ISO 22753:2021

Molecular biomarker analysis — Method for the statistical evaluation of analytical results obtained in testing sub-sampled groups of genetically modified seeds and grains — General requirements

This document describes general requirements, procedures and performance criteria for evaluating the content of genetically modified (GM) seeds/grains in a lot by a group testing strategy that includes qualitative analysis of sub-sampled groups followed by statistical evaluation of the results. This document is applicable to group testing strategy estimating the GM content on a percentage seed/grain basis for purity estimation, testing towards a given reject/accept criterion and for cases where seed/grain lots are carrying stacked events. This document is not applicable to processed products. NOTE Description of the use of group testing strategy are available in References [1], [7], [8], [18], [19] and [20].

Analyse moléculaire de biomarqueurs — Méthode pour l'évaluation statistique des résultats d'analyse obtenus lors des essais de sous-échantillons multiples de semences et de graines génétiquement modifiées — Exigences générales

Le présent document décrit les exigences générales, les modes opératoires et les critères de performance applicables à l’évaluation de la teneur en semences/graines génétiquement modifiées (GM) dans un lot par une stratégie d’analyse de groupe qui comprend l’analyse qualitative de sous-échantillons multiples puis l’évaluation statistique des résultats. Le présent document est applicable à la stratégie d’analyse de groupe permettant d’estimer la teneur en OGM sur un pourcentage de semences/graines afin d’en estimer la pureté, d'évaluer si un critère de rejet/d'acceptation défini est respecté et de déterminer les cas où des lots de semences/graines contiennent un empilement d’événements. Le présent document n’est pas applicable aux produits transformés. NOTE Une description de l’utilisation de la stratégie d’analyse de groupe est donnée dans les Références [1], [7], [8], [18], [19] et [20].

General Information

• Status: Published
• Publication Date: 26-Aug-2021
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Drafting Committee: ISO/TC 34/SC 16/WG 9 - Subsampling of seeds and grains
• Current Stage: 6060 - International Standard published
• Start Date: 27-Aug-2021
• Due Date: 12-Jul-2021
• Completion Date: 27-Aug-2021

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 22753
First edition
2021-08
Corrected version
2022-11
Molecular biomarker analysis —
Method for the statistical evaluation of
analytical results obtained in testing
sub-sampled groups of genetically
modified seeds and grains — General
requirements
Analyse moléculaire de biomarqueurs — Méthode pour l'évaluation
statistique des résultats d'analyse obtenus lors des essais de sous-
échantillons multiples de semences et de graines génétiquement
modifiées — Exigences générales
Reference number
ISO 22753:2021(E)
© ISO 2021
---------------------- Page: 1 ----------------------
ISO 22753:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on

the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below

or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
© ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 22753:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction .................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ..................................................................................................................................................................................... 1

3 Terms and definitions .................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 4

4.1 General ........................................................................................................................................................................................................... 4

4.2 Preparation of seed/grain groups ......................................................................................................................................... 4

4.3 Detection methods for the qualitative analysis of GM seed/grain in seed/grain

groups ............................................................................................................................................................................................................. 5

4.4 Statistical evaluation ........................................................................................................................................................................ 5

5 Reagents ........................................................................................................................................................................................................................ 6

6 Apparatus and equipment ......................................................................................................................................................................... 6

7 Design of testing plan ..................................................................................................................................................................................... 6

7.1 General ........................................................................................................................................................................................................... 6

7.2 Single-stage testing plan ............................................................................................................................................................... 6

7.3 Double-stage testing plan ............................................................................................................................................................. 7

8 Selection of qualitative methods ........................................................................................................................................................ 8

8.1 General ........................................................................................................................................................................................................... 8

8.2 Performance criteria ......................................................................................................................................................................... 8

9 Interpretation .........................................................................................................................................................................................................8

10 Expression of results ....................................................................................................................................................................................10

10.1 Classification of a seed/grain lot into “accept” or “reject” category .................................................... 10

10.2 Estimation of the level of molecular biomarker in the seed/grain lot ............................................... 10

11 Test report ...............................................................................................................................................................................................................10

Annex A (informative) Terms and definitions comparison table ......................................................................................12

Annex B (informative) Implementation of the method to evaluate GMO content in seeds/

grains example ....................................................................................................................................................................................................14

Annex C (informative) Estimation of the limit of detection for a testing plan to detect GM

seeds/grains in seed lots ..........................................................................................................................................................................21

Annex D (informative) Experimental determination of maximum group size ...................................................24

Bibliography .............................................................................................................................................................................................................................25

iii
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ISO 22753:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www.iso.org/patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to

the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see

www.iso.org/iso/foreword.html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,

Horizontal methods for molecular biomarker analysis.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www.iso.org/members.html.
This corrected version of ISO 22753:2021 incorporates the following corrections:
— Formula C.1 has been corrected.
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ISO 22753:2021(E)
Introduction

Seed and grain testing is used throughout the world to commercially define the purity of seed and grain

lots.

Commercial requirements for labelling agricultural products with genetically modified organism (GMO)

content at a specified threshold level both as a seed/grain contaminant and a food ingredient have

become common to satisfy regulations and consumer demands. Conformance with these specifications

is evaluated at various points of the supply chain, often starting with the harvested grain.

Quantitative real-time polymerase chain reaction (PCR) can be used to determine the GMO content by

analysis of the ratio of GMO DNA copy numbers to plant-species specific DNA copy numbers followed by

a conversion to genetically modified (GM) mass fraction.

Multiple events stacked in a crop, such as those generated by crossing two or more single events,

are widely used in agricultural production. A stacked event seed or grain containing GMO DNA

corresponding to two or more GM events commingled in lot cannot be differentiated by quantitative

PCR alone from multiple seeds within the lot each containing a single GM event. Consequently, if the

actual measured GMO arises only from GM stacked event seeds, GM content measured by quantitative

real-time PCR of a single sample will lead to an overestimation of the actual number of GM seeds or

grains present.

The group testing strategy described in this document provides a reliable alternative to estimate the

GM content on the basis of the fact that whole seeds/grains are the sample material.

The process described in this document can provide a method to accurately estimate the percentages

of GM seeds/grains in a lot irrespective of the presence of stacked event seeds/grains. GM content is

determined for representative subsampled groups of seed/grain from a lot and statistically analysed.

© ISO 2021 – All rights reserved
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INTERNATIONAL STANDARD ISO 22753:2021(E)
Molecular biomarker analysis — Method for the statistical
evaluation of analytical results obtained in testing sub-
sampled groups of genetically modified seeds and grains —
General requirements
1 Scope

This document describes general requirements, procedures and performance criteria for evaluating

the content of genetically modified (GM) seeds/grains in a lot by a group testing strategy that includes

qualitative analysis of sub-sampled groups followed by statistical evaluation of the results.

This document is applicable to group testing strategy estimating the GM content on a percentage seed/

grain basis for purity estimation, testing towards a given reject/accept criterion and for cases where

seed/grain lots are carrying stacked events.
This document is not applicable to processed products.

NOTE Description of the use of group testing strategy are available in References [1], [7], [8], [18], [19] and

[20].
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 16577, Molecular biomarker analysis — Terms and definitions

ISO 21572, Foodstuffs — Molecular biomarker analysis — Immunochemical methods for the detection and

quantification of proteins

ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — General requirements and definitions
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16577 and the following apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
absolute PCR limit of detection
absolute polymerase chain reaction limit of detection
absolute PCR LOD

lowest nominal (average) number of target copies in the template volume distributed to individual PCRs

that would allow for an acceptable probability of detecting the target
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ISO 22753:2021(E)
3.2
AQL
acceptable quality limit

level of impurity that is acceptable to the producer and that production practices can support

3.3
consumer risk
consumer (beta) risk
probability of accepting a lot at the lower quality limit (3.10)
3.4
deviant seed/grain

considered non-conforming based on the presence or absence of a specific trait or characteristic

Note 1 to entry: For the purpose of this document, a deviant seed is considered to possess a GM characteristic

that is not expected or is unintended based on the expected or known GM characteristics of the seed/grain.

3.5
false negative rate
FNR

probability that a known positive (seed/grain group) test sample (3.20) has been classified as negative

by the method

Note 1 to entry: The false negative rate is the number of misclassified known positives divided by the total

number of positive test samples (3.20).

[SOURCE: ISO 16577:2016, 3.63, modified — the abbreviation has been added, “positive test sample”

has been changed to “positive (seed/grain group) test sample”, and the formula has been deleted.]

3.6
false positive rate
FPR

probability that a known negative (seed/grain group) test sample (3.20) has been classified as positive

by the method

Note 1 to entry: The false positive rate is the number of misclassified known negatives divided by the total

number of negative test samples (3.20).

[SOURCE: ISO 16577:2016, 3.65, modified — the abbreviation has been added, “negative test sample”

has been changed to “negative (seed/grain group) test sample”, and the formula has been deleted.]

3.7
group size
number of seeds/grains comprising a group
3.8
group testing

statistical evaluation of analyte contents based on qualitative analysis results (i.e. positive or negative)

from each seed/grain group in the test sample (3.20)
3.9
laboratory sample
sample or subsample(s) received by the laboratory

Note 1 to entry: The seed/grain sample received is expected to represent the seed/grain lot (3.18).

[SOURCE: ISO 16577:2016, 3.89, modified — Note 1 to entry has been added.]
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ISO 22753:2021(E)
3.10
LQL
lower quality limit
highest impurity that is acceptable to the consumer
Note 1 to entry: This can be equivalent to the threshold (3.22).
3.11
mass fraction

ratio of GM seeds/grains relative to the total seeds/grains corresponding to mass ratio

3.12
number of deviant seed/grain groups

number of seed/grain groups (3.17) including one or more deviant seeds/grains (3.4)

3.13
operating characteristic curve
OC curve

graph plotting the percentage of deviant seeds/grains and the probability of acceptance respectively

on the horizontal and the vertical axes and used in quality control to determine the probability of

accepting seed/grain lots (3.18) in a testing plan (3.21)
3.14
producer risk
producer (alpha) risk
probability of rejecting a lot at the AQL (3.2)
3.15
representative sample

sampling units (samples or groups) that have been extracted from a lot with the process ensuring all

sampling units of the lots have an equal probability of being selected and not altered in any way that

would change the analytical result
Note 1 to entry: The extraction process can be a multi-stage process.
3.16
reject/accept criterion

maximum number of deviant seed/grain groups (3.12) that can be detected in the test sample (3.20) of an

acceptable seed/grain lot (3.18)
3.17
seed/grain group
group

determined number of seeds/grains prepared from a seed/grain test sample (3.20) by representative

sampling
3.18
seed/grain lot
lot
population for which sampling is intended to estimate the measured parameter
3.19
stacked event

accumulation of two or more transformation events as a result of traditional breeding and/or successive

transformation steps)

Note 1 to entry: In the context of this document a stacked event refers to a stack in which the two or more events

are not genetically linked.
[SOURCE: ISO 16577:2016, 3.197, modified — Note 1 to entry has been added.]
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ISO 22753:2021(E)
3.20
test sample

sample prepared for testing or analysis, the whole quantity or part of it being used for testing or

analysis at one time
Note 1 to entry: The test sample is prepared from the laboratory sample (3.9).

