Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Simposio Internacional sobre Detección de OGM25 y 26 de octubre 2011, México
Implementation of the ISO and JRC standards in GMO analysis laboratories
Mathieu Rolland 1 - Emmanuelle Pic 2
1 GEVES - BioGEVES, 2 Anses - Plant Health Laboratory
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
The European regulatoryframework :
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
The European regulatoryframework :
Main regulations :
- Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC
- Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed
- Regulation (EC) No 1830/2003 of the European Parliament and of the Council of 22 September 2003 concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending Directive 2001/18/EC
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
The European regulatoryframework :
To submit an application, the petitioner has to provide:
- The sequence of the event- The description of a detection method (based on qPCR)- Some reference material
Petitioner : submission of application
National Competent Authority
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
The European regulatoryframework :
Petitioner : submission of application
National Competent Authority
European Food Safety Authority
Scientific opinion of EFSA
6 monthsMember States
consultation(3 months)
Validation of the detectionmethod by the European
reference laboratory
Decision of the EU :authorization or refusal
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
The European regulatoryframework :
European Network ofGMO Laboratories
France :
Petitioner : submission of application
National Competent Authority
European Food Safety Authority
Validation of the detectionmethod by the European
reference laboratory
A network of three official laboratories
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
The European regulatoryframework :
Plant Health Laboratory
Petitioner : submission of application
National Competent Authority
European Food Safety Authority
Validation of the detectionmethod by the European
reference laboratory
European Network ofGMO Laboratories
France :
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
The French Network of official laboratories :
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
GEVES: Group for the study and control of varieties and seeds
(National Institute for Agricultural Research)
Ministry for Food, Agriculture and Fisheries (The French Association for
Seeds and Seedlings)
A public Interest Group (GIP)
An official and unique organization in France
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Two main tasks
Analysis of 4 different types :
- Detection of GMOs
- Detection of pathogens
- Genetic marking of varieties
-The analysis of biochemical constituents
Participation in research programs:
- Expertise at the international level : adaptation, optimization and validation of protocols
- Participate in and monitor inter-laboratory networks
- Scientific publications, posters, oral communications…
Accreditation ISO 17025cofracsince 2004
ACCREDITATION
N°1-1540
WWW.COFRAC.FR
BioGEVES: Laboratory of molecular biology and biochemistry of GEVES
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
French Agency for Food, Environmental and Occupational Health & Safety
Ministry for
health
Ministry for food, agriculture and
fisheries
Ministry for consumer
affairs
Ministry for ecology and sustainable
development
Ministry for labour
Parliament
Vote of the annual grant, annual report
5 supervisory ministriesState goals & resources
contract
Involvement in several national plans:
environmental health, occupational health,
Ecophyto 2018, Cancer Plan, etc.
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
- human health and safety in the fields of environment, work and food,- protection of animal health and welfare, as well as plant health.
Assess nutritional and health risks and benefits
Monitor health safety
Conduct, coordinate and initiate research
projectsCoordinate the R 31 network of scientific
organisations
Collaborate with European agencies(EFSA, ECHA , EEA, EU-OSHA, ECDC and EMA)
Authorise marketing of veterinary medicinal
products
Provide training and information and contribute
to public debate
Provide laboratory reference missions
Recommend public health measures
Anses contributes to ensuring :
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
- human health and safety in the fields of environment, work and food,- protection of animal health and welfare, as well as plant health.
Assess nutritional and health risks and benefits
Monitor health safety
Conduct, coordinate and initiate research
projectsCoordinate the R 31 network of scientific
organisations
Collaborate with European agencies(EFSA, ECHA , EEA, EU-OSHA, ECDC and EMA)
Authorise marketing of veterinary medicinal
products
Provide training and information and contribute
to public debate
Provide laboratory reference missions
Recommend public health measures
Anses contributes to ensuring :
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
12 reference and research laboratories
Bacteriology, Virology and GMO
unit (Angers)
Plant Health Laboratory : 6 sites (Montpellier, Nancy, Rennes, Saint-Pierre (Reunion Island), Clermont-Ferrand and Angers)
Accreditation ISO 17025 by the COFRAC for the detection and quantification of some GMO events
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Standards for the detection of genetically modified organisms:
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
ISO standards :
- ISO 24276:2006 "Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - General requirements and definitions"
- ISO 21571:2005 "Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Nucleic acid extraction"
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
- ISO 21569:2005 "Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Qualitative nucleic acid based methods"
- ISO 21570:2005 "Foodstuffs - Methods of analysis for the detection of genetically modified organisms and derived products - Quantitative nucleic acid based methods"
ISO standards :
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
- Guide technique d'accréditation COFRACLAB GTA 02, March 2008
- Guide d'application AFNORGA V03-042, November 2006
In France : guides to helpapply the ISO standards
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Implementation of a new method
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
- Compendium of reference methods for GMO analysis
- http://gmo-crl.jrc.ec.europa.eu/gmomethods/
Methods description :
Intellectual Property aspects
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
- Compendium of reference methods for GMO analysis
- http://gmo-crl.jrc.ec.europa.eu/gmomethods/
Intellectual Property aspects
CropLife International licences
Methods description :
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Unit of certification : mass/mass or copy number/copy number
Certified Reference Materials :
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Specificity :- Theoretical evaluation (FastA, Blast etc. on GenBank, EMBL etc.)- Experimental evaluation (preferably on CRM) :
Qualitative analysis :
Events
Method GA21 NK603 MON810 TC1507 Bt11 …
GA21
NK603
MON810
TC1507
Bt11 …
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Limit of Detection (LOD) :
Target copy number
Haploid genome copy number
Relative GMO content (%)
Number of repetitions
60 60,000 0.1 6 (ISO) or 10 (JRC)
40 60,000 0.07 6 (ISO) or 10 (JRC)
20 60,000 0.03 6 (ISO) or 10 (JRC)
10 60,000 0.02 6 (ISO) or 10 (JRC)
5 60,000 0.01 6 (ISO) or 10 (JRC)
0.1 60,000 - 6
- Independent dilutions (each level) of GMO DNA in non-GMO DNA- LOD = lowest level where 6 (or 10) repetitions give a positive result- 0.1 copy level : the number of positive results must be 1 (control of the method used to quantify DNA)
Qualitative analysis :
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Quantitative analysis :
Specificity and LOD : idem qualitative analysis.Limit of Quantification (LOQ) :
Target copy number
Haploid genome copy number
Relative GMO content (%)
Number of repetitions
400 60,000 0.67 6 (ISO) or 10 (JRC)
300 60,000 0.50 6 (ISO) or 10 (JRC)
200 60,000 0.33 6 (ISO) or 10 (JRC)
100 60,000 0.17 6 (ISO) or 10 (JRC)
50 60,000 0.08 6 (ISO) or 10 (JRC)
0.1 60,000 - 6
Same principle as for LOD estimation but :- target copy numbers ~ 10 times higher- LOQ = level where RSDr 25 %
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Quantitative analysis :
Linearity :- At least 5 levels prepared by independent dilutions of GMO DNA in non-GMO DNA (The dynamic range must cover the values corresponding to the expected use)
- For each level, 4 independent experiments and 2 PCR by experiment.
