simposio internacional sobre detección de ogm 25 y 26 de octubre 2011, méxico

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


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



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





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



European Network ofGMO Laboratories

France :






A network of three official laboratories



Plant Health Laboratory






European Network ofGMO Laboratories

France :


The French Network of official laboratories :


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


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


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.


- 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 :


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


Standards for the detection of genetically modified organisms:


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"


- 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 :


- 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


Implementation of a new method


- Compendium of reference methods for GMO analysis

- http://gmo-crl.jrc.ec.europa.eu/gmomethods/

Methods description :

Intellectual Property aspects


- Compendium of reference methods for GMO analysis

- http://gmo-crl.jrc.ec.europa.eu/gmomethods/

Intellectual Property aspects

CropLife International licences

Methods description :


Unit of certification : mass/mass or copy number/copy number

Certified Reference Materials :


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 …


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 :


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 %



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)



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)




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.







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.


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.


Thank you very much !

Muchas gracias !

[email protected]

[email protected]

simposio internacional sobre detección de ogm 25 y 26 de octubre 2011, méxico

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