Note 2 to entry: The test sample is expected to represent the laboratory sample (3.9).

[SOURCE: ISO 16577:2016, 3.210, modified — Note 1 to entry and Note 2 to entry have been added.]

3.21
testing plan

plan specifying group testing (3.8) conditions including group size (3.7), the number of seed/grain groups

(3.17) and the number of deviant seed/grain groups (3.12) in test sample (3.20) resulting in rejection of

seed/grain lot (3.18)
3.22
threshold
maximum acceptable content of GMO presence in a lot
Note 1 to entry: This can be a prescribed value.

Note 2 to entry: Thresholds can be expressed in mass fraction (3.11) with the proviso that an uncertainty factor

is involved in the conversion to a seed/grain percentage threshold.
4 Principle
4.1 General

In this method, the test sample is divided into a predetermined number of groups. Each group consists

of a determined number of seed/grain and is tested qualitatively for the presence or absence of a GM

target. A statistical evaluation is performed on the number of GM positive groups relative to the total

number of seed/grain groups to determine the GM content in mass fraction.

A statistical calculation determines the optimal testing conditions, namely, the number of seeds/grains

per group (group size), the number of seed/grain groups, and the maximum number of GMO positive

seed/grain groups for seed/grain lot acceptance. Alternatively, a statistical calculation provides an

estimate of the percentage by number of the GM seeds/grains in a lot, according to a given testing plan.

4.2 Preparation of seed/grain groups
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ISO 22753:2021(E)
Key
1 bulk seed/grain lot
2 laboratory sample
3 test sample
4 seed/grain groups
5 deviant seed/grain
NOTE Each group is represented as an array on the right.

Figure 1 — Sampling illustration of the obtention of seed/grain groups from a bulk seed/grain

lot

The process of forming seed/grain groups from a series of sampling steps starting with the bulk seed/

grain lot is shown in Figure 1, (1).

Although the procedures for obtaining a laboratory sample from a seed/grain lot is not the subject

of this document, a laboratory sample (2) from a seed/grain lot shall be obtained appropriately. The

procedures can be designed according to the References [3], [6], [10], [11], [12], [15], [19] and [23].

The laboratory sample shall be thoroughly mixed and divided/reduced to create the test sample (3).

Likewise, the test sample shall be thoroughly mixed (i.e. homogeneous) and divided into seed/grain

groups (each group represented as an array in Figure 1, (4)) following simple random sampling

principles. The seed/grain groups can vary in size from one single seed/grain up to the complete test

sample (i.e. a single bulk). In most cases, multiple seed/grain groups are created from the test sample.

A determined number of seeds/grains can either be obtained by weighing or a volumetric measurement,

where an approximation of number is made based on a determined conversion factor (e.g. thousand

seeds/grains weight). For the case that weight is used to obtain the seed/grain groups, the operator

shall have an estimate of the variability introduced by using weight rather than seed/grain count.

The group testing procedure described in Clause 7 is carried out on the collective qualitative (positive

or negative) results for each seed/grain group.

4.3 Detection methods for the qualitative analysis of GM seed/grain in seed/grain

groups
[21]

In general, GMO detection methods are categorized into two classes . The first class of assays targets

a nucleic acid sequence for detecting GMO presence. The second class includes methods for detecting a

specified protein that confers a specific transgenic trait. Detection methods from either or both classes

should be selected considering fitness-for-purpose. Guidance on the selection of qualitative methods is

[4]
provided in Clause 8. Further details can be found in ISO 21569 and ISO 21572.
4.4 Statistical evaluation

Sampling and measurement uncertainty shall be considered. Sampling uncertainty can be adequately

[18][2]

considered using the binomial distribution . The FPR and the FNR of the qualitative assay should

[2]
be considered . The LOD of the applied detection method should be considered.

The group testing described here can be used to set reject/accept criteria based on a given threshold by

GMO content, as well as to estimate the GMO content and associated upper and lower confidence limits.

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ISO 22753:2021(E)
5 Reagents
All reagents used in the analysis should be those specified in the method.
Otherwise, all reagents should be of molecular biology grade.

These reagents shall be stored and used as recommended by the supplier or according to the laboratory

quality assurance specifications. It can also be appropriate to aliquot the reaction solutions required

for the analytical method in order to avoid subjecting them to repeated freeze–thaw cycles, or to reduce

the chances of cross contamination or both. Further details shall refer to ISO 24276 and ISO 21572.

6 Apparatus and equipment

The laboratory should use properly maintained equipment suitable for the methods employed.

Further details shall refer to ISO 24276 and ISO 21572.
7 Design of testing plan
7.1 General

The number of seeds/grains tested, the reject/accept criteria, the sample preparation steps and the

method used for testing shall be determined depending on the analytical purpose.

In seed/grain sample classification, it can be determined whether the number of deviant seeds/grains

or seed/grain groups is above a given reject/accept criterion or not. Then, it can be decided to reject or

accept the seed/grain lot based on the test results.
A basic testing plan for group testing consists of three fundamental parameters:
a) the number of seed/grain groups;
b) the size of the seed/grain groups;

c) the maximum number of deviant seed/grain groups for seed/grain lot acceptance (reject/accept

criterion).

The risks associated with the AQL and the LQL are the producer (alpha) and consumer (beta) risks

respectively, and together with the FPR and FNR allow the design of an appropriate testing plan.

The OC curve can be used to develop a testing plan. Explanations for the estimation of the LOD for a

zero deviant testing plan, the effect of the genome size on the group size if methods targeting DNA are

applied, and the effect of the individual seed size on the sample preparation are given in Annex C.

Annex D provides guidance on the determination of the maximum group size whatever analytical

method is used in the laboratory.
[16]

NOTE Seedcalc is a statistical program (Microsoft Excel spreadsheet application) that is freely available

from the International Seed Testing Association and has procedures to facilitate the design. Seedcalc is located

on the ISTA website.
7.2 Single-stage testing plan

A single-stage testing plan consists of one testing stage. Groups are taken from the test sample and

evaluated once, and a decision is then made based on the results to accept or reject the seed/grain

test sample. In a single-stage testing plan, a specified number of individual seeds/grains or seed/

grain groups shall be selected randomly from the test sample and tested. Depending on the number

of deviants detected and the maximum number of deviants specified in the plan, the seed/grain lot is

either accepted or rejected.
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ISO 22753:2021(E)

The probability that an individual seed/grain or seed/grain group is deviant, p , can be calculated as

given in Formula (1):
pP=−11=−()1−p (1)
where
P is the probability that there are no deviant seeds/grains in the group;
p is the true unknown impurity in the seed/grain lot;

m is the number of individual seeds/grains in a seed/grain group (if seeds/grains are tested indi-

vidually, m = 1).

Then, the probability that a lot will be accepted, P(a) is calculated as given in Formula (2):

 
ni−
P()a = pp()1− (2)
 
∑ b b
 
i=0
where
P(a) is the probability that a lot will be accepted;
n is the number of individual seeds/grains or seed/grain groups tested;
c is the maximum number of deviant seed/grain groups for acceptance.
By combining Formulae (1) and (2), P(a) is a function of p, n, m and c.

After n, m and c are determined, an OC curve can be drawn by plotting p and P(a) on the x-axis and

y-axis, respectively.
7.3 Double-stage testing plan

A double-stage testing plan is generally set up so that additional seed/grain groups are tested in the

second stage. Initial seed/grain groups are taken from the test sample and tested. Based on this test

result, three different decisions can be made:
a) accept the seed/grain lot;
b) reject the seed/grain lot; or
c) draw a second set of seed/grain groups from the test sample and retest.

The test results from the first and second stages of testing are combined and used to determine whether

the seed/grain lot is accepted or rejected (see Figure B.1). In Annex B examples for implementation of a

double-stage testing plan to evaluate GMO content in seeds/grains are provided. Subclause B.1 can also

be applied for cases where seed/grain lots are carrying stacked events.
Some additional terms are defined as follows:

— n , the number of independent seed/grain groups to be tested in the first stage;

— n , the number of independent seed/grain groups to be tested in the second stage;

— c , the maximum number of allowable deviant seed/grain groups for acceptance in the first stage;

— c , the minimum number of deviant seed/grain groups that will result in rejection at the first stage;

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ISO 22753:2021(E)

— c , the maximum number of deviant seed/grain groups in the first and second stages combined

allowed for acceptance;
— d , the number of deviant seed/grain groups in the first stage;
— d , the number of deviant seed/grain groups in the second stage.
P(a) is calculated as given in Formula (3):
n n n
c   c −1    ci-   
11ni−−ni 2 nj−
1 i 2 i 3 j
11 2
P()a = pp()11− + pp()− × pp()1−
 
     
∑ b b ∑  b b ∑ b b 
i=0 ic=+1 j=0
i i j
       
(3)
8 Selection of qualitative methods
8.1 General

An analytical method shall be chosen to meet the purpose of testing. The performance characteristics

of the method should be determined before application in seed/grain testing.

Analytical methods have been developed to detect specific genes encoding transgenic traits or specific

characteristics expressed by specific genes in seeds/grains. Nucleic-acid-based methods such as PCR

[4][5]

are available that detect specific DNA sequences encoding elements, constructs or GMO events .

Protein-based methods such as ELISA and lateral flow immunoassays require a specific antibody for

detecting a specific GM protein (
...

INTERNATIONAL ISO
STANDARD 22753
First edition
2021-08
Molecular biomarker analysis —
Method for the statistical evaluation of
analytical results obtained in testing
sub-sampled groups of genetically
modified seeds and grains — General
requirements
Analyse moléculaire de biomarqueurs — Méthode pour l'évaluation
statistique des résultats d'analyse obtenus lors des essais de sous-
échantillons multiples de semences et de graines génétiquement
modifiées — Exigences générales
Reference number
ISO 22753:2021(E)
ISO 2021
---------------------- Page: 1 ----------------------
ISO 22753:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO 22753:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 4

4.1 General ........................................................................................................................................................................................................... 4

4.2 Preparation of seed/grain groups .......................................................................................................................................... 5

4.3 Detection methods for the qualitative analysis of GM seed/grain in seed/grain groups ....... 5

4.4 Statistical evaluation .......................................................................................................................................................................... 5

5 Reagents ........................................................................................................................................................................................................................ 6

6 Apparatus and equipment .......................................................................................................................................................................... 6

7 Design of testing plan ...................................................................................................................................................................................... 6

7.1 General ........................................................................................................................................................................................................... 6

7.2 Single-stage testing plan ................................................................................................................................................................. 6

7.3 Double-stage testing plan .............................................................................................................................................................. 7

8 Selection of qualitative methods ......................................................................................................................................................... 8

8.1 General ........................................................................................................................................................................................................... 8

8.2 Performance criteria .......................................................................................................................................................................... 8

9 Interpretation .......................................................................................................................................................................................................... 8

10 Expression of results .....................................................................................................................................................................................10

10.1 Classification of a seed/grain lot into “accept” or “reject” category ......................................................10

10.2 Estimation of the level of molecular biomarker in the seed/grain lot .................................................10

11 Test report ................................................................................................................................................................................................................10

Annex A (informative) Terms and definitions comparison table ........................................................................................12

Annex B (informative) Implementation of the method to evaluate GMO content in seeds/

grains example.....................................................................................................................................................................................................14

Annex C (informative) Estimation of the limit of detection for a testing plan to detect GM

seeds/grains in seed lots ..........................................................................................................................................................................21

Annex D (informative) Experimental determination of maximum group size ......................................................24

Bibliography .............................................................................................................................................................................................................................25

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ISO 22753:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,

Horizontal methods for molecular biomarker analysis.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2021 – All rights reserved
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ISO 22753:2021(E)
Introduction

Seed and grain testing is used throughout the world to commercially define the purity of seed and grain

lots.

Commercial requirements for labelling agricultural products with genetically modified organism (GMO)

content at a specified threshold level both as a seed/grain contaminant and a food ingredient have

become common to satisfy regulations and consumer demands. Conformance with these specifications

is evaluated at various points of the supply chain, often starting with the harvested grain.

Quantitative real-time polymerase chain reaction (PCR) can be used to determine the GMO content by

analysis of the ratio of GMO DNA copy numbers to plant-species specific DNA copy numbers followed by

a conversion to genetically modified (GM) mass fraction.

Multiple events stacked in a crop, such as those generated by crossing two or more single events,

are widely used in agricultural production. A stacked event seed or grain containing GMO DNA

corresponding to two or more GM events commingled in lot cannot be differentiated by quantitative

PCR alone from multiple seeds within the lot each containing a single GM event. Consequently, if the

actual measured GMO arises only from GM stacked event seeds, GM content measured by quantitative

real-time PCR of a single sample will lead to an overestimation of the actual number of GM seeds or

grains present.

The group testing strategy described in this document provides a reliable alternative to estimate the

GM content on the basis of the fact that whole seeds/grains are the sample material.

The process described in this document can provide a method to accurately estimate the percentages

of GM seeds/grains in a lot irrespective of the presence of stacked event seeds/grains. GM content is

determined for representative subsampled groups of seed/grain from a lot and statistically analysed.

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INTERNATIONAL STANDARD ISO 22753:2021(E)
Molecular biomarker analysis — Method for the statistical
evaluation of analytical results obtained in testing sub-
sampled groups of genetically modified seeds and grains —
General requirements
1 Scope

This document describes general requirements, procedures and performance criteria for evaluating

the content of genetically modified (GM) seeds/grains in a lot by a group testing strategy that includes

qualitative analysis of sub-sampled groups followed by statistical evaluation of the results.

This document is applicable to group testing strategy estimating the GM content on a percentage seed/

grain basis for purity estimation, testing towards a given reject/accept criterion and for cases where

seed/grain lots are carrying stacked events.
This document is not applicable to processed products.

NOTE Description of the use of group testing strategy are available in References [1], [7], [8], [18], [19] and

[20].
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 16577, Molecular biomarker analysis — Terms and definitions

ISO 21572, Foodstuffs — Molecular biomarker analysis — Immunochemical methods for the detection and

quantification of proteins

ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — General requirements and definitions
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16577 and the following apply.

ISO and IEC maintain terminology databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
absolute PCR limit of detection
absolute polymerase chain reaction limit of detection
absolute PCR LOD

lowest nominal (average) number of target copies in the template volume distributed to individual PCRs

that would allow for an acceptable probability of detecting the target
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ISO 22753:2021(E)
3.2
AQL
acceptable quality limit

level of impurity that is acceptable to the producer and that production practices can support

3.3
consumer risk
consumer (beta) risk
probability of accepting a lot at the lower quality limit (3.10)
3.4
deviant seed/grain

considered non-conforming based on the presence or absence of a specific trait or characteristic

Note 1 to entry: For the purpose of this document, a deviant seed is considered to possess a GM characteristic

that is not expected or is unintended based on the expected or known GM characteristics of the seed/grain.

3.5
false negative rate
FNR

probability that a known positive (seed/grain group) test sample (3.20) has been classified as negative

by the method

Note 1 to entry: The false negative rate is the number of misclassified known positives divided by the total

number of positive test samples (3.20).

[SOURCE: ISO 16577:2016, 3.63, modified — the abbreviation has been added, “positive test sample”

has been changed to “positive (seed/grain group) test sample”, and the formula has been deleted.]

3.6
false positive rate
FPR

probability that a known negative (seed/grain group) test sample (3.20) has been classified as positive

by the method

Note 1 to entry: The false positive rate is the number of misclassified known negatives divided by the total

number of negative test samples (3.20).

[SOURCE: ISO 16577:2016, 3.65, modified — the abbreviation has been added, “negative test sample”

has been changed to “negative (seed/grain group) test sample”, and the formula has been deleted.]

3.7
group size
number of seeds/grains comprising a group
3.8
group testing

statistical evaluation of analyte contents based on qualitative analysis results (i.e. positive or negative)

from each seed/grain group in the test sample (3.20)
3.9
laboratory sample
sample or subsample(s) received by the laboratory

Note 1 to entry: The seed/grain sample received is expected to represent the seed/grain lot (3.18).

[SOURCE: ISO 16577:2016, 3.89, modified — Note 1 to entry has been added.]
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ISO 22753:2021(E)
3.10
LQL
lower quality limit
highest impurity that is acceptable to the consumer
Note 1 to entry: This can be equivalent to the threshold (3.22).
3.11
mass fraction

ratio of GM seeds/grains relative to the total seeds/grains corresponding to mass ratio

3.12
number of deviant seed/grain groups

number of seed/grain groups (3.17) including one or more deviant seeds/grains (3.4)

3.13
operating characteristic curve
OC curve

graph plotting the percentage of deviant seeds/grains and the probability of acceptance respectively

on the horizontal and the vertical axes and used in quality control to determine the probability of

accepting seed/grain lots (3.18) in a testing plan (3.21)
3.14
producer risk
producer (alpha) risk
probability of rejecting a lot at the AQL (3.2)
3.15
representative sample

sampling units (samples or groups) that have been extracted from a lot with the process ensuring all

sampling units of the lots have an equal probability of being selected and not altered in any way that

would change the analytical result
Note 1 to entry: The extraction process can be a multi-stage process.
3.16
reject/accept criterion

maximum number of deviant seed/grain groups (3.12) that can be detected in the test sample (3.20) of an

acceptable seed/grain lot (3.18)
3.17
seed/grain group
group

determined number of seeds/grains prepared from a seed/grain test sample (3.20) by representative

sampling
3.18
seed/grain lot
lot
population for which sampling is intended to estimate the measured parameter
3.19
stacked event

accumulation of two or more transformation events as a result of traditional breeding and/or successive

transformation steps)

Note 1 to entry: In the context of this document a stacked event refers to a stack in which the two or more events

are not genetically linked.
[SOURCE: ISO 16577:2016, 3.197, modified — Note 1 to entry has been added.]
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ISO 22753:2021(E)
3.20
test sample

sample prepared for testing or analysis, the whole quantity or part of it being used for testing or

analysis at one time
Note 1 to entry: The test sample is prepared from the laboratory sample (3.9).

Note 2 to entry: The test sample is expected to represent the laboratory sample (3.9).

[SOURCE: ISO 16577:2016, 3.210, modified — Note 1 to entry and Note 2 to entry have been added.]

3.21
testing plan

plan specifying group testing (3.8) conditions including group size (3.7), the number of seed/grain groups

(3.17) and the number of deviant seed/grain groups (3.12) in test sample (3.20) resulting in rejection of

seed/grain lot (3.18)
3.22
threshold
maximum acceptable content of GMO presence in a lot
Note 1 to entry: This can be a prescribed value.

Note 2 to entry: Thresholds can be expressed in mass fraction (3.11) with the proviso that an uncertainty factor

is involved in the conversion to a seed/grain percentage threshold.
4 Principle
4.1 General

In this method, the test sample is divided into a predetermined number of groups. Each group consists

of a determined number of seed/grain and is tested qualitatively for the presence or absence of a GM

target. A statistical evaluation is performed on the number of GM positive groups relative to the total

number of seed/grain groups to determine the GM content in mass fraction.

A statistical calculation determines the optimal testing conditions, namely, the number of seeds/grains

per group (group size), the number of seed/grain groups, and the maximum number of GMO positive

seed/grain groups for seed/grain lot acceptance. Alternatively, a statistical calculation provides an

estimate of the percentage by number of the GM seeds/grains in a lot, according to a given testing plan.

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ISO 22753:2021(E)
4.2 Preparation of seed/grain groups
Key
1 bulk seed/grain lot
2 laboratory sample
3 test sample
4 seed/grain groups
5 deviant seed/grain
NOTE Each group is represented as an array on the right.

Figure 1 — Sampling illustration of the obtention of seed/grain groups from a bulk seed/grain

lot

The process of forming seed/grain groups from a series of sampling steps starting with the bulk seed/

grain lot is shown in Figure 1, (1).

Although the procedures for obtaining a laboratory sample from a seed/grain lot is not the subject

of this document, a laboratory sample (2) from a seed/grain lot shall be obtained appropriately. The

procedures can be designed according to the References [3], [6], [10], [11], [12], [15], [19] and [23].

The laboratory sample shall be thoroughly mixed and divided/reduced to create the test sample (3).

Likewise, the test sample shall be thoroughly mixed (i.e. homogeneous) and divided into seed/grain

groups (each group represented as an array in Figure 1, (4)) following simple random sampling

principles. The seed/grain groups can vary in size from one single seed/grain up to the complete test

sample (i.e. a single bulk). In most cases, multiple seed/grain groups are created from the test sample.

A determined number of seeds/grains can either be obtained by weighing or a volumetric measurement,

where an approximation of number is made based on a determined conversion factor (e.g. thousand

seeds/grains weight). For the case that weight is used to obtain the seed/grain groups, the operator

shall have an estimate of the variability introduced by using weight rather than seed/grain count.

The group testing procedure described in Clause 7 is carried out on the collective qualitative (positive

or negative) results for each seed/grain group.

4.3 Detection methods for the qualitative analysis of GM seed/grain in seed/grain

groups
[21]

In general, GMO detection methods are categorized into two classes . The first class of assays targets

a nucleic acid sequence for detecting GMO presence. The second class includes methods for detecting a

specified protein that confers a specific transgenic trait. Detection methods from either or both classes

should be selected considering fitness-for-purpose. Guidance on the selection of qualitative methods is

[4]
provided in Clause 8. Further details can be found in ISO 21569 and ISO 21572.
4.4 Statistical evaluation

Sampling and measurement uncertainty shall be considered. Sampling uncertainty can be adequately

[18][2]

considered using the binomial distribution . The FPR and the FNR of the qualitative assay should

[2]
be considered . The LOD of the applied detection method should be considered.
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ISO 22753:2021(E)

The group testing described here can be used to set reject/accept criteria based on a given threshold by

GMO content, as well as to estimate the GMO content and associated upper and lower confidence limits.

5 Reagents
All reagents used in the analysis should be those specified in the method.
Otherwise, all reagents should be of molecular biology grade.

These reagents shall be stored and used as recommended by the supplier or according to the laboratory

quality assurance specifications. It can also be appropriate to aliquot the reaction solutions required

for the analytical method in order to avoid subjecting them to repeated freeze–thaw cycles, or to reduce

the chances of cross contamination or both. Further details shall refer to ISO 24276 and ISO 21572.

6 Apparatus and equipment

The laboratory should use properly maintained equipment suitable for the methods employed.

Further details shall refer to ISO 24276 and ISO 21572.
7 Design of testing plan
7.1 General

The number of seeds/grains tested, the reject/accept criteria, the sample preparation steps and the

method used for testing shall be determined depending on the analytical purpose.

In seed/grain sample classification, it can be determined whether the number of deviant seeds/grains

or seed/grain groups is above a given reject/accept criterion or not. Then, it can be decided to reject or

accept the seed/grain lot based on the test results.
A basic testing plan for group testing consists of three fundamental parameters:
a) the number of seed/grain groups;
b) the size of the seed/grain groups;

c) the maximum number of deviant seed/grain groups for seed/grain lot acceptance (reject/accept

criterion).

The risks associated with the AQL and the LQL are the producer (alpha) and consumer (beta) risks

respectively, and together with the FPR and FNR allow the design of an appropriate testing plan.

The OC curve can be used to develop a testing plan. Explanations for the estimation of the LOD for a

zero deviant testing plan, the effect of the genome size on the group size if methods targeting DNA are

applied, and the effect of the individual seed size on the sample preparation are given in Annex C.

Annex D provides guidance on the determination of the maximum group size whatever analytical

method is used in the laboratory.
[16]

NOTE Seedcalc is a statistical program (Microsoft Excel spreadsheet application) that is freely available

from the International Seed Testing Association and has procedures to facilitate the design. Seedcalc is located

on the ISTA website.
7.2 Single-stage testing plan

A single-stage testing plan consists of one testing stage. Groups are taken from the test sample and

evaluated once, and a decision is then made based on the results to accept or reject the seed/grain

test sample. In a single-stage testing plan, a specified number of individual seeds/grains or seed/

6 © ISO 2021 – All rights reserved
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ISO 22753:2021(E)

grain groups shall be selected randomly from the test sample and tested. Depending on the number

of deviants detected and the maximum number of deviants specified in the plan, the seed/grain lot is

either accepted or rejected.

The probability that an individual seed/grain or seed/grain group is deviant, p , can be calculated as

given in Formula (1):
pP=−11=− 1−p (1)
where
P is the probability that there are no deviant seeds/grains in the group;
p is the true unknown impurity in the seed/grain lot;

m is the number of individual seeds/grains in a seed/grain group (if seeds/grains are tested indi-

vidually, m = 1).

Then, the probability that a lot will be accepted, P(a) is calculated as given in Formula (2):

 
ni−
P()a = pp()1− (2)
 
∑ b b
 
i=0
where
P(a) is the probability that a lot will be accepted;
n is the number of individual seeds/grains or seed/grain groups tested;
c is the maximum number of deviant seed/grain groups for acceptance.
By combining Formulae (1) and (2), P(a) is a function of p, n, m and c.

After n, m and c are determined, an OC curve can be drawn by plotting p and P(a) on the x-axis and

y-axis, respectively.
7.3 Double-stage testing plan

A double-stage testing plan is generally set up so that additional seed/grain groups are tested in the

second stage. Initial seed/grain groups are taken from the test sample and tested. Based on this test

result, three different decisions can be made:
a) accept the seed/grain lot;
b) reject the seed/grain lot; or
c) draw a second set of seed/grain groups from the test sample and retest.

The test results from the first and second stages of testing are combined and used to determine whether

the seed/grain lot is accepted or rejected (see Figure B.1). In Annex B examples for implementation of a

double-stage testing plan to evaluate GMO content in seeds/grains are provided. Subclause B.1 can also

be applied for cases where seed/grain lots are carrying stacked events.
Some additional terms are defined as follows:

— n , the number of independent seed/grain groups to be tested in the first stage;

— n , the number of independent seed/grain groups to be tested in the second stage;

— c , the maximum number of allowable deviant seed/grain groups for acceptance in the first stage;

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ISO 22753:2021(E)

— c , the minimum number of deviant seed/grain groups that will result in rejection at the first stage;

— c , the maximum number of deviant seed/grain groups in the first and second stages combined

allowed for acceptance;
— d , the number of deviant seed/grain groups in the first stage;
— d , the number of deviant seed/grain groups in the second stage.
P(a) is calculated as given in Formula (3):
n n n
       
c c −1 ci-
11ni−−ni 2 nj−
1 i 2 i 3 j
11 2
P()a = pp()11− + pp()− × pp()1−
      
∑ b b ∑  b b ∑ b b 
i=0 ic=+1 j=0
i i j
       
(3)
8 Selection of qualitative methods
8.1 General

An analytical method shall be chosen to meet the purpose of testing. The performance characteristics

of the method should be determined before application in seed/grain testing.

Analytical methods have been developed to detect specific genes encoding transgenic traits or specific

characteristics expressed by specific genes in seeds/grains. Nucleic-acid-based methods such as PCR

[4][5]

are available that detect specific DNA sequences encoding elements, constructs or GMO events .

Protein-based methods such as ELISA and lateral flow immunoassays require a specific antibody for

detecting a specific GM protein (see ISO 21572).
8.2 Performance criteria

The analytical methods applied for the test plan protocol shall detect at least one GM seed/grain in a

group with high probability of detection. Refer to Reference [2].
In the case of PCR, detection methods shall be chosen to meet the purpose
...

NORME ISO
INTERNATIONALE 22753
Première édition
2021-08
Analyse moléculaire de
biomarqueurs — Méthode pour
l'évaluation statistique des résultats
d'analyse obtenus lors des essais
de sous-échantillons multiples de
semences et de graines génétiquement
modifiées — Exigences générales
Molecular biomarker analysis — Method for the statistical evaluation
of analytical results obtained in testing sub-sampled groups of
genetically modified seeds and grains — General requirements
Numéro de référence
ISO 22753:2021(F)
© ISO 2021
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ISO 22753:2021(F)
DOCUMENT PROTÉGÉ PAR COPYRIGHT
© ISO 2021

Tous droits réservés. Sauf prescription différente ou nécessité dans le contexte de sa mise en œuvre, aucune partie de cette

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être demandée à l’ISO à l’adresse ci-après ou au comité membre de l’ISO dans le pays du demandeur.

