SIST EN ISO 24276:2006

SLOVENSKI STANDARD
SIST EN ISO 24276:2006
01-april-2006
Živila – Analitske metode za odkrivanje gensko spremenjenih organizmov in
njihovih produktov – Splošne zahteve in definicije (ISO 24276:2006)
Foodstuffs - Methods of analysis for the detection of genetically modified organisms and
derived products - General requirements and definitions (ISO 24276:2006)
Lebensmittel - Verfahren zum Nachweis von gentechnisch modifizierten Organismen und
ihren Produkten - Allgemeine Anforderungen und Definitionen (ISO 24276:2006)
Produits alimentaires - Méthodes d'analyse pour la détection des organismes
génétiquement modifiés et des produits dérivés - Exigences générales et définitions (ISO
24276:2006)
Ta slovenski standard je istoveten z: EN ISO 24276:2006
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
SIST EN ISO 24276:2006 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 24276:2006

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SIST EN ISO 24276:2006
EUROPEAN STANDARD
EN ISO 24276
NORME EUROPÉENNE
EUROPÄISCHE NORM
February 2006
ICS 67.050

English Version
Foodstuffs - Methods of analysis for the detection of genetically
modified organisms and derived products - General
requirements and definitions (ISO 24276:2006)
Produits alimentaires - Méthodes d'analyse pour la Lebensmittel - Verfahren zum Nachweis von gentechnisch
détection des organismes génétiquement modifiés et des modifizierten Organismen und ihren Produkten -
produits dérivés - Exigences générales et définitions (ISO Allgemeine Anforderungen und Definitionen (ISO
24276:2006) 24276:2006)
This European Standard was approved by CEN on 23 January 2006.
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 Central Secretariat 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 Central Secretariat has the same status as the official
versions.
CEN members are the national standards bodies of Austria, Belgium, Cyprus, Czech Republic, Denmark, Estonia, Finland, France,
Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania,
Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: rue de Stassart, 36  B-1050 Brussels
© 2006 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 24276:2006: E
worldwide for CEN national Members.

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SIST EN ISO 24276:2006

EN ISO 24276:2006 (E)





Foreword


This document (EN ISO 24276:2006) has been prepared by Technical Committee CEN/TC 275
"Food analysis - Horizontal methods", the secretariat of which is held by DIN, in collaboration
with Technical Committee ISO/TC 34 "Agricultural food products".

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 August 2006, and conflicting national
standards shall be withdrawn at the latest by August 2006.

According to the CEN/CENELEC Internal Regulations, the national standards organizations of
the following countries are bound to implement this European Standard: Austria, Belgium,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary,
Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.


2

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SIST EN ISO 24276:2006

INTERNATIONAL ISO
STANDARD 24276
First edition
2006-02-01


Foodstuffs — Methods of analysis for
the detection of genetically modified
organisms and derived products —
General requirements and definitions
Produits alimentaires — Méthodes d'analyse pour la détection des
organismes génétiquement modifiés et des produits dérivés —
Exigences générales et définitions





Reference number
ISO 24276:2006(E)
©
ISO 2006

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SIST EN ISO 24276:2006
ISO 24276:2006(E)
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ii © ISO 2006 – All rights reserved

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SIST EN ISO 24276:2006
ISO 24276:2006(E)
Contents Page
Foreword. iv
Introduction . v
1 Scope. 1
2 Normative references. 1
3 Terms and definitions. 1
3.1 General definitions . 1
3.2 Terms relative to extraction and purification of DNA . 5
3.3 Terms referring to DNA amplification and PCR. 5
3.4 Definitions referring to DNA and PCR controls. 5
3.5 Terms relative to reference materials. 7
3.6 Terms relative to quantitation . 7
3.7 Terms relative to GMOs . 7
4 Application to the relevant International Standards . 7
4.1 General. 7
4.2 Guidance for the user on the selection of methods. 8
4.3 Performance characteristics. 9
5 General laboratory and procedural requirements. 10
5.1 General. 10
5.2 Use of controls. 10
5.3 Laboratory organization. 12
6 Interpretation and expression of results. 13
6.1 General. 13
6.2 Interpretation of controls . 13
6.3 Expression of a negative result. 14
6.4 Expression of a positive result. 14
6.5 Expression of ambiguous results . 14
6.6 Quality assurance requirements. 15
7 Test report. 15
Bibliography . 16

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SