ISO/FDIS 21569-1

ISO/DISFDIS 21569-1
ISO/TC 34/SC 16
Secretariat: ANSI
Date: 2026-08-1210-14
Horizontal methods for molecular biomarker analysis — Methods of
analysis for the detection of genetically modified organisms and
derived products —
Part 1:
Qualitative nucleic acid based methods
Méthodes horizontales d’analyse de biomarqueurs moléculaires — Méthodes d’analyse pour la détection des
organismes génétiquement modifiés et des produits dérivés —
Partie 1: Méthodes qualitatives basées sur l’utilisation des acides nucléiques
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ii
Contents
Foreword . iv
Introduction . v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 General. 2
4.2 PCR amplification . 2
4.3 Detection and confirmation of PCR products . 2
5 Reagents . 2
6 Apparatus and equipment . 3
7 Procedure . 3
7.1 General. 3
7.2 Quality, integrity and amplifiability of nucleic acid extracts . 3
7.3 Performance criteria . 4
7.4 Aspects of PCR design . 4
7.5 PCR target descriptions . 6
7.6 Controls . 6
7.7 PCR set-up, detection and confirmation of PCR products . 6
8 Interpretation . 6
8.1 General. 6
8.2 Verification . 7
9 Expression of results and quality assurance . 7
9.1 General. 7
9.2 Expression of a negative result . 7
9.3 Expression of a positive result . 8
9.4 Quality assurance requirements . 8
10 Test report . 8
Bibliography . 9

iii
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
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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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
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This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 16,
Horizontal methods for molecular biomarker analysis, in collaboration with the European Committee for
Standardization (CEN) Technical Committee CEN/TC 275, Food analysis - Horizontal methods, in accordance
with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
This secondfirst edition cancels and replaces the first edition (ISO 21569:2005),, which has been technically
revised. It also incorporates the Amendment ISO 21569:2005/Amd 1:2013.
The main changes are as follows:
— Removed allthe annexes have been moved from this document and published them as separate new
document to ISO/TS 21569-11; (under preparation);
— the text has been updated to reflect the latest state of the art (i.e. inclusion of real-time PCR);
— Clause 9clarified on the expression of results and quality assurance clause has been clarified with specific
requirements;
— editorially revised.
A list of all parts in the ISO 21569 series can be found on the ISO website.
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
Introduction
The search for a genetically modified origin of ingredients is performed by means of the following successive
(or simultaneous) steps. :
— After sample collection, nucleic acids are extracted from the test portion.
— Extracted nucleic acids can be further purified, simultaneously or after the extraction process.
— Afterwards, they are quantified (if necessary), diluted (if necessary) and subjected to analytical
procedures (such as polymerase chain reaction (PCR)).
[1] [2]
These steps are detailed in this document and in ISO 21570 and ISO 21571 .
Further information about general requirements and definitions involving the steps cited above are collected
[3]
in ISO 24276 .
The qualitative detection of DNA target sequences is performed in order to obtain a yes or no answer to the
question of whether a certain target sequence is detected or not relative to appropriate controls and within
the detection limits of the analytical method used and test portion analysed.
[4]
The specificity of the methods, as described in ISO/TS 21569-11 (under preparation) and the other parts
of this series, ranges from screening methods to detect common DNA sequences characteristic of genetically
modified organisms (GMOs),) to specific identification of a genetic construct or a specific transformation
event.
v
Horizontal methods for molecular biomarker analysis — Methods of
analysis for the detection of genetically modified organisms and
derived products —
Part 1:
Qualitative nucleic acid based methods
1 Scope
This document specifies a procedure to qualitatively detect genetically modified organisms (GMOs) and
derived products by analysing the nucleic acids extracted from the sample under study. The main focus is on
polymerase chain reaction (PCR)-based amplification methods.
It specifies general requirements for the specific detection and identification of target nucleic acid sequences
(DNA) and for the confirmation of the identity of the amplified DNA sequence.
Guidance, minimum requirements and performance criteria given in this document are intended to ensure
that comparable, accurate and reproducible results are obtained in different laboratories.
This document is applicable to food matrices, but it. It is also applicable to other matrices (e.g. feed and plant
samples from the environment).
[4]
Specific examples of methods are provided in ISO/TS 21569-11 (under preparation) and the other parts of
this series.
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 21571, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Nucleic acid extraction
ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — General requirements and definitions
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 24276 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/
4 Principle
4.1 General
Qualitative analysis consists of specific detection of target nucleic acid sequences in the test samples. Each
method shall specify the target sequence.
A qualitative result shall clearly demonstrate the presence or absence of the genetic element under study,
relative to appropriate controls.
NOTE Detection limits and size of the test portion are critical aspects of a method.
PCR methods for qualitative analysis are conventional PCR and real-time PCR. Conventional PCR monitors the
amplification of a targeted DNA at its end and requires a post-PCR analysis step. In real-time PCR, the
amplification is monitored during the PCR, either by using a sequence-specific probe consitingconsisting of an
fluorescent-labelled oligonucleotide or by using non-specific fluorescent dyes that intercalate with any
double-stranded DNA.
4.2 PCR amplification
Amplification of the target sequence occurs in vitro through a reaction catalysed by a DNA polymerase in the
[5] [6]
presence of oligonucleotide primers and deoxyribonucleoside triphosphates in a defined reaction buffer.
An important prerequisite for the amplification of the target sequence is that the reaction mixture contains no
polymerase inhibitors. Amplification of the DNA is a cyclical process consisting of:
— denaturation of the double-stranded DNA into single-stranded nucleic acid by means of heating;
— annealing of the primers to the target sequence at a suitable temperature;
— extension of the primers, which are bound to both single strands, by a DNA polymerase suitable for PCR,
at an appropriate temperature.
4.3 Detection and confirmation of PCR products
Conventional PCR products are detected by gel electrophoresis or an appropriate alternative, if necessary,
after isolation by means of a suitable separation procedure. The identity of any detected target sequence
should be verified by an appropriate technique (e.g. by DNA sequence analysis, by hybridization or by
restriction enzyme analysis).
In the case of real-time PCR analysis, amplification and detection occur simultaneously.
5 Reagents
The reaction solutions required for the analytical method should be stored at approximately –20 °C if not
specified otherwise.
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, and/or to reduce chances of cross contamination.
5.1 Target DNA/control.
5.2 Water.
5.3 Deoxyribonucleoside triphosphate (dNTP) solution, containing dATP, dCTP, dGTP, and dTTP /
/dUTP.
NOTE The use of dUTP can interfere with restriction enzyme analyses of PCR products.
5.4 PCR buffer solution.
The PCR buffer solution is usually delivered with the DNA polymerase, which can, but does not always, include
MgCl in a concentration specified by the manufacturer. The final MgCl concentrations are method specific
2 2
and are therefore listed in the method. Ready-to-use reagents can be commercially available. The
manufacturer’s instructions for use should be considered.
5.5 Thermostable DNA polymerase.
5.6 Forward primer.
5.7 Reverse primer.
5.8 Fluorescent labelled oligonucleotide probe or intercalating fluorescent dye in the case of real-
time PCR.
6 Apparatus and equipment
The information on apparatus and equipment shall be given in the specific method.
7 Procedure
7.1 General
For the basic principl
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