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Publié en Suisse
© ISO 2021 – Tous droits réservés
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ISO 22753:2021(F)
Sommaire Page

Avant-propos .............................................................................................................................................................................................................................iv

Introduction .................................................................................................................................................................................................................................v

1 Domaine d’application ................................................................................................................................................................................... 1

2 Références normatives ..................................................................................................................................................................................1

3 Termes et définitions ...................................................................................................................................................................................... 1

4 Principe.......................................................................................................................................................................................................................... 4

4.1 Généralités ................................................................................................................................................................................................. 4

4.2 Préparation des groupes de semences/graines ........................................................................................................ 5

4.3 Méthodes de détection pour l’analyse qualitative de semences/graines GM dans

des groupes de semences/graines ......................................................................................................................................... 5

4.4 Évaluation statistique ...................................................................................................................................................................... 6

5 Réactifs ........................................................................................................................................................................................................................... 6

6 Appareillage et matériel ..............................................................................................................................................................................6

7 Conception du plan d’essai ........................................................................................................................................................................6

7.1 Généralités ................................................................................................................................................................................................. 6

7.2 Plan d’essai à une étape .................................................................................................................................................................. 7

7.3 Plan d’essai à deux étapes ............................................................................................................................................................. 7

8 Sélection des méthodes qualitatives .............................................................................................................................................. 8

8.1 Généralités ................................................................................................................................................................................................. 8

8.2 Critères de performance ................................................................................................................................................................ 9

9 Interprétation .........................................................................................................................................................................................................9

10 Expression des résultats ..........................................................................................................................................................................11

10.1 Classification d’un lot de semences/graines dans la catégorie «accepté» ou «rejeté» ........ 11

10.2 Estimation de la teneur en biomarqueur moléculaire dans le lot de semences/

graines ........................................................................................................................................................................................................ 11

11 Rapport d’essai ...................................................................................................................................................................................................11

Annexe A (informative) Tableau de comparaison des termes et définitions ........................................................12

Annexe B (informative) Exemple d’application de la méthode pour évaluer la teneur

en OGM dans les semences/graines ..............................................................................................................................................14

Annexe C (informative) Estimation de la limite de détection pour un plan d’essai conçu

pour détecter les semences/graines GM dans des lots de semences .........................................................22

Annexe D (informative) Détermination expérimentale de la taille de groupe maximale ......................26

Bibliographie ...........................................................................................................................................................................................................................28

iii
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ISO 22753:2021(F)
Avant-propos

L'ISO (Organisation internationale de normalisation) est une fédération mondiale d'organismes

nationaux de normalisation (comités membres de l'ISO). L'élaboration des Normes internationales est

en général confiée aux comités techniques de l'ISO. Chaque comité membre intéressé par une étude

a le droit de faire partie du comité technique créé à cet effet. Les organisations internationales,

gouvernementales et non gouvernementales, en liaison avec l'ISO participent également aux travaux.

L'ISO collabore étroitement avec la Commission électrotechnique internationale (IEC) en ce qui

concerne la normalisation électrotechnique.

Les procédures utilisées pour élaborer le présent document et celles destinées à sa mise à jour sont

décrites dans les Directives ISO/IEC, Partie 1. Il convient, en particulier, de prendre note des différents

critères d'approbation requis pour les différents types de documents ISO. Le présent document a

été rédigé conformément aux règles de rédaction données dans les Directives ISO/IEC, Partie 2 (voir

www.iso.org/directives).

L'attention est attirée sur le fait que certains des éléments du présent document peuvent faire l'objet de

droits de propriété intellectuelle ou de droits analogues. L'ISO ne saurait être tenue pour responsable

de ne pas avoir identifié de tels droits de propriété et averti de leur existence. Les détails concernant

les références aux droits de propriété intellectuelle ou autres droits analogues identifiés lors de

l'élaboration du document sont indiqués dans l'Introduction et/ou dans la liste des déclarations de

brevets reçues par l'ISO (voir www.iso.org/brevets).

Les appellations commerciales éventuellement mentionnées dans le présent document sont données

pour information, par souci de commodité, à l’intention des utilisateurs et ne sauraient constituer un

engagement.

Pour une explication de la nature volontaire des normes, la signification des termes et expressions

spécifiques de l'ISO liés à l'évaluation de la conformité, ou pour toute information au sujet de l'adhésion

de l'ISO aux principes de l’Organisation mondiale du commerce (OMC) concernant les obstacles

techniques au commerce (OTC), voir www.iso.org/avant-propos.

Le présent document a été élaboré par le comité technique ISO/TC 34, Produits alimentaires, sous-

comité SC 16, Méthodes horizontales pour l'analyse moléculaire de biomarqueurs.

La présente version française de l'ISO 22753:2021 correspond à la version anglaise publiée le 2021-08

et corrigée le 2022-11.

Il convient que l’utilisateur adresse tout retour d’information ou toute question concernant le présent

document à l’organisme national de normalisation de son pays. Une liste exhaustive desdits organismes

se trouve à l'adresse www.iso.org/fr/members.html.
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ISO 22753:2021(F)
Introduction

Les essais sur les semences et les graines sont utilisés dans le monde entier pour définir commercialement

la pureté des lots de semences et graines.

Les exigences commerciales relatives à l’étiquetage des produits agricoles ayant une teneur en

organismes génétiquement modifiés (OGM) à un seuil spécifié, à la fois sous forme de contaminant pour

les semences/graines et sous forme d’ingrédient alimentaire, se sont généralisées afin de respecter les

réglementations et satisfaire les demandes des consommateurs. Le respect de ces exigences est évalué

à différents stades de la chaîne d’approvisionnement, qui commence souvent par la récolte des graines.

La réaction de polymérisation en chaîne (PCR) quantitative en temps réel peut être utilisée pour

déterminer la teneur en OGM par l’analyse du rapport entre le nombre de copies d’ADN de l’OGM et

le nombre de copies d’ADN spécifique de l’espèce végétale puis la conversion en fraction massique

génétiquement modifiée (GM).

De multiples événements empilés pendant une récolte, notamment ceux générés par le croisement

d’au moins deux événements individuels, sont couramment utilisés dans le domaine de la production

agricole. Les semences ou graines à empilement d’événements contenant de l’ADN d’OGM correspondant

à au moins deux événements GM conjugués ne peuvent pas être différenciées, par une PCR quantitative

seule, des semences multiples au sein du lot contenant chacun un événement GM individuel. Par

conséquent, si l’OGM réel mesuré provient uniquement de semences à empilement d’événements GM,

la teneur en OGM mesurée par PCR quantitative en temps réel d’un seul échantillon entraînera une

surestimation du nombre réel de semences ou graines GM présentes.

La stratégie d'analyse de groupe, décrite dans le présent document, constitue une solution fiable

pour estimer la teneur en OGM en se basant sur le fait que des semences/graines entières constituent

l'échantillon.

Le processus décrit dans le présent document peut fournir une méthode permettant d’estimer avec

précision les pourcentages de semences/graines GM dans un lot, indépendamment de la présence

de semences/graines à empilement d’événements. La teneur en OGM est déterminée pour les sous-

échantillons multiples représentatifs de semences/graines provenant d’un lot, puis analysée d’un point

de vue statistique.
© ISO 2021 – Tous droits réservés
---------------------- Page: 5 ----------------------
NORME INTERNATIONALE ISO 22753:2021(F)
Analyse moléculaire de biomarqueurs — Méthode pour
l'évaluation statistique des résultats d'analyse obtenus
lors des essais de sous-échantillons multiples de semences
et de graines génétiquement modifiées — Exigences
générales
1 Domaine d’application

Le présent document décrit les exigences générales, les modes opératoires et les critères de performance

applicables à l’évaluation de la teneur en semences/graines génétiquement modifiées (GM) dans un lot

par une stratégie d’analyse de groupe qui comprend l’analyse qualitative de sous-échantillons multiples

puis l’évaluation statistique des résultats.

Le présent document est applicable à la stratégie d’analyse de groupe permettant d’estimer la teneur

en OGM sur un pourcentage de semences/graines afin d’en estimer la pureté, d'évaluer si un critère

de rejet/d'acceptation défini est respecté et de déterminer les cas où des lots de semences/graines

contiennent un empilement d’événements.
Le présent document n’est pas applicable aux produits transformés.

NOTE Une description de l’utilisation de la stratégie d’analyse de groupe est donnée dans les Références [1],

[7], [8], [18], [19] et [20].
2 Références normatives

Les documents suivants sont cités dans le texte de sorte qu’ils constituent, pour tout ou partie de leur

contenu, des exigences du présent document. Pour les références datées, seule l’édition citée s’applique.

Pour les références non datées, la dernière édition du document de référence s'applique (y compris les

éventuels amendements).

ISO 16577, Analyse de biomarqueurs moléculaires — Vocabulaire pour les méthodes d’analyse de

biomarqueurs moléculaires dans l’agriculture et la production agroalimentaire

ISO 21572, Produits alimentaires— Analyse des biomarqueurs moléculaires — Méthodes immunochimiques

pour la détection et la quantification des protéines

ISO 24276, Produits alimentaires — Méthodes d'analyse pour la détection des organismes génétiquement

modifiés et des produits dérivés — Exigences générales et définitions
3 Termes et définitions

Pour les besoins du présent document, les termes et définitions donnés dans l’ISO 16577 ainsi que les

suivants s’appliquent.

L’ISO et l’IEC tiennent à jour des bases de données terminologiques destinées à être utilisées en

normalisation, consultables aux adresses suivantes:

— ISO Online browsing platform: disponible à l’adresse https:// www .iso .org/ obp

— IEC Electropedia: disponible à l’adresse https:// www .electropedia .org/
© ISO 2021 – Tous droits réservés
---------------------- Page: 6 ----------------------
ISO 22753:2021(F)
3.1
limite de détection PCR absolue
limite de détection absolue de la réaction de polymérisation en chaîne
LOD PCR absolue

plus petit nombre nominal (moyen) de copies cibles dans le volume de matrice distribué dans chaque

PCR qui offrirait une probabilité acceptable de détection de la cible
3.2
NQA
niveau de qualité acceptable

niveau d’impureté qui est acceptable pour le producteur et que les méthodes de production peuvent

supporter
3.3
risque du consommateur
risque du consommateur (bêta)
probabilité d’acceptation d’un lot à la limite de qualité inférieure (3.10)
3.4
graine/semence déviante

graine/semence considérée comme non conforme en raison de la présence ou de l’absence d’un trait ou

d’une caractéristique particulière

Note 1 à l'article: Pour les besoins du présent document, est considérée comme déviante une semence qui possède

une caractéristique GM inattendue ou imprévue par rapport aux caractéristiques GM connues ou attendues de la

semence/graine.
3.5
taux de faux négatifs
TFN

probabilité qu’un échantillon pour essai (3.20) positif (groupe de semences/graines) connu ait été classé

comme négatif par la méthode

Note 1 à l'article: Le taux de faux négatifs est le nombre de positifs connus mal classés divisé par le nombre total

d’échantillons pour essai (3.20) positifs.

[SOURCE: ISO 16577:2016, 3.63, modifiée — l’abréviation a été ajoutée, «échantillon pour essai positif»

a été remplacé par «échantillon pour essai (groupe de semences/graines) positif» et la formule a été

supprimée.]
3.6
taux de faux positifs
TFP

probabilité qu’un échantillon pour essai (3.20) négatif (groupe de semences/graines) connu ait été classé

comme positif par la méthode

Note 1 à l'article: Le taux de faux positifs est le nombre de négatifs connus mal classés divisé par le nombre total

d’échantillons pour essai (3.20) négatifs.