R2 = 1y = -3.22 x + 49.5
Calculation of :- R2 - slope
Performance of the method :- R2 ≥ 0.98- -3.1 ≥ slope ≥ -3.6 (ideally -3.32)
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Quantitative analysis :
Accuracy (trueness and precision) :
Definition of minimum performance requirements for analytical methods of GMO testing 10/2008 (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm)
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Quantitative analysis :
Accuracy (trueness and precision) :
Verification of analytical methods for GMO testing when implementing interlaboratory validated methods 07/2011 (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm)
TruenessProcedure: The trueness should be determined at a level close to the level set in legislation (e.g. 0.9 %threshold), or according to the intended use of the method, and additionally at a level close to theLOQ.Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testingof samples. At least results from 16 PCR replicates should be obtained. Acceptance criterion: The trueness shall be within ± 25 % of the accepted reference value or a Z-scorewithin the range of 2 and -2 should be obtained.
Precision - Relative Repeatability Standard Deviation (RSDr)Procedure: Repeatability can be determined in a similar way as described under Trueness. It iscalculated from PCR replicates run under repeatability conditions (see Terminology). Repeatabilityshould be available for all tested GM-levels.Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testingof samples. At least 16 single test results should be obtained. Acceptance criterion: The relative repeatability standard deviation should be ≤ 25%, over the dynamicrange of the method.
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Quantitative analysis :
Accuracy (trueness and precision) :
Verification of analytical methods for GMO testing when implementing interlaboratory validated methods 07/2011 (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm)
TruenessProcedure: The trueness should be determined at a level close to the level set in legislation (e.g. 0.9 %threshold), or according to the intended use of the method, and additionally at a level close to theLOQ.Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testingof samples. At least results from 16 PCR replicates should be obtained. Acceptance criterion: The trueness shall be within ± 25 % of the accepted reference value or a Z-scorewithin the range of 2 and -2 should be obtained.
Precision - Relative Repeatability Standard Deviation (RSDr)Procedure: Repeatability can be determined in a similar way as described under Trueness. It iscalculated from PCR replicates run under repeatability conditions (see Terminology). Repeatabilityshould be available for all tested GM-levels.Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testingof samples. At least 16 single test results should be obtained. Acceptance criterion: The relative repeatability standard deviation should be ≤ 25%, over the dynamicrange of the method.
The trueness should be determined at a level close to the level set in legislation (e.g. 0.9 % threshold), or according to the intended use of the method, and additionally at a level close to the LOQ.
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Quantitative analysis :
Accuracy (trueness and precision) :
Verification of analytical methods for GMO testing when implementing interlaboratory validated methods 07/2011 (http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm)
TruenessProcedure: The trueness should be determined at a level close to the level set in legislation (e.g. 0.9 %threshold), or according to the intended use of the method, and additionally at a level close to theLOQ.Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testingof samples. At least results from 16 PCR replicates should be obtained. Acceptance criterion: The trueness shall be within ± 25 % of the accepted reference value or a Z-scorewithin the range of 2 and -2 should be obtained.
Precision - Relative Repeatability Standard Deviation (RSDr)Procedure: Repeatability can be determined in a similar way as described under Trueness. It iscalculated from PCR replicates run under repeatability conditions (see Terminology). Repeatabilityshould be available for all tested GM-levels.Testing conditions (reaction volume, PCR machine, etc.) should be the same as during routine testingof samples. At least 16 single test results should be obtained. Acceptance criterion: The relative repeatability standard deviation should be ≤ 25%, over the dynamicrange of the method.
The trueness shall be within ± 25 % of the accepted reference value or a Z-score within the range of 2 and -2 should be obtained.
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Summary
Favorable context for the EU official labs :
- Validation of the methods by the JRC- Access to the methods- Access to certified reference materials- In France: access to guides helping the Implementation of new methods
Context of low contamination levels
But : only applies to events for which an application has been submitted.
Mathieu Rolland - BioGEVES Emmanuelle Pic – ANSES 25 -27 octubre 2011, México
Thank you very much !
Muchas gracias !