soya) ERM-BF411 (Bt-176 maize) ERM-BF412 (Bt-11 maize) ERM-BF413 and ERM-BF413k (MON 810 maize) ERM-BF414 (GA21 maize) ERM-BF415 (NK603 maize) ERM-BF416 (MON 863 maize) ERM-BF417 (MON 863 x MON 810 maize) ERM-BF418 (1507 maize) ERM-BF419 (H7-1 sugar beet) ERM-BF420 (3272 maize) ERM-BF421 (EH92-527-1 potato) ERM-BF422 (281-24-236 x 3006-210-23 cotton) ERM-BF423 (MIR604 maize) ERM-BF424 (59122 maize) ERM-BF425 (356043 soya) ERM-BF426 (305423 soya) ERM-BF427 (98140 maize) ERM-BF428 (GHB119 cotton) ERM-BF429 (T304-40 cotton) ERM-BF430 (AM04-1020 potato) ERM-BF431 (AV43-6-G7 potato) ERM-BF432 (DAS-684164 soya) ERM-BF433 (DAS-40278-9 maize) ERM-BF434 (73496 rapeseed) ERM-BF435 (PH05-026-0048 potato) ERM-BF436 (DAS-44406-6 soya) ERM-BF437 (DAS-81419-2 soya) ERM-BF438 (VCO-Ø1981-5 maize) * ERM-BF439 (4114 maize) * * in production

World–wide distribution to GMO testing laboratories

GMO analysis: Workflow

2. Extraction – Quantification – inhibition check

3. Screening – Identification of GM

4. Quantification (event and taxon)

1. Sample preparation

Am I a GMO?

Qualitative

Presence/absence

Taxon identification

Verification of DNA amplicability

Evaluation of GMO presence

(Screening)

GMO identification (event-specific)

Quantitative

The need for decision-support tools in traceability of GMOs

The number of new GMOs is increasing constantly

The development of screening approaches using element- or

construct-specific detection methods are crucial for:

1. Time and cost effective routine testing strategies by GMO

testing laboratories

2. Responses to non-authorised GMO alerts, for which event-

specific methods are usually not available.

16 3 November 2015

Due to the increased number of approved GM events, screening for P35s and tNOS alone is no longer

sufficient!

Most commonly used promoters in GM plants Most commonly used terminators in GM plants

Letting go of the past

Identification?