[SOURCE: ISO 16577:2016, 3.65, modifiée — l’abréviation a été ajoutée, «échantillon pour essai négatif»

a été remplacé par «échantillon pour essai (groupe de semences/graines) négatif» et la formule a été

supprimée.]
3.7
taille du groupe
nombre de semences/graines contenues dans un groupe
© ISO 2021 – Tous droits réservés
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ISO 22753:2021(F)
3.8
essai de groupe

évaluation statistique de la teneur en analyte à partir de résultats d’analyse qualitative (c’est-à-dire

positifs ou négatifs) pour chaque groupe de semences/graines de l’échantillon pour essai (3.20)

3.9
échantillon pour laboratoire
échantillon ou sous-échantillon(s) reçu(s) par le laboratoire

Note 1 à l'article: Il est attendu que l'échantillon de semence/graine reçu soit représentatif du lot de semences/

graines (3.18).
[SOURCE: ISO 16577:2016, 3.89, modifiée — La Note 1 à l’article a été ajoutée.]
3.10
LQL
limite de qualité inférieure
niveau d’impureté le plus élevé qui est acceptable pour le consommateur
Note 1 à l'article: Ce terme peut être équivalent au seuil (3.22).
3.11
fraction massique

rapport entre les semences/graines GM et les semences/graines totales correspondant au rapport de

masse
3.12
nombre de groupes de graines/semences déviantes

nombre de groupes de semences/graines (3.17) comprenant une ou plusieurs semences/graines déviantes

(3.4)
3.13
courbe caractéristique opérationnelle
courbe OC

représentation graphique du pourcentage de semences/graines déviantes et de la probabilité

d’acceptation situés respectivement sur les axes horizontal et vertical et utilisée dans le cadre de

contrôles qualité afin de déterminer la probabilité d’acceptation de lots de semences/graines (3.18) d’un

plan d’essai (3.21)
3.14
risque du producteur
risque du producteur (alpha)
probabilité de rejet d’un lot au NQA (3.2)
3.15
échantillon représentatif

unités d’échantillonnage (échantillons ou groupes) qui ont été extraites d’un lot au moyen d’un processus

garantissant que toutes les unités d’échantillonnage ont les mêmes chances d’être sélectionnées et n’ont

pas été modifiées au point de fausser le résultat d’analyse

Note 1 à l'article: Le processus d’extraction peut être un processus à multiples étapes.

3.16
critère de rejet/d’acceptation

nombre de groupes de semences/graines déviantes (3.12) maximal qui peut être détecté dans l’échantillon

pour essai (3.20) d’un lot de semences/graines (3.18) acceptables
© ISO 2021 – Tous droits réservés
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ISO 22753:2021(F)
3.17
groupe de semences/graines
groupe

nombre déterminé de semences/graines préparées à partir d’un échantillon pour essai (3.20) de

semences/graines par échantillonnage représentatif
3.18
lot de semences/graines
lot
population destinée à être échantillonnée pour estimer le paramètre mesuré
3.19
empilement d’événements

accumulation d’au moins deux événements de transformation à la suite d’un croisement traditionnel et/

ou d’étapes de transformation génétique successives

Note 1 à l'article: Dans le contexte du présent document, un empilement d'événements désigne un empilement

dans lequel les deux événements ou plus n’ont pas de liens génétiques.
[SOURCE: ISO 16577:2016, 3.197, modifiée — La Note 1 à l’article a été ajoutée.]
3.20
échantillon pour essai

échantillon préparé pour essai ou analyse, la quantité totale ou une partie de celui-ci étant utilisée pour

l’essai ou l’analyse en une seule fois

Note 1 à l'article: L’échantillon pour essai est préparé à partir d’un échantillon pour laboratoire (3.9).

Note 2 à l'article: Il est attendu que l’échantillon pour essai soit représentatif de l’échantillon pour laboratoire

(3.9).

[SOURCE: ISO 16577:2016, 3.210 modifiée — La Note 1 à l'article et la Note 2 à l’article ont été ajoutées.]

3.21
plan d’essai

plan spécifiant les conditions de l’essai de groupe (3.8), y compris la taille du groupe (3.7), le nombre de

groupes de semences/graines (3.17) et le nombre de groupes de semences/graines déviantes (3.12) au sein

de l’échantillon pour essai (3.20) à la suite du rejet d’un lot de semences/graines (3.18)

3.22
seuil
teneur maximale acceptable d’un OGM dans un lot
Note 1 à l'article: Il peut s’agit d’une valeur prescrite.

Note 2 à l'article: Les seuils peuvent être exprimés en fraction massique (3.11), à condition qu’un facteur

d’incertitude soit utilisé pour la conversion en seuil de pourcentage de semences/graines.

4 Principe
4.1 Généralités

Dans cette méthode, l'échantillon pour essai est divisé en un nombre de groupes prédéterminé.

Chaque groupe comprend un nombre déterminé de semences/graines et est analysé qualitativement

par rapport à la présence ou l’absence d’une cible GM. Une évaluation statistique est effectuée sur le

nombre de groupes positifs aux OGM par rapport au nombre total de groupes de semences/graines afin

de déterminer la teneur en OGM en fraction massique.

Un calcul statistique détermine les conditions d’essai optimales, à savoir le nombre de semences/

graines par groupe (taille du groupe), le nombre de groupes de semences/graines et le nombre maximal

de groupes de semences/graines positifs aux OGM pour l’acceptation du lot de semences/graines. Un

© ISO 2021 – Tous droits réservés
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ISO 22753:2021(F)

calcul statistique fournit également une estimation du pourcentage en nombre de semences/graines

GM d’un lot, en fonction d’un plan d’essai défini.
4.2 Préparation des groupes de semences/graines
Légende
1 lot de semences/graines en vrac
2 échantillon pour laboratoire
3 échantillon pour essai
4 groupes de semences/graines
5 graine/semence déviante
NOTE Chaque groupe est représenté sous la forme d’une rangée à droite.

Figure 1 — Illustration schématique de l’obtention de groupes de semences/graines à partir

d’un lot de semences/graines en vrac

Le processus de formation de groupes de semences/graines à partir d’une série d'étapes

d'échantillonnage, en commençant par le lot de semences/graines en vrac, est illustré à la Figure 1, (1).

Bien que les modes opératoires permettant d’obtenir un échantillon pour laboratoire à partir d’un lot

de semences/graines ne soient pas abordés par le présent document, un échantillon pour laboratoire (1)

provenant d’un lot de semences/graines doit être obtenu de manière appropriée. Les modes opératoires

peuvent être conçus d’après les Références [3], [6], [10], [11], [12], [15], [19] et [23].

L’échantillon pour laboratoire doit être soigneusement mélangé et divisé/réduit pour créer l’échantillon

pour essai (3). De la même manière, l’échantillon pour essai doit être soigneusement mélangé (c’est-à-

dire, homogénéisé) et divisé en groupes de semences/graines [chaque groupe étant représenté sous la

forme d’une rangée à la Figure 1, (4)] en respectant les principes de l’échantillonnage aléatoire simple.

Les groupes de semences/graines peuvent varier en taille (d’une seule semence/graine à un échantillon

pour essai complet, soit un seul volume). Dans la plupart des cas, plusieurs groupes de semences/

graines sont créés à partir de l’échantillon pour essai.

Un nombre déterminé de semences/graines peut être obtenu par pesage ou par mesure de volume,

auquel cas une approximation du nombre est effectuée sur la base d’un facteur de conversion déterminé

(par exemple, poids de mille semences/graines). Dans le cas où ce poids est utilisé pour obtenir les

groupes de semences/graines, l’opérateur doit avoir une estimation de la variabilité introduite par

l’utilisation du poids plutôt que par le dénombrement des semences/graines.

Le mode opératoire de l’essai de groupe, décrit à l’Article 7 est effectué sur les résultats collectifs

qualitatifs (positifs ou négatifs) pour chaque groupe de semences/graines.
4.3 Méthodes de détection pour l’analyse qualitative de semences/graines GM dans
des groupes de semences/graines
[21]

En général, les méthodes de détection d’OGM sont divisées dans deux catégories . La première

catégorie d’analyse cible une séquence d’acide nucléique pour détecter la présence d’OGM. La seconde

fait appel à des méthodes de détection d’une protéine spécifiée qui confère un trait transgénique

spécifique. Il convient de sélectionner les méthodes de détection parmi l’une des catégories ou les deux

catégories, en tenant compte de leur adéquation avec l’objectif. Des recommandations sur la sélection

© ISO 2021 – Tous droits réservés
---------------------- Page: 10 ----------------------
ISO 22753:2021(F)

des méthodes qualitatives sont fournies à l’Article 8. Des informations supplémentaires sont données

[4]
dans l’ISO 21569 et dans l’ISO 21572.
4.4 Évaluation statistique

L’incertitude d’échantillonnage et de mesure doit être prise en compte. L'incertitude d'échantillonnage

[18][2]

peut être adéquatement prise en compte en utilisant la distribution binomiale . Il convient de tenir

[2]

compte du TFP et du TFN de l’analyse qualitative . Il convient de tenir compte de la LOD de la méthode

de détection appliquée.

L’essai de groupe décrit ici peut être utilisé pour définir les critères de rejet/d’acceptation en fonction

d’un seuil défini par la teneur en OGM, ainsi que pour estimer la teneur en OGM et les limites de confiance

supérieure et inférieure associées.
5 Réactifs

Il convient que tous les réactifs utilisés lors de l’analyse soient ceux spécifiés dans la méthode.

En l'absence de spécification, il convient que tous les réactifs soient de qualité analytique reconnue.

Ces réactifs doivent être conservés et utilisés selon les recommandations du fournisseur ou

conformément aux spécifications d’assurance qualité du laboratoire. Il peut également être approprié

d’aliquoter les solutions réactionnelles requises pour la méthode d’analyse afin d’éviter de les soumettre

à des cycles répétés de congélation-décongélation et/ou de réduire les risques de contamination croisée.

Pour plus d’informations, voir l’ISO 24276 et l’ISO 21572.
6 Appareillage et matériel

Il convient que le laboratoire utilise un matériel correctement entretenu et adapté aux méthodes

employées.
Pour plus d’informations, voir l’ISO 24276 et l’ISO 21572.
7 Conception du plan d’essai
7.1 Généralités

Le nombre de semences/graines soumises à essai, les critères de rejet/d’acceptation, les étapes de

préparation des échantillons et la méthode d’analyse utilisée doivent être déterminés en fonction de

l’objectif de l’analyse.

