Detection methods in support to the EU legislation on...
Transcript of Detection methods in support to the EU legislation on...
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)
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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)
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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
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BEA
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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
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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
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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
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+
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!
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
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• 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
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• 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!
New perspectives
• New information about GMOs • New strategies for GMO characterization (unauthorized) • Development of new detection methods