MON863 BT176 BT11 DAS59122 NK603 T25

MON40-3-2 A2704-12 MON89788 A5547-127 DP-305423 DP-356043

GA21 MON810 DAS1507 3272 MIR604 MON88017

P35S T-Nos NPTII

LL25

MON531

3006x281

CP4 EPSPS PAT

Screening strategy: the matrix approach

Crop GM Event

GM elements and constructs

element 1 element 2 element 3 construct 1 construct 2

cotton GM Event 1 + + + - -

cotton GM Event 2 + + - - -

cotton GM Event 3 - - + - -

cotton GM Event 4 - + - + -

maize GM Event 5 + - - - +

maize GM Event 6 - + - - +

maize GM Event 7 - - - - -

maize GM Event 8 + + - + -

maize GM Event 9 + + + + -

soybean GM Event 10 - + + - -

soybean GM Event 11 + - - + +

soybean GM Event 12 - - + - +

soybean GM Event 13 + - - + -

4) Quantification of

GMO found

GMO Event 1507 Maize (DAS-01507-1) Maize - + - - + - Auth

GMO Event 176 Maize (SYN-EV176-9 ) Maize - - + - + - LLP

GMO Event 3272 Maize (SYN-E3272-5) Maize - - - - - + LLP

GMO Event 59122 Maize (DAS-59122-7) Maize - + - - + - Auth

GMO Event 98140 Maize (DP-098140-6) Maize - - - - + - LLP

GMO Event Bt11 Maize (SYN-BT011-1) Maize - + - + + + Auth

GMO Event DAS-40278-9 Maize (DAS-40278-9) Maize - - - - - - LLP

GMO Event GA21 Maize (MON-00021-9) Maize - - - - - + Auth

GMO Event MIR162 Maize (SYN-IR162-4) Maize - - - - - + Auth

GMO Event MIR604 Maize (SYN-IR604-5) Maize - - - - - + Auth

GMO Event MON810 Maize (MON-00810-6) Maize - - - - + - Auth

GMO Event MON863 Maize (MON-00863-5) Maize - - - - + + Auth

GMO Event MON87460 Maize (MON-87460-4) Maize - - - - + + LLP

GMO Event MON88017 Maize (MON-88017-3) Maize + - - - + + Auth

GMO Event MON89034 Maize (MON-89034-3) Maize - - - - + + Auth

GMO Event NK603 Maize (MON-00603-6) Maize + - - - + + Auth

GMO Event T25 Maize (ACS-ZM003-2) Maize - + - - + - Auth

GMO Event GT73 Rapeseed (MON-00073-7) Rapeseed + - - - - - Auth

GMO Event Ms1 Rapeseed (ACS-BN004-7) Rapeseed - - + - - + LLP2

GMO Event MS8 Rapeseed (ACS-BN005-8) Rapeseed - - + - - + Auth

GMO Event Rf1 Rapeseed (ACS-BN001-4) Rapeseed - - + - - + LLP2

GMO Event Rf2 Rapeseed (ACS-BN002-5) Rapeseed - - + - - + LLP2

GMO Event RF3 Rapeseed (ACS-BN003-6) Rapeseed - - + - - + Auth

GMO Event T45 Rapeseed (ACS-BN008-2) Rapeseed - + - - + - Auth

GMO Event Topas 19/2 Rapeseed (ACS-BN007-1) Rapeseed - + - - + - LLP2

GMO Event 305423 Soybean (DP-305423-1) Soybean - - - - - - LLP

GMO Event 356043 Soybean (DP-356043-5) Soybean - - - - + - Auth

GMO Event 40-3-2 Roundup Ready Soybean (MON-04032-6) Soybean - - - - + + Auth

GMO Event A2704-12 Soybean (ACS-GM005-3) Soybean - + - - + - Auth

GMO Event A5547-127 Soybean (ACS-GM006-4) Soybean - + - - + - Auth

GMO Event CV127 Soybean (BPS-CV127-9) Soybean - - - - - - LLP

GMO Event FG72 Soybean (MST-FG072-3) Soybean - - - - - + LLP

GMO Event MON87701 Soybean (MON-87701-2) Soybean - - - + - - Auth

GMO Event MON89788 Soybean (MON-89788-1) Soybean + - - - - - Auth

MA

IZ

E E

VE

NT

S

CA

NO

LA

S

OY

BEA

N

Screening results: the matrix approach

1) Taxon identification

and verification of

DNA amplification

2) Evaluation of GMO

presence

3) Identification of

GMO:

Use necessary event-

specific methods

Towards the future and harmonization: adopting new screening tools

Pre-Spotted Plates (PSP)

Ready-to-use and multi-target detection system

Multiplexing assays

22

Pre-Spotted Plates (PSP) for GMO detection

PSP

RTi-PCR plastic support whose wells are spotted with dried primers &

probes targeting chosen DNA sequences (GMO detection assays)

Advantage

Perform up to 96 assays in a single experiment, with limited steps

Multi-target ready-to-use PCR plates

23

Screening-PSP

6 Element-sp. Methods 7 Taxon-sp. Methods 3 Event-sp. Methdos

Event-sp. PSP

All GM events from 4 species listed in EU register Maize, Soybean, Oilseed rape and Cotton

1 2 3 4 5 6 7 8 9 10 11 12

A HMG E3272 E98140 BT11 BT176 DAS 40278 DAS 59122 GA21 MIR162 MIR604 MON810 MON863

Samp

le 1

B MON 87460 MON 88017 MON 89034 NK603 T25 TC1507 LEC A2704 A5547 CV127 DP 305423 DP 356043

C FG72 GTS 40-3-2 MON 87701 MON 89788 CruA GT73 MS1 MS8 RF1 RF2 RF3 T45

D Topas 19/2 Sah7 E281 E3006 GHB119 GHB614 LL Cotton25 MON 1445 MON 15985 MON531 MON 88913 T304