Lors de la classification des échantillons de semences/graines, il est possible de déterminer si le nombre

de semences/graines déviantes ou groupes de semences/graines est supérieur ou non à un critère de

rejet/d’acceptation défini. Il peut ensuite être décidé de rejeter ou d’accepter le lot de semences/graines

d’après les résultats d’essai.

Un plan d’essai de base pour l’essai de groupe comprend trois paramètres fondamentaux:

a) le nombre de groupes de semences/graines;
b) la taille des groupes de semences/graines;

c) le nombre maximal de groupes de graines/semences déviantes pour l’acceptation du lot de

semences/graines (critère de rejet/d’acceptation).

Les risques associés au NQA et à la LQL sont respectivement les risques du producteur (alpha) et du

consommateur (bêta) qui, avec le TFP et le TFN, permettent de concevoir un plan d’essai approprié.

© ISO 2021 – Tous droits réservés
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ISO 22753:2021(F)

La courbe OC peut être utilisée pour élaborer un plan d’essai. Des explications concernant l’estimation

de la LOD pour un plan d’essai déviant au zéro, l’effet de la taille du génome sur la taille du groupe si des

méthodes de ciblage de l’ADN sont appliquées, et l’effet de la taille de chaque semence sur la préparation

des échantillons sont données à l’Annexe C.

L’Annexe D fournit des recommandations sur la détermination de la taille maximale du groupe, quelle

que soit la méthode d’analyse utilisée au sein du laboratoire.
[16]

NOTE Seedcalc est un logiciel statistique (tableur Microsoft Excel) disponible gratuitement auprès de

l’International Seed Testing Association et intègre des fonctions permettant de faciliter la conception. Seedcalc

est disponible sur le site web de l’ISTA.
7.2 Plan d’essai à une étape

Un plan d’essai à une étape comprend une étape d’essai. Des groupes sont prélevés dans l’échantillon

d’essai et évalués une fois. Une décision est ensuite prise, en fonction des résultats, d’accepter ou de

rejeter l’échantillon pour essai de semence/graine. Dans un plan d’essai à une étape, un nombre spécifié

...

FINAL
INTERNATIONAL ISO/FDIS
DRAFT
STANDARD 22753
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
Method for the statistical evaluation of
Voting begins on:
2021­05­31 analytical results obtained in testing
sub-sampled groups of genetically
Voting terminates on:
2021­07­26
modified seeds and grains — General
requirements and definitions
RECIPIENTS OF THIS DRAFT ARE INVITED TO
SUBMIT, WITH THEIR COMMENTS, NOTIFICATION
OF ANY RELEVANT PATENT RIGHTS OF WHICH
THEY ARE AWARE AND TO PROVIDE SUPPOR TING
DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
Reference number
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/FDIS 22753:2021(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN­
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. ISO 2021
---------------------- Page: 1 ----------------------
ISO/FDIS 22753:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting

on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address

below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH­1214 Vernier, Geneva
Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
---------------------- Page: 2 ----------------------
ISO/FDIS 22753:2021(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 4

4.1 General ........................................................................................................................................................................................................... 4

4.2 Preparation of seed/grain groups .......................................................................................................................................... 5

4.3 Detection methods for the qualitative analysis of GM seed/grain in seed/grain groups ....... 5

4.4 Statistical evaluation .......................................................................................................................................................................... 5

5 Reagents ........................................................................................................................................................................................................................ 6

6 Apparatus and equipment .......................................................................................................................................................................... 6

7 Design of testing plan ...................................................................................................................................................................................... 6

7.1 General ........................................................................................................................................................................................................... 6

7.2 Single­stage testing plan ................................................................................................................................................................. 6

7.3 Double­stage testing plan .............................................................................................................................................................. 7

8 Selection of qualitative methods ......................................................................................................................................................... 8

8.1 General ........................................................................................................................................................................................................... 8

8.2 Performance criteria .......................................................................................................................................................................... 8

9 Interpretation .......................................................................................................................................................................................................... 8

10 Expression of results .....................................................................................................................................................................................10

10.1 Classification of a seed/grain lot into “accept” or “reject” category ......................................................10

10.2 Estimation of the level of molecular biomarker in the seed/grain lot .................................................10

11 Test report ................................................................................................................................................................................................................10

Annex A (informative) Terms and definitions comparison table ........................................................................................12

Annex B (informative) Implementation of the method to evaluate GMO content in seeds/

grains example.....................................................................................................................................................................................................14

Annex C (informative) Estimation of the limit of detection for a testing plan to detect GM

seeds/grains in seed lots ..........................................................................................................................................................................21

Annex D (informative) Experimental determination of maximum group size ......................................................25

Bibliography .............................................................................................................................................................................................................................26

© ISO 2021 – All rights reserved iii
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ISO/FDIS 22753:2021(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non­governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO's adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,

Horizontal methods for molecular biomarker analysis.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2021 – All rights reserved
---------------------- Page: 4 ----------------------
ISO/FDIS 22753:2021(E)
Introduction

Seed and grain testing is used throughout the world to commercially define the purity of seed and grain

lots.

Commercial requirements for labelling agricultural products with genetically modified organism (GMO)

content at a specified threshold level both as a seed/grain contaminant and a food ingredient have

become common to satisfy regulations and consumer demands. Conformance with these specifications

is evaluated at various points of the supply chain, often starting with the harvested grain.

Quantitative real-time polymerase chain reaction (PCR) can be used to determine the GMO content by

analysis of the ratio of GMO DNA copy numbers to plant-species specific DNA copy numbers followed by

a conversion to genetically modified (GM) mass fraction.

Multiple events stacked in a crop, such as those generated by crossing two or more single events,

are widely used in agricultural production. A stacked event seed or grain containing GMO DNA

corresponding to two or more GM events commingled in lot cannot be differentiated by quantitative

PCR alone from multiple seeds within the lot each containing a single GM event. Consequently, if the

actual measured GMO arises only from GM stacked event seeds, GM content measured by quantitative

real­time PCR of a single sample will lead to an overestimation of the actual number of GM seeds or

grains present.

The group testing strategy described in this document provides a reliable alternative to estimate the

GM content on the basis of the fact that whole seeds/grains are the sample material.

The process described in this document can provide a method to accurately estimate the percentages

of GM seeds/grains in a lot irrespective of the presence of stacked event seeds/grains. GM content is

determined for representative subsampled groups of seed/grain from a lot and statistically analysed.

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FINAL DRAFT INTERNATIONAL STANDARD ISO/FDIS 22753:2021(E)
Molecular biomarker analysis — Method for the statistical
evaluation of analytical results obtained in testing sub-
sampled groups of genetically modified seeds and grains —
General requirements and definitions
1 Scope

This document describes general requirements, procedures and performance criteria for evaluating

the content of genetically modified (GM) seeds/grains in a lot by a group testing strategy that includes

qualitative analysis of sub-sampled groups followed by statistical evaluation of the results.

This document is applicable to group testing strategy estimating the GM content on a percentage seed/

grain basis for purity estimation, testing towards a given reject/accept criterion and for cases where

seed/grain lots are carrying stacked events.
This document is not applicable to processed products.

NOTE Description of the use of group testing strategy are available in References [1], [7], [8], [18], [19] and

[20].
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 16577, Molecular biomarker analysis — Terms and definitions

ISO 21572, Foodstuffs — Molecular biomarker analysis — Immunochemical methods for the detection and

quantification of proteins

ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and

derived products — General requirements and definitions
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 16577 and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
absolute PCR limit of detection
absolute polymerase chain reaction limit of detection
absolute PCR LOD

lowest nominal (average) number of target copies in the template volume distributed to individual PCRs

that would allow for an acceptable probability of detecting the target
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3.2
AQL
acceptable quality limit

level of impurity that is acceptable to the producer and that production practices can support

3.3
consumer risk
consumer (beta) risk
probability of accepting a lot at the lower quality limit (3.10)
3.4
deviant seed/grain

considered non-conforming based on the presence or absence of a specific trait or characteristic

Note 1 to entry: For the purpose of this document, a deviant seed is considered to possess a GM characteristic

that is not expected or is unintended based on the expected or known GM characteristics of the seed/grain.

3.5
false negative rate
FNR

probability that a known positive (seed/grain group) test sample (3.20) has been classified as negative

by the method

Note 1 to entry: The false negative rate is the number of misclassified known positives divided by the total

number of positive test samples (3.20).

[SOURCE: ISO 16577:2016, 3.63, modified — the abbreviation has been added, “positive test sample”

has been changed to “positive (seed/grain group) test sample”, and the formula has been deleted.]

3.6
false positive rate
FPR

probability that a known negative (seed/grain group) test sample (3.20) has been classified as positive

by the method

Note 1 to entry: The false positive rate is the number of misclassified known negatives divided by the total

number of negative test samples (3.20).

[SOURCE: ISO 16577:2016, 3.65, modified — the abbreviation has been added, “negative test sample”

has been changed to “negative (seed/grain group) test sample”, and the formula has been deleted.]

3.7
group size
number of seeds/grains comprising a group
3.8
group testing

statistical evaluation of analyte contents based on qualitative analysis results (i.e. positive or negative)

from each seed/grain group in the test sample (3.20)
3.9
laboratory sample
sample or subsample(s) received by the laboratory

Note 1 to entry: The seed/grain sample received is expected to represent the seed/grain lot (3.18).

[SOURCE: ISO 16577:2016, 3.89, modified — Note 1 to entry has been added.]
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ISO/FDIS 22753:2021(E)
3.10
LQL
lower quality limit
highest impurity that is acceptable to the consumer
Note 1 to entry: This can be equivalent to the threshold (3.22).
3.11
mass fraction

ratio of GM seeds/grains relative to the total seeds/grains corresponding to mass ratio

3.12
number of deviant seed/grain groups

number of seed/grain groups (3.17) including one or more deviant seeds/grains (3.4)

3.13
operating characteristic curve
OC curve

graph plotting the percentage of deviant seeds/grains and the probability of acceptance respectively

on the horizontal and the vertical axes and used in quality control to determine the probability of

accepting seed/grain lots (3.18) in a testing plan (3.21)
3.14
producer risk
producer (alpha) risk
probability of rejecting a lot at the AQL (3.2)
3.15
representative sample

sampling units (samples or groups) that have been extracted from a lot with the process ensuring all

sampling units of the lots have an equal probability of being selected and not altered in any way that

would change the analytical result
Note 1 to entry: The extraction process can be a multi-stage process.
3.16
reject/accept criterion

maximum number of deviant seed/grain groups (3.12) that can be detected in the test sample (3.20) of an

acceptable seed/grain lot (3.18)
3.17
seed/grain group
group

determined number of seeds/grains prepared from a seed/grain test sample (3.20) by representative

sampling
3.18
seed/grain lot
lot
population for which sampling is intended to estimate the measured parameter
3.19
stacked event

accumulation of two or more transformation events as a result of traditional breeding and/or successive

transformation steps)

Note 1 to entry: In the context of this document a stacked event refers to a stack in which the two or more events

are not genetically linked.
[SOURCE: ISO 16577:2016, 3.197, modified — Note 1 to entry has been added.]
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ISO/FDIS 22753:2021(E)
3.20
test sample

sample prepared for testing or analysis, the whole quantity or part of it being used for testing or

analysis at one time
Note 1 to entry: The test sample is prepared from the laboratory sample (3.9).

Note 2 to entry: The test sample is expected to represent the laboratory sample (3.9).

[SOURCE: ISO 16577:2016, 3.210, modified — Note 1 to entry and Note 2 to entry have been added.]

3.21
testing plan

plan specifying group testing (3.8) conditions including group size (3.7), the number of seed/grain groups

(3.17) and the number of deviant seed/grain groups (3.12) in test sample (3.20) resulting in rejection of

seed/grain lot (3.18)
3.22
threshold
maximum acceptable content of GMO presence in a lot
Note 1 to entry: This can be a prescribed value.

Note 2 to entry: Thresholds can be expressed in mass fraction (3.11) with the proviso that an uncertainty factor

is involved in the conversion to a seed/grain percentage threshold.
4 Principle
4.1 General

In this method, the test sample is divided into a predetermined number of groups. Each group consists

of a determined number of seed/grain and is tested qualitatively for the presence or absence of a GM

target. A statistical evaluation is performed on the number of GM positive groups relative to the total

number of seed/grain groups to determine the GM content in mass fraction.

A statistical calculation determines the optimal testing conditions, namely, the number of seeds/grains

per group (group size), the number of seed/grain groups, and the maximum number of GMO positive

seed/grain groups for seed/grain lot acceptance. Alternatively, a statistical calculation provides an

estimate of the percentage by number of the GM seeds/grains in a lot, according to a given testing plan.

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ISO/FDIS 22753:2021(E)
4.2 Preparation of seed/grain groups
Key
1 bulk seed/grain lot
2 laboratory sample
3 test sample
4 seed/grain groups
5 deviant seed/grain
NOTE Each group is represented as an array on the right.

Figure 1 — Sampling illustration of the obtention of seed/grain groups from a bulk seed/grain

lot

The process of forming seed/grain groups from a series of sampling steps starting with the bulk seed/

grain lot is shown in Figure 1, (1).

Although the procedures for obtaining a laboratory sample from a seed/grain lot is not the subject

of this document, a laboratory sample (2) from a seed/grain lot shall be obtained appropriately. The

procedures can be designed according to the References [3], [6], [10], [11], [12], [15], [19] and [23].

The laboratory sample shall be thoroughly mixed and divided/reduced to create the test sample (3).

Likewise, the test sample shall be thoroughly mixed (i.e. homogeneous) and divided into seed/grain

groups (each group represented as an array in Figure 1, (4)) following simple random sampling

principles. The seed/grain groups can vary in size from one single seed/grain up to the complete test

sample (i.e. a single bulk). In most cases, multiple seed/grain groups are created from the test sample.

A determined number of seeds/grains can either be obtained by weighing or a volumetric measurement,

where an approximation of number is made based on a determined conversion factor (e.g. thousand

seeds/grains weight). For the case that weight is used to obtain the seed/grain groups, the operator

shall have an estimate of the variability introduced by using weight rather than seed/grain count.

The group testing procedure described in Clause 7 is carried out on the collective qualitative (positive

(e) or negative) results for each seed/grain group.

4.3 Detection methods for the qualitative analysis of GM seed/grain in seed/grain

groups
[21]

In general, GMO detection methods are categorized into two classes . The first class of assays targets

a nucleic acid sequence for detecting GMO presence. The second class includes methods for detecting a

specified protein that confers a specific transgenic trait. Detection methods from either or both classes

should be selected considering fitness-for-purpose. Guidance on the selection of qualitative methods is

[4]
provided in Clause 8. Further details can be found in ISO 21569 and ISO 21572.
4.4 Statistical evaluation

Sampling and measurement uncertainty shall be considered. Sampling uncertainty can be adequately

[18][2]

considered using the binomial distribution . The FPR and the FNR of the qualitative assay should

[2]
be considered . The LOD of the applied detection method should be considered.
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ISO/FDIS 22753:2021(E)

The group testing described here can be used to set reject/accept criteria based on a given threshold by

GMO content, as well as to estimate the GMO content and associated upper and lower confidence limits.

5 Reagents
All reagents used in the analysis should be those specified in the method.
Otherwise, all reagents should be of molecular biology grade.

These reagents shall be stored and used as recommended by the supplier or according to the laboratory

quality assurance specifications. It can also be appropriate to aliquot the reaction solutions required

for the analytical method in order to avoid subjecting them to repeated freeze–thaw cycles, or to reduce

the chances of cross contamination or both. Further details shall refer to ISO 24276 and ISO 21572.

6 Apparatus and equipment

The laboratory should use properly maintained equipment suitable for the methods employed.

Further details shall refer to ISO 24276 and ISO 21572.
7 Design of testing plan
7.1 General

The number of seeds/grains tested, the reject/accept criteria, the sample preparation steps and the

method used for testing shall be determined depending on the analytical purpose.

In seed/grain sample classification, it can be determined whether the number of deviant seeds/grains

or seed/grain groups is above a given reject/accept criterion or not. Then, it can be decided to reject or

accept the seed/grain lot based on the test results.
A basic testing plan for group testing consists of three fundamental parameters:
a) the number of seed/grain groups;
b) the size of the seed/grain groups;

c) the maximum number of deviant seed/grain groups for seed/grain lot acceptance (reject/accept

criterion).

The risks associated with the AQL and the LQL are the producer (alpha) and consumer (beta) risks

respectively, and together with the FPR and FNR allow the design of an appropriate testing plan.

The OC curve can be used to develop a testing plan. Explanations for the estimation of the LOD for a

zero deviant testing plan, the effect of the genome size on the group size if methods targeting DNA are

applied, and the effect of the individual seed size on the sample preparation are given in Annex C.

Annex D provides guidance on the determination of the maximum group size whatever analytical

method is used in the laboratory.
[16]

NOTE Seedcalc is a statistical program (Microsoft Excel spreadsheet application) that is freely available

from the International Seed Testing Association and has procedures to facilitate the design. Seedcalc is located

on the ISTA website.
7.2 Single-stage testing plan

A single­stage testing plan consists of one testing stage. Groups are taken from the test sample and

evaluated once, and a decision is then made based on the results to accept or reject the seed/grain

test sample. In a single-stage testing plan, a specified number of individual seeds/grains or seed/

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grain groups shall be selected randomly from the test sample and tested. Depending on the number

of deviants detected and the maximum number of deviants specified in the plan, the seed/grain lot is

either accepted or rejected.

The probability that an individual seed/grain or seed/grain group is deviant, p , can be calculated as

given in Formula (1):
pP=−11=− 1−p (1)
where
P is the probability that there are no deviant seeds/grains in the group;
p is the true unknown impurity in the seed/grain lot;

m is the number of individual seeds/grains in a seed/grain group (if seeds/grains are tested indi­

vidually, m = 1).

Then, the probability that a lot will be accepted, P(a) is calculated as given in Formula (2):

 
ni−
P()a = pp()1− (2)
 
∑ b b
 
i=0
where
P(a) is the probability that a lot will be accepted;
n is the number of individual seeds/grains or seed/grain groups tested;
c is the maximum number of deviant seed/grain groups for acceptance.
By combining Formulae (1) and (2), P(a) is a function of p, n, m and c.

After n, m and c are determined, an OC curve can be drawn by plotting p and P(a) on the x-axis and

y-axis, respectively.
7.3 Double-stage testing plan

A double-stage testing plan is generally set up so that additional seed/grain groups are tested in the

second stage. Initial seed/grain groups are taken from the test sample and tested. Based on this test

result, three different decisions can be made:
a) accept the seed/grain lot;
b) reject the seed/grain lot; or
c) draw a second set of seed/grain groups from the test sample and retest.

The test results from the first and second stages of testing are combined and used to determine whether

the seed/grain lot is accepted or rejected (see Figure B.1). In Annex B examples for implementation of a

double­stage testing plan to evaluate GMO content in seeds/grains are provided. Subclause B.1 can also

be applied for cases where seed/grain lots are carrying stacked events.
Some additional terms are defined as follows:

— n , the number of independent seed/grain groups to be tested in the first stage;

— n , the number of independent seed/grain groups to be tested in the second stage;

— c , the maximum number of allowable deviant seed/grain groups for acceptance in the first stage;

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— c , the minimum number of deviant seed/grain groups that will result in rejection at the first stage;

— c , the maximum number of deviant seed/grain groups in the first and second stages combined

allowed for acceptance;
— d , the number of deviant seed/grain groups in the first stage;
— d , the number of deviant seed/grain groups in the second stage.
P(a) is calculated as given in Formula (3):
n n n2
       
c c −1 ci-
11ni−−ni nj−
1 i 2 i 3 j
11 2
P()a = pp()11− + pp()− × 1pp()−
      
∑ b b ∑  b b ∑ b b 
i=0 ic=+1 j=0
i i j
       
(3)
8 Selection of qualitative methods
8.1 General

An analytical method shall be chosen to meet the purpose of testing. The performance characteristics

of the method should be determined before application in seed/grain testing.

Analytical methods have been developed to detect specific genes encoding transgenic traits or specific

characteristics expressed by specific genes in seeds/grains. Nucleic-acid-based methods such as PCR

[4][5]
are available that detect specific DNA sequences encod
...