SIST EN ISO 22753:2023
(Main)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 (ISO 22753:2021, 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 (ISO 22753:2021, Corrected version 2022-11)
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].
Untersuchung auf molekulare Biomarker - Verfahren zur statistischen Auswertung von Analyseergebnissen aus der Untersuchung von Untergruppen von gentechnisch verändertem Saatgut und Getreide - Allgemeine Anforderungen (ISO 22753:2021, Korrigierte Fassung 2022-11)
Dieses Dokument beschreibt allgemeine Anforderungen, Verfahren und Leistungskriterien für die Bewertung des Gehalts an gentechnisch verändertem (GV )Saatgut/Getreide in einer Partie mittels einer Gruppenunter¬suchungsstrategie, die die qualitative Analyse von Untergruppen und die anschließende statistische Auswertung der Ergebnisse umfasst.
Dieses Dokument ist anwendbar auf Strategien zur Gruppenuntersuchung, bei denen der GVO-Gehalt auf Grundlage der Saatgut /Getreideprozentanteile zur Einschätzung der Reinheit geschätzt wird, auf Strategien, bei denen mit dem Ziel eines Ablehn /Annahmekriteriums untersucht wird, und in Fällen von Saatgut /Getreidepartien, die kombinierte Ereignisse enthalten.
Dieses Dokument ist nicht anwendbar auf verarbeitete Erzeugnisse.
ANMERKUNG Beschreibungen der Anwendung von Gruppenuntersuchungsstrategien finden sich in den Literaturhinweisen [1] [7] [8] [18] [19] und [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 (ISO 22753:2021, Version corrigée 2022-11)
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].
Analiza molekularnih biomarkerjev - Metoda za statistično vrednotenje rezultatov analiz, pridobljenih pri preskušanju podvzorcev skupin gensko spremenjenih semen in zrn - Splošne zahteve (ISO 22753:2021, popravljena verzija 2022-11)
Ta dokument opisuje splošne zahteve, postopke in merila za ocenjevanje vsebnosti gensko spremenjenih (GS) semen/zrn v seriji s strategijo skupinskega preskušanja, ki vključuje kvalitativno analizo podvzorcev skupin, ki ji sledi statistična ocena rezultatov.
Dokument se uporablja za strategijo skupinskega preskušanja, pri katerem se vsebnost GS ocenjuje na podlagi odstotka semena/zrna za oceno čistosti, preskušanja v skladu z danim merilom za zavrnitev/sprejem in za primere, ko serije semena/zrna vsebujejo naložene dogodke.
Ta dokument se ne uporablja za predelane proizvode.
OPOMBA: Opis uporabe strategije preskušanja skupin je na voljo v referencah [1], [7], [8], [18], [19] in [20].
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 22753:2023
01-februar-2023
Analiza molekularnih biomarkerjev - Metoda za statistično vrednotenje rezultatov
analiz, pridobljenih pri preskušanju podvzorcev skupin gensko spremenjenih
semen in zrn - Splošne zahteve (ISO 22753:2021, popravljena verzija 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 (ISO 22753:2021, Corrected version 2022-11)
Untersuchung auf molekulare Biomarker - Verfahren zur statistischen Auswertung von
Analyseergebnissen aus der Untersuchung von Untergruppen von gentechnisch
verändertem Saatgut und Getreide - Allgemeine Anforderungen (ISO 22753:2021,
Korrigierte Fassung 2022-11)
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 (ISO 22753:2021, Version
corrigée 2022-11)
Ta slovenski standard je istoveten z: EN ISO 22753:2022
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN ISO 22753:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 22753:2023
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SIST EN ISO 22753:2023
EN ISO 22753
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2022
EUROPÄISCHE NORM
ICS 67.050
English Version
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 (ISO 22753:2021, Corrected version
2022-11)
Analyse moléculaire de biomarqueurs - Méthode pour Untersuchung auf molekulare Biomarker - Verfahren
l'évaluation statistique des résultats d'analyse obtenus zur statistischen Auswertung von Analyseergebnissen
lors des essais de sous-échantillons multiples de aus der Untersuchung von Untergruppen von
semences et de graines génétiquement modifiées - gentechnisch verändertem Saatgut und Getreide -
Exigences générales (ISO 22753:2021, Version corrigée Allgemeine Anforderungen (ISO 22753:2021,
2022-11) Korrigierte Fassung 2022-11)
This European Standard was approved by CEN on 14 November 2022.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 22753:2022 E
worldwide for CEN national Members.
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SIST EN ISO 22753:2023
EN ISO 22753:2022 (E)
Contents Page
European foreword . 3
Endorsement notice . 3
2
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SIST EN ISO 22753:2023
EN ISO 22753:2022 (E)
European foreword
The text of ISO 22753:2021, Corrected version 2022-11 has been prepared by Technical Committee
ISO/TC 34 "Food products” of the International Organization for Standardization (ISO) and has been
taken over as EN ISO 22753:2022 by Technical Committee CEN/TC 275 “Food analysis - Horizontal
methods” the secretariat of which is held by DIN.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2023, and conflicting national standards shall be
withdrawn at the latest by May 2023.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 22753:2021, Corrected version 2022-11 has been approved by CEN as
EN ISO 22753:2022 without any modification.
3
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SIST EN ISO 22753:2023
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SIST EN ISO 22753:2023
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
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SIST EN ISO 22753:2023
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: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
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SIST EN ISO 22753:2023
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
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SIST EN ISO 22753:2023
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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SIST EN ISO 22753:2023
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.
v
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SIST EN ISO 22753:2023
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SIST EN ISO 22753:2023
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
1
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SIST EN ISO 22753:2023
ISO 22753:2021(E)
3.2
AQL
A
QL
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.]
2
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SIST EN ISO 22753:2023
ISO 22753:2021(E)
3.10
LQL
L
QL
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.]
3
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SIST EN ISO 22753:2023
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
4
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SIST EN ISO 22753:2023
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
...
SLOVENSKI STANDARD
oSIST prEN ISO 22753:2022
01-oktober-2022
Analiza molekularnih biomarkerjev - Metoda za statistično vrednotenje rezultatov
analiz, pridobljenih pri preskušanju podvzorcev skupin gensko spremenjenih
semen in zrn - Splošne zahteve (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 (ISO 22753:2021)
Untersuchung auf molekulare Biomarker - Verfahren zur statistischen Auswertung von
Analyseergebnissen aus der Untersuchung von Untergruppen von gentechnisch
verändertem Saatgut und Getreide - Allgemeine Anforderungen (ISO 22753:2021)
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 (ISO 22753:2021)
Ta slovenski standard je istoveten z: prEN ISO 22753
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
oSIST prEN ISO 22753:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 22753:2022
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oSIST prEN ISO 22753:2022
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
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oSIST prEN ISO 22753:2022
ISO 22753:2021(E)
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oSIST prEN ISO 22753:2022
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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oSIST prEN ISO 22753:2022
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.
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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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oSIST prEN ISO 22753:2022
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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3.2
AQL
A
QL
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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3.10
LQL
L
QL
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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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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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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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/
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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
b
given in Formula (1):
m
pP=−11=− 1−p (1)
()
b
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):
c
n
ni−
i
P()a = pp()1− (2)
∑ b b
i
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;
1
— n , the numb
...
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