E HMG E3272 E98140 BT11 BT176 DAS 40278 DAS 59122 GA21 MIR162 MIR604 MON810 MON863

Samp

le 2

F MON 87460 MON 88017 MON 89034 NK603 T25 TC1507 LEC A2704 A5547 CV127 DP 305423 DP 356043

G FG72 GTS 40-3-2 MON 87701 MON 89788 CruA GT73 MS1 MS8 RF1 RF2 RF3 T45

H Topas 19/2 Sah7 E281 E3006 GHB119 GHB614 LL Cotton25 MON 1445 MON 15985 MON531 MON 88913 T304

1 2 3 4 5 6 7 8 9 10 11 12

A HMG p35s HMG p35s HMG p35s HMG p35s HMG p35s HMG p35s

B Lec tNOS Lec tNOS Lec tNOS Lec tNOS Lec tNOS Lec tNOS

C CruA CTP2-EPSPS

CruA CTP2-EPSPS

CruA CTP2-EPSPS

CruA CTP2-EPSPS

CruA CTP2-EPSPS

CruA CTP2-EPSPS

D Sah7 PAT Sah7 PAT Sah7 PAT Sah7 PAT Sah7 PAT Sah7 PAT

E UGP BAR UGP BAR UGP BAR UGP BAR UGP BAR UGP BAR

F PLD Cry1Ab PLD Cry1Ab PLD Cry1Ab PLD Cry1Ab PLD Cry1Ab PLD Cry1Ab

G GS CV127 GS CV127 GS CV127 GS CV127 GS CV127 GS CV127

H DAS-

40278

DP-305423

DAS-40278

DP-305423

DAS-40278

DP-305423

DAS-40278

DP-305423

DAS-40278

DP-305423

DAS-40278

DP-305423

Replicate 1 Replicate 2 Replicate 1 Replicate 2 Positive Negative

Sample 1 Sample 2 Controls

Development of a GMO Screening System

24

+

I. Which assays?

II. Assay standardization for use on PSP (and re-assessment of method

performance)

III. Building a screening strategy: combine PSP with a Decision Support System

+

Web!

Best identification

strategy!

25

Digital Droplet PCR

Digital Array Chip: integrated fluidic circuit

• 12 sample input ports and panels • 765 partitions per panel

Sample inputs

Chip

Pressure

Accumulator

Sample inlets

Partition

Pressure valve

0

1000

2000

3000

4000

5000

6000

0 100 200 300 400 500 600 700 800 900

Number of positive partitions (H )

Nu

mb

er

of

co

pie

s o

f ta

rget

DN

A (

T)

Binomial approximation T = number of copies of target DNA H = positives partitions (hits) C = total number of partitions

As H increases, there is an increase in the number of partitions containing more than one copy of the target DNA

dPCR: identified pros

29

• No need for standard curves

• Less sensitive to inhibition

• Applicability to difficult matrices (to be further investigated)

• High sensitivity, precision, trueness

• Cost effectiveness

• Multiplexing

ENGL WG on dPCR

31

• Transferability of existing qPCR methods into a digital PCR format

• Accreditation • Applicability to difficult matrices • Applicability to analytical areas other than GM

food/feed • Definition and assessment of relevant method

performance criteria • Multiplexing

From: Loman et al. High-throughput bacterial genome sequencing: an embarrassment of choice, a world of opportunity. Nature Rev Micr, 2012

NGS = Next Generation Sequencing

Ability to read DNA:

• in a very fast and cheap manner

(massive parallel sequencing)

• in every field related to DNA, i.e.

related to Life Science

NGS in

labs

Reduction of costs Increased throughput

NGS in

labs

From billions to trillions!

Advantages of NGS

From millions to hundreds!

NGS applications in the GMO field

New perspectives

• New information about GMOs • New strategies for GMO characterization (unauthorized) • Development of new detection methods

Thank you for your attention

www.jrc.ec.europa.eu