gmo committee - ista · pdf file• feedback to gmo committee for on-going activity ....

GMO Committee Chair: Cheryl Dollard – Canada Canadian Food Inspection Agency Vice Chair: René Mathis – France GEVES Committee Report 2010 – 2013

2013 Committee Members

Name Country Responsibiltiy Cheryl Dollard Canada Chair, PT WG Lead René Mathis France Vice-Chair, Rules and Handbook WG Lead Elizabeth Bates Belgium Accreditation Program Sofia Ben Tahar France Lutz Grohmann Germany David Grothaus USA Benjamin Kaufman USA Information Exchange – Workshops WG Lead Enrico Noli Italy Handbook – Co-Lead (w. R. Mathis) Jean-Louis Laffont France Kirk Remund USA Stacked Events – WG Lead Ana Laura Vicario Argentina Contact - New Technologies Bruno Zaccomer France Publication of PT Results – WG Lead Dabing Zhang China Tajinder Grewal Canada Andrea Jonitz Germany Christoph Haldemann Switzerland Past Chair (2007-2011) Guy Van Den Eede Italy Elena Perri Italy Audit Checklist – WG Lead Ray Shilito USA New member – 2013!!

2013-06-15 –GMO TCOM

GMO TCOM Working Groups • Work and projects are organized by Working Groups (WGs) – each with 4-10

members coordinated by a WG Lead; • Members work in several groups at a given time.

Working Group Lead Members

GMO Proficiency Tests C. Dollard E. Bates, A.Jonitz, R. Mathis, B.Zaccomer, C.Haldemann, J.-L. Laffont; R. Shilito, ISTA Technical Coordinator (N. Ettel )

Accreditation Program E. Bates S.Ben Tahar, L.Grohmann, D.Grothaus, E. Noli, E. Perri, A.-L. Vicario

Info Exchange: Workshops B. Kaufman C. Dollard, E.Noli, G.Van den Eede, B. Zaccomer, D.Zhang, C. Haldemann, J.-L. Laffont, K. Remund

Info Exchange: Webpage “Information platform for GM Seed”

Vacant (formerly C. Leonhardt)

T. Grewal, M. Crisin Ichim, B. Kaufman, E. Noli, G. Van den Eede, D. Zhang

Identification of stacked Genes K. Remund J.-L. Laffont, B. Zaccomer, C. Haldemann

Publication of results of the GMO PT

B. Zaccomer B. Zaccomer, E. Bates, C. Dollard, B. Kaufman, J.-L. Laffont, K. Remund, C. Haldemann

Establishment of an audit checklist

E. Perri E. Bates, S. Ben Tahar, R. Mathis

Rules-Handbook R. Mathis/E. Noli

E. Bates, C. Dollard, B. Kaufman, K. Remund, B. Zaccomer, C. Haldeman, J.-L. Laffont, A.-L. Vicario

2013-06-15 –GMO TCOM

Working group members:

Cheryl Dollard (Lead), Elizabeth Bates, Nadine Ettel,

Christoph Haldemann, Andrea Jonitz, Jean-Louis Laffont, René Mathis , Ray Shilito, Bruno Zaccomer

GMO PT Program 2010-2013

Progress Report: Proficiency Test

PT Year Crop # of Events

# of samples in a set

# seeds per sample

# of sample sets prepared

# participants - Member labs

# participants - Non-member

labs

PT14 2010/11 Soybean 1 12 3000 40 23 17 PT15 2011 Flax 1 8 1500 38 17 18 PT16 2012 Maize 1 9 1800 65 35 18 PT17 2012 Soybean 2 8 2000 48 22 23 PT18 2013 Maize 2 8 2500 In progress… In progress… In progress…

PT19 Soy 2013 PT20 Cotton 2014 PT21 Alfalfa 2014

Rounds completed within the last Tri-annum:

Upcoming rounds:

Two SOPS developed describing all steps required to execute a single PT round: “Administration of GMO PT program” “Sample preparation for GMO PT program” Still have many challenges: Sourcing GM and Non-GM Seed; Funding to cover associated costs

2013-06-15 – Cheryl Dollard

2013-06-15 – Cheryl Dollard

0102030405060

A

B

C

BMP05

101520253035

A

B

C

BMP

Quantification

Performance Trends:

Progress Report: Proficiency Test

Success of the Performance Based Approach for GMO Testing within ISTA:

13 laboratories are accredited for GMO Testing

Qualitative Detection


Elena Perri (Lead), Elizabeth Bates, Sofia Ben Tahar,

René Mathis

Establishment of an audit check list for GMO Testing Laboratories

Background vISTA has developed an Accreditation Standard vThe current version of the ISTA International Rules for Seed Testing is

an integral part of the Standard (ISTA Accreditation Standard 1.2). vThis is referred both to: - standard methods (the Rules include detailed description of the technical procedure and quality requirements) - PBA methods – GMO testing (the Rules describe only general criteria)

2013-06-15 – Elena Perri

Aims of the WG v to discuss the “minimum requirements” for GMO Accreditation by

ISTA: how the theoretical requirements should be implemented by the lab

v to cooperate with the ISTA Accreditation Department in order to provide a useful aid to the ISTA auditors

Main Topics discussed

• Management Requirements and Quality assurance system control

• Staff • Methods • Technical requirements: ü controls ü samples handling and environmental

conditions ü equipment and goods (relevant for the

quality of the tests) ü testing


What was done

• the GMO check list was completed and linked to the relevant ISTA Laboratory Accreditation Standard

• the minimum requirements for each topic of the check list (where applicable) were identified

• to be discussed with the ISTA auditors already asked to audit GMO laboratories (system and technical)

• to be provided the ISTA Accreditation Department and to be asked for a feedback

The draft check list is now ready:


Chronicles of GMO TCOM/Information Exchange Working Group: Workshops

2011- 2013 Beni Kaufman (Lead), C. Dollard, E.Noli, G.Van den Eede, B. Zaccomer, D.Zhang, C. Haldemann, J.-L. Laffont, K. Remund

2013-06-15 – Beni Kaufman

Date Organisation Event Place

July, 2011

ISTA/ The National Institute of Ecology (INE) , SEMARNAT, Mexico,

Autonomous Metropolitan University

Statistical Aspects of GMO

Detection

Mexico City, Mexico

Dec, 2011 ISTA/Shanghai Jiao Tong University

Biotechnology Trait Detection Shanghai,

China

Oct, 2012 ISTA/LaRAS – University Bologna ISTA GMO Auditors Training Bologna,

Italy


Workshops 2011- 2013

Location • LaRAS – University of Bologna, 9-11 October 2012 Organization • Rasha El-Khadem, Rita Zecchinelli, Enrico Noli • Lecturers: Christoph Haldemann, Jean-Louis Laffont, Enrico Noli Purposes • Train ISTA Auditors on GMO testing issues • Feedback to GMO Committee for on-going activity Pre-workshop Questionnaire • To assess Auditors background and experiences • To consider expectations and take suggestions

ISTA GMO Auditors Workshop

Program Theory: GMO Introduction; Testing objectives;PCR methods basics;

Seedcalc; Laboratory Accreditation - Method evaluation; Reporting results ;ISTA Proficiency Tests on GMO

Practice: Demo by LaRAS staff: Workflow in GMO testing by Quantitative rt PCR and qualitative PCR


~60 participants representing regulating bodies, academia, and industry. From 11 countries; Including:

Mexico Workshop: • Mexican Secretariat of Environment and Natural Resources (SEMARNAT) • Ministry of Agriculture, Livestock, Rural Development, Fisheries and Food

(SAGARPA) • The National Metrology Center (CENAM) • The Inter Ministerial Commission on Biosafety of Genetically Modified

(CIBIOGEM) • The National Institute for Forestry, Agriculture and Livestock (INIFAP) • The National Center for Genetic Resources • The Center for Scientific Research of Yucatan (CICY) • Researches from UAM campus Xochimilco • The National Seed Institute of Uruguay • ANSES GEVES - Plant Health Laboratory in France, • The Saskatchewan Research Council, Canada • Pioneer Hi Bred International, Monsanto, BASF Plant Science, Bayer

Crop Science Canada and Kenya Seed Company.

China Workshop: 中国农业科学院北京畜牧兽医研究所

LongPing High technology company Bioseed Research India Pvt. Ltd.

浙江省农科院农业部转基因植物环境安全监督检验测试中

心（成都） Korea Seed & Variety Service

Bayer Crop Science Croatian Centre for Agriculture, Food and

Rural Affairs, Institute for Seed and Seedlings

深圳市农业科技促进中心 Embrapa - Soybean

SGS company Embrapa Food Technology

Ti-Shiue Biotech Inc. 中国农业科学院北京畜牧兽医研究所

Industrial Technology Research Institute SGS company

Korea Seed & Variety Service 黑龙江省农科院农产品质量安全研究所杜邦先锋种子公司 - Pioneer Hi-Bred

深圳市农业科技促进中心 J R Simplot Company



René Mathis (Lead), Enrico Noli (Co-Lead - Handbook Development), Cheryl Dollard, Elizabeth Bates, Jean-

Louis Laffont, Beni Kaufman, Christoph Haldemann, Kirk Remund, Bruno Zaccomer, Ana-Laura Vicario, Elena Perri

GMO Rules Chapter

Working Group: Rule chapter on GMO testing

Outline of Presentation: • The objective • The schedule (and work done) • The content • Where we are now

2013-06-15 – René Mathis

Working Group: Rule chapter on GMO testing • The objective:

- To have a chapter dealing with GMO testing in ISTA‘s rules - To keep uniformity in GM seed testing

• Why having a chapter ?

• ISTA is involved in GMO testing on seeds • GMO testing is an increasing activity for seed trade • Laboratories need guidance about ISTA‘s principles and technical

aspects of GMO testing. • Answer ISTA‘s strategy decision (2001 : to establish a GMO chapter)

• Characteristics

• This chapter stays general on all aspects of GMO seed testing • This chapter is based on already existing ISTA‘s papers • Doesn‘t provide protocols (according to PBA) • Is linked to a Hand book for the detailed technical considerations • Is not creating new ISTA position / statement about GMO analysis


Working Group: Rule chapter on GMO testing - The chapter on testing for seeds of GMO gathers ISTA‘s existing practices to get all the

information in one document - Enhancing existing process (formerly chapter 8) by clarifying ambiguous parts and filling gaps - Associated Technical details and guidance will be presented in a Handbook.

Chapter 8

GMO Handbook (In progress)

ISTA’s position paper

(Strategy regarding Methods for GM seeds) Chapter 19:

Testing for seeds of GMO

Chapter 2

ISTA’s PD Evaluation (GM presence)

ISTA’s PD Evaluation (GM purity)


(View regarding the units for reporting)

ISTA’s paper Principles and Conditions

for Lab Accreditation under PBA



• The process (and work done) - 9 members WG

• E. Bates, C. Dollard, E. Noli, C. Haldeman, B. Kaufman, J.-L. Laffont, R. Mathis, K. Remund, B.Zaccomer,

- Launched by july 2011, work start from january 2012 - Working at distance: separately + phone calls (1/month) - 2 meetings (in presence) 4 days total

• 1 day meeting at ISTA congress 2012 (Venlo) • 3 days specific meeting at Toulouse (FR) february 2013

- First draft of the chapter

• October 2012: a first draft is shared with the GMO TCOM • Draft modified according to TCOM comments • November 2012 : Draft sent to ISTA Technical Coordinator

- Final draft of the chapter

• Feb 2013:chapter was edited to be more precise, then shared with the GMO TCOM • Adapted according to TCOM comments • March 2013 sent to ISTA Technical Coordinator + Rules Committee Chair


• The CONTENT • Additional chapter in ISTA rules : Chapter 19 • Testing for Seeds of Genetically Modified Organisms

- 19.1 Object - 19.2 Definitions - 19.3 General principles - 19.4 Procedure - 19.5 Testing approaches - 19.6 Calculation and expression of results - 19.7 Reporting results - 19.8 References


Working Group: Rule chapter on GMO testing:

• 19.1 Object - The object of testing for seeds of

genetically modified organisms (GMOs) is to give guidelines to detect, quantify or confirm the presence of GMO seeds in seed lots.

- These guidelines can be applied to testing adventitious presence (AP) of genetically modified organisms (GMOs) and GMO trait purity testing

• Main points

• It is about seed testing

• It covers AP and purity


Working Group: Rule chapter on GMO testing: Content

• 19.2 Definitions - Adventitious presence ; Analyte ;Certified

reference material ; Genetically modified organism ; GMO event ; GMO trait ; Limit of detection ; Limit of quantification ; Performance-based approach ; Proficiency test ; Seed bulk ; Seed group ; Transgenic ; Reference material



• Main points:

• Internationally used and accepted terminology in the field of GMOs

• 19.3 General principles • The ISTA strategy regarding methods for the detection, identification and

quantification of genetically modified seeds in conventional seed lots is available on the ISTA web site at: https://www.seedtest.org/upload/cms/user/42Int-M-I200142ISTAPositionPaperonGMOapproved14112001-update1.pdf

• This chapter describes testing for adventitious presence of GM seeds and GMO trait purity. Currently there is no universal threshold for GM seeds in conventional seed lots, or of regulated GM seed in deregulated GM seed, or a specified level of GMO purity in a seed lot; the establishment of reliable methods for the detection, identification and quantification of GMO content is therefore very important. Different technologies, strategies and methods for GMO testing are continuously evolving and new methods being developed. The quality of these test results depends much more on methodology, equipment and training than in other classical seed testing methods. This makes the standardization of GMO testing very difficult. The ISTA approach has targeted the uniformity in GMO testing results, not by the uniformity in testing methodology, but by using a performance-based approach (PBA). The PBA requires that laboratories demonstrate that the GMO detection, identification or quantification methods that they are using on seed samples for reporting results on ISTA Certificates meet acceptable standards set by ISTA. These standards include, among others, sampling, testing and reporting. In order for a laboratory to be recognised as ISTA accredited for GMO testing, it will need to ensure that documented evidence of validation and reliability of the laboratory is available to the ISTA auditors. The evidence must include:

• -performance data based on seed samples for the event and species for which the laboratory is seeking ISTA accreditation, and

• -participation in an ISTA GMO proficiency test including the specific event and species, if available.

• Main points:

• Refers to ISTA’s position paper (nov-2001)

• Targetting uniformity with the performance based approach:

• PDE • PT



• 19.3 General principles

• This requirement will ensure the reliability of the analysis and the final test result reported on the ISTA Certificate. The PBA gives seed testing laboratories the choice to use different technological approaches, e.g. bioassays, protein-based methods and DNA-based methods.

• For further information, see the ISTA Principles and Conditions for Laboratory Accreditation under the Performance Based Approach (see

• Generally, GMO tests that are used to assess GMO trait purity are identical to the tests used for testing for AP of GM seeds. However, there are differences in the testing steps as well as in the objectives. This chapter addresses these distinctions whenever they apply.

• Main points:

• Refers to ISTA’s principles for laboratory accreditation under PBA



- 19.4 Procedure

• Adventitious presence of GMO testing and GMO trait purity testing are “two sides of the same coin”; both applications make use of the same tests, and follow a very similar work flow (Figure 1). The expected results differ in the two applications. In GMO AP testing, most of the time the expected outcome is “not detected” or a low estimate of the proportion of GMO present. In GMO trait purity testing, the expected result is the quantification of a high percentage of presence of the specified trait.

• The methods used for these analyses can be classified and characterized in a number of ways. According to the level at which the analysis occurs, tests can be conducted at the DNA level (19.5.1), protein level (19.5.2) or organism level, as in bioassays (19.5.3).

• The appropriate approach to GMO testing is chosen according to the question which the test is attempting to answer (see Figure 1). A qualitative question, e.g. “Is there any GM seed in the sample?” can be answered by applying a qualitative test (see 19.5.1.2 and 19.5.2.2), while a quantitative question, e.g. “How much GM seed is there in a seed lot?” can be answered by using either a quantitative test (see 19.5.1.3) or a group-testing approach (Remund et al., 2001), also known as the semi-quantitative method (which relies on qualitative tests of seed groups). Another classification that applies only to DNA-based methods is in relation to the specificity of the method, as described further in section 19.4.1.

• Both AP GMO testing and GMO trait purity testing can be performed on individual seeds or on seed bulks, although each application will require a different sampling and testing scheme. Seed bulk testing is more common in AP GMO testing, where the detection target is a transgenic protein or a DNA segment. GMO trait purity tests are usually performed on a representative sample of individual seeds or seedlings, and target the GMO trait or, similarly, the protein or the DNA. However, when performed on seed bulks, the test is performed at the DNA level to detect the absence of transgenic DNA, and targets the uninterrupted insertion site (Battistini and Noli, 2009)

• Main points • (See fig1 scheme) • Similar workflow for AP or

purity • Expected results differ • Different “levels” of analysis

• AP or purity testing will require different testing schemes



• 19.4 Procedure

• Main points:

• (See fig 1 scheme) • Similar workflow for AP or

purity • Expected results differ • Different “levels” of analysis:

• DNA, Protein, Organism

• AP and purity testing will require different testing schemes, ex:

• Seed bulk testing is more common in AP GMO testing

• purity tests are usually performed on a representative sample of individual seeds



• 19.4 Procedure con’t • 19.4.1 Sample size

• Chapter 2: Sampling gives definitions of various sample types, including primary, composite, submitted and working samples, as well as guidelines for obtaining seed lot samples that represent the properties of the seed lot. These definitions and guidelines apply also to GMO testing. The working sample is the portion of the submitted sample that is actually tested by the testing method (as defined in Chapter 2). The size of the working sample depends on given threshold requirements, the method capability and the degree of required statistical confidence, and can be determined using appropriate statistical tools (e.g. SeedCalc (19.6.3)). The sample submitted to the laboratory must therefore be at least the size of the working sample, but more realistically larger than the working sample. For more information regarding sampling, see Chapter 2.

• The in terms of limit of detection, in ordsizes of seed bulks and groups must be consistent with the performance of the analytical method er to allow the detection of even one GM seed. For quantitative methods, the size of the sample must be consistent with the limit of quantification, to allow the quantification of even one GM seed in the sample.

• 19.4.2 Personnel and equipment • Many of the procedures used for GMO testing are composed of several stages (e.g. seed planting or grinding, DNA or

protein extraction, detection of the target analyte, and reporting of results) which can be carried out by different personnel in the laboratory (see Figure 1). The laboratory must show that personnel are adequately trained in the procedures that they are carrying out, and that they understand the overall workflow of the procedures and their contribution to that workflow. Each part of the workflow and the equipment must be adequately validated, verified or calibrated before useAppropriate equipment and facilities must be provided for the use of the chosen methods. For biomolecular assays (DNA and protein), apparatus for grinding and analyte extraction are necessary, as well as equipment dedicated to the detection of the target analyte.

• For DNA-based detection, it is important to prevent contamination, and the use of separate rooms for certain manipulations is preferred.

• For protein-based detection, care must be taken to avoid degradation of the matrix and the extracted analyte.

• For bioassays, care must be taken to ensure the provision of controlled germination conditions adequate to allow the expression of the trait.19.4.3 Test conditions

• Tests must be carried out under conditions of the ISTA Accreditation Standard quality framework. This includes, but is not limited to the following:

• – Analysts involved in this testing must have the documented skills and training in the corresponding procedures. • – All equipment must be appropriate to the techniques used. Scheduled maintenance, verification, and calibration of the

instrumentation used must be carried out. • – The spatial arrangements and organization of the testing area must prevent contamination. • – Reagents of appropriate grade and certified reference materials (when available) must be used. • – Appropriate controls must be used to validate the testing results.

• Main points • Chapter 2 definitions and

guidelines apply also to GMO testing

• The size of the working sample depends on given threshold requirements, the method capability and the degree of required statistical confidence

• The laboratory must show that personnel are adequately trained

• Appropriate equipment and facilities must be provided for the use of the chosen methods



• 19.5 Testing approaches • 19.5.1 DNA-based methods

- 19.5.1.1 General principles of DNA-based testing - 19.5.1.2 End-point qualitative PCR - 19.5.1.3 Real-time PCR - 19.5.1.4 Other technologies

• 19.5.2 Protein-based methods - 19.5.2.1 General principles of protein-based testing - 19.5.2.2 Lateral flow strip test - 19.5.2.3 Enzyme-linked immunosorbent assay

• 19.5.3 Bioassays - 19.5.3.1 General principles of bioassays - 19.5.3.2 Scoring of GMO presence

• Main points

• DNA

• Protein

• Bioassays



• 19.5 Testing approaches • 19.5.1 DNA-based methods

• 19.5.1.1 General principles of DNA-based testing • DNA-based testing requires a series of steps which can be carried out by different laboratory

personnel and which should all show evidence of validation and being fit for purpose for the testing being carried out. The steps are the following:

• – examination of the seed sample; • – grinding of the seed to produce a homogenous matrix; • – subsampling and DNA extraction; • – DNA amplification; • – detection of the amplified DNA. • Because of the amplification step, it is important that the laboratory ensures adequate

protection against contamination by seed dust, extracted DNA or amplified DNA for each tested sample. Appropriate control samples (e.g. environmental, blank or negative controls) must be used. If available, it is recommended to use certified reference materials

• In the case of methods using the polymerase chain reaction (PCR), several types of testing can be done that will differ in the level of selectivity and specificity.

• – In GMO screening, primers are chosen that amplify individual genetic elements frequently found in a number of different GMO events. The detection of such targets suggests the presence of GMO, but does not represent by itself conclusive evidence.

• – In construct-specific PCR, the primers are chosen such that the amplification target spans genetic elements not usually combined in nature, providing a strong indication of the presence of a GMO event that includes that construct.

• – In event-specific testing, the primers are designed to detect the unique integration site of a specific transformation event. Thus, a positive result is indicative of the presence of that particular event.

• Whatever the type of method selected and its origin, internally developed or publicly available, its performance must be evaluated according to the PBA requirements and following the procedures as directed by the ISTA GMO Committee.

• Main points:

• Mention Technical considerations related to the different steps of the test

• Whatever the type of the method used its performance must be evaluated according to PBA requirements

• Explains the principle of the techniques

• Mention possible other technologies



• 19.5 Testing approaches • 19.5.1.2 End-point qualitative PCR •

• 19.5.1.2 End-point qualitative PCR • In end-point PCR, the standard steps of PCR are carried out, with detection of PCR

products at the end of the process. This detection step can be the electrophoresis of the amplified DNA molecules on gel or the measurement of fluorescence associated with the PCR reaction. With electrophoresis, the test is scored as positive if a band of the appropriate size is observed on the gel, and negative if no band is observed. With fluorescence detection, the test is scored by comparison to the fluorescence measurement of appropriate positive and negative control samples.

• 19.5.1.3 Real-time PCR • During real-time PCR, DNA amplification activates fluorochromes attached to the primers

or probes. This activation can be measured in real time and can give an estimate of the number of DNA molecules being amplified in each cycle

• DNA amplification can also be measured by activation of intercalating fluorescent dyes. In this case, special attention to false-positive results must be paid, since the activation of intercalating dyes can be associated with amplification of non-specific PCR products

• Real-time PCR can be qualitative or quantitative. • In qualitative real-time PCR tests, the test is scored positive if fluorescence above the

defined baseline is detected before a given PCR cycle (usually set by amplification of a known GMO control DNA).

• In quantitative real-time PCR tests, the assay is designed to quantify the target against a standard curve produced from reference material. The experimental set-up and reporting of results must follow accepted statistically sound methods such as those suggested in the GMO Handbook.

• 19.5.1.4 Other technologies • The descriptions in section 19.4.1.3 apply to technologies (primer and probe sets, methods and

equipment used for amplification and detection as well as for quantification) that are widely used in laboratories carrying out GMO testing.

• Other methods are currently being developed for use in GMO detection. Use of these methods can also be included in ISTA’s PBA as long as the laboratory develops and maintains adequate validation data for the methods used.

• Main points:







• 19.5 Testing approaches • 19.5.2 Protein-based methods

• In order to detect single proteins in seeds, the seeds need to be ground and extracted with a suitable buffer. The detection of proteins using an immunoassay in a complex mixture such as that obtained by extraction of seed powder requires a number of precautions. The detectable protein content may vary due to the protein itself, the extraction process and buffer and the type of seed used. Particular difficulties are well known (e.g. oil content of oilseed rape, gossypol in cotton seeds, varietal differences, seed maturity, seed moisture) and the laboratory should have validated the extraction and detection methods for each seed matrix by spike and recovery tests (see GMO Method Handbook). Proteins are generally rapidly degraded. The extraction should be carried out at room temperature, or below, and after extraction the mixture should be used quickly or stored at low temperature. When using commercial lateral flow strip tests (19.5.2.2) or ELISA kits (19.5.2.3), it is important to refer to the assay conditions as defined by the test kit suppliers. Moreover, these assay conditions must be internally validated in the laboratory conditions, systematically include positive and negative controls in each test and follow the ISTA Principles and Conditions for Laboratory Accreditation under the Performance Based Approach (see http://www.seedtest.org/upload/cms/user/ISTAMethodValidationforSeedTesting-V1.01.pdf).

• It is not recommended to use protein-based tests for quantification of GMO, as the variations in sample type (e.g. germplasm, seed maturity) and in extraction and detection methods can result in target protein content variation in the protein extract and cause difficulty in estimating the GMO content. Protein detection is not always event-specific, as several events may contain the same protein (e.g. NK603/MON88017; MON810/Bt11), but the careful use of multiple methods may allow a good indication of which event is being detected

• 19.5.2.2 Lateral flow strip test • The lateral flow strip test consists of an immunoassay in which globulins or antibodies are immobilized on a

capillary paper. The strip is dipped into the protein extract. The presence of the target protein (the antigen) is represented by the appearance of at least two bands, a negative result only by a control band. The result can be scored only if the control band is visible. A maximum time of reading must be defined to avoid false-positive scoring, due to unspecific staining which can occur after a long reaction time.

• 19.5.2.3 Enzyme-linked immunosorbent assay • The enzyme-linked immunosorbent assay (ELISA) is a sensitive immunoassay that uses an enzyme linked

to an antibody or antigen as a marker for the detection of the specific trait protein through a colorimetric reaction.

• Main points:







http://www.seedtest.org/upload/cms/user/ISTAMethodValidationforSeedTesting-V1.01.pdf

• 19.5.3.1 General principles of bioassays • Bioassays are tests based on visual assessment of phenotypic effects of treatments on seeds

or seedlings. The most common use of bioassays is to determine the presence of seed which carries herbicide-resistance traits. In this case the seeds or seedlings are exposed to herbicide, and the expected effect on the plant is lack of normal development when the seeds do not contain the herbicide-resistance trait. All seeds or plants that continue to germinate or grow normally are scored as positive for the GMO trait. The appropriate concentration of herbicide must be determined per crop and growth stage. It is important to consider that bioassays determine the presence of a GMO trait, but cannot determine the presence of any specific event, as in many crops multiple events exist with the same herbicide-resistant phenotype. Therefore, in such cases herbicide bioassays can only be used to screen for the presence of GMO, but cannot detect the presence of a particular event.

• 19.5.3.2 Scoring of GMO presence • Standardized methods of scoring and analysing the results for the herbicide testing should be

in place. This should include statistical considerations of the numbers of seeds used and scored. The result must take into consideration the germination percentage

• 19.5 Testing approaches • 19.5.3 Bioassays

• Main points:







• 19.6 Calculation and expression of results • 19.6.1 Consideration of the testing objective

• The applicant must clearly state the specific testing objective, as

this is critical in defining the testing approach and in calculating and expressing results. Possible testing objectives include:

• – reporting the presence or absence of a GMO in the seed lot;

• – estimating the proportion of the GMO present in the seed lot with the associated measurement uncertainty.

• The methods described in 19.5 produce either qualitative, i.e., detected (GM trait observed) or not detected (GM trait not observed), or quantitative results. Both types of results can be statistically analysed to meet the testing objective, but the data analysis methods and associated calculation tools differ.

• To assess for the presence of two or more stacked events in the same seed, testing individual seed is the appropriate approach. When seed are tested in bulk, the presence of stacked events cannot be demonstrated. However, some statistical tools such as the one proposed by ISTA in SeedCalc Stack9 can estimate the percentage of seeds that could have two or three stacked events.

• Main points

• The testing objective must be stated

• Statistical analysis of qualitative results or quantitative results can be made

• But will require different statistical methods



• 19.6 Calculation and expression of results • 19.6.2 Units of measurement

• The calculation and expression of results depend on the testing objectives, testing methods and the associated units of measurement. The aim or request of the applicant will need to be carefully considered. In order to cope with the different objectives and circumstances where quantification of seeds with GMO traits is required, and in concordance with the PBA, it is acceptable to report quantitative test results using any one of the following units:

• a) % in number of seeds: the estimate of the percentage of GM seeds in the seed lot. In addition to individual testing, the percentage in number of seeds is the unit to be used when a group testing approach is chosen; e.g. with SeedCalc (see 19.6.3).

• b) % in mass of seeds: the estimate of the percentage of GMO content by mass. This unit should be used when a standard curve is prepared using certified reference material certified by % mass (g/kg).

• c) % DNA copies: the estimate of the percentage of GMO content by number of copies. This unit should be used when a standard curve is prepared using certified reference material certified by % DNA copies.

• All these three units are acceptable for preparing ISTA Certificates for reporting results by accredited laboratories. The acceptance of more than one unit can avoid raising the difficult question of converting factors. A simple mechanical conversion between units is complex or even impossible

• Whatever the unit used to express results, the resulting GM estimate should be methodologically meaningful, that is, a laboratory using quantitative real-time PCR should not report a value that is lower than its validated limit of quantification.

• Moreover, in quantitative real-time PCR, results should be biologically meaningful. The lab should pay attention to results that are lower than 1 divided by the size of the working sample.

• 19.6.3 ISTA tools for calculation of results • Remund et al. (2001) and Laffont et al. (2005) provided statistical tools for qualitative and

quantitative testing methods which are implemented in the SeedCalc MS Excel workbook (available on the ISTA web site).

• Main points . • According to ISTA’s position

paper on units (approved 2009): • It is acceptable to report

quantitative test results using: % number of seeds; % mass of seeds; %DNA copies

• The resulting GM estimate should be methodologically meaningful,

• ex: using quantitative real-time PCR should not report a value that is lower than its validated limit of quantification

• Appropriate statistical tool must be used ie SeedCalc



• 19.7 Reporting results

• The result of a genetically modified organism test must be

reported under ‘Other determinations’ as follows:

• – the request of the applicant;

• – the name and scope (with reference to the target) of the method(s) used;

• – a description of the working sample (e.g. pure seed fraction, inert matter present, other seeds present, washed seed);

• – the number of seeds in the working sample;

• – a description and the source of the reference material used (e.g. certified reference material, provider);

• – the limit of detection of the method (when testing seed groups or seed bulk);

• – the limit of quantification of the method (when testing seed bulk with a quantitative method)

• Main points

• Like other chapter there is a guidance to report the results

• Results can be: • Qualitative • Quantitative from

qualitative tests on multiple pools

• Quantitative test from a bulk



• 19.7 Reporting results • 19.7.1 Qualitative test results • Suggested phrases for reporting the detection of test targets depending upon

the result are as follows: • a) If the test target(s) was(were) not detected: ‘ The test target was not

detected.’ • b) If the test target(s) was (were) detected: ‘The test target was detected.’

• 19.7.2 Quantitative results obtained by multiple qualitative tests of

individuals or groups of seeds or seedlings • Results should be reported relative to the percentage of seeds or seedlings

showing the test target specified by the applicant. The total number of seeds tested, the number of groups, and the number of seeds per group must be reported. Suggested phrases for reporting such results depending upon the result are as follows:

• a) If the test target(s) was (were) not detected: ‘The test target(s) was (were) not detected.’

• b) If the test target(s) was (were) detected: ‘The % of seeds in the lot with the test target(s) was determined to be …%, with a 95% confidence interval of […%, …%].’ or ‘For the test target(s) specified by the applicant, the seed lot meets the specification of ...% (maximum or minimum) with …% confidence.’

• If the results do not show evidence that the seed lot meets a given specification with some confidence, then the applicant will report the point estimate with the 95% confidence intervaI.

• Main points







Working Group: Rule chapter on GMO testing: Content • 19.7 Reporting results • 19.7.3 Quantitative measurements of GMO in

bulk samples • Results should be reported relative to the percentage of the test target

specified by the applicant by mass or number of DNA copies. The testing plan (e.g. number of replicate seed samples, number of replicate flour samples per seed sample, number of extracts per flour sample, number of replicate measurements per extract) must be indicated.

• Required phrases for reporting depending upon the results are as follows:

• a) If the test target was not detected (no signal or below the limit of detection): ‘The test target was not detected at a level above the limit of detection.

• b) If the test target was detected at a level above the limit of detection and below the limit of quantification: ‘The test target was detected at a level below the limit of quantification of the method used.’

• c) If seeds showing the test target were found at a level above the limit of quantification: ‘The test target(s) percentage in the seed lot was determined to be …% by mass or number of copies, with a 95% confidence interval of […%,…%]‘ or ‘For the test target(s) specified by the applicant, the seed lot meets the specification of ...% (maximum or minimum) by mass or number of copies with …% confidence.

• If the results do not show evidence that the seed lot meets a given specification with some confidence, then the applicant will report the point estimate with the 95% confidence intervaI.

• Main points






• Conclusions about the content -The chapter on testing for seeds of GMO gathers ISTA‘s

existing practices to get all the information in one document

-Enhances the existing process (formerly chapter 8) by clarifying ambiguous parts and filling gaps

-Associated Technical details and guidance will be presented in a Handbook




Chapter 8

GMO Handbook (In progress)


(Strategy regarding Methods for GM seeds) Chapter 19:

Testing for seeds of GMO

Chapter 2

ISTA’s PD Evaluation (GM presence)

ISTA’s PD Evaluation (GM purity)


(View regarding the units for reporting)

ISTA’s paper Principles and Conditions

for Lab Accreditation under PBA


• Where we are now :

• GMO rule chapter will be submitted to the vote of delegates - (at the present ISTA congress 2013)

• Working on the related Handbook (on GMO testing on seeds) - Same WG members

• More people will be involved (from ISTA accreditation dept; ECOM) - Phones conferences + a full day meeting in Antalya


RUL COM GMO-TCOM VOTING delegates ECOM



Input 1

Input 2

Input 3



• THANKS !

2013-06-15 – René Mathis GMO Rules WG-2012

Thank You • ISTA Secretariat

- Special thanks to Agnes Hegedüs, Nadine Ettel, Martina Haefeli, Jonathan Taylor, Ronnie Don, Rasha El-Khadem, Patricia Muschick and many others

• ISTA Executive Committee - Special thanks to our ECOM representative: Rita Zecchinelli and Rules Chair:

Steve Jones

• Industry Supporters - Dow, DuPont Pioneer, Bayer CropScience, Monsanto, Syngenta, Limagrain - Special thanks to Elizabeth Bates, Benoit Mayes of Bayer CropScience and the

Canadian Food Inspection Agency for last minute provision and purity checks of seed used in PT15 and16 respectively.

• German Government for Financial support of the PT program - Special thanks to LTZ-Agustenberg, Andrea Jontz and Uwe Bertrand for PT

sample preparation

• ISTA members and Congress participants

2013-06-15 –GMO TCOM

Open Meeting: GMO TCOM

•Saturday, June 15, 2013

•20:30 – 22:00 •Room: Lal A3

2013-06-15 –GMO TCOM

gmo committee - ista · pdf file• feedback to gmo committee for on-going activity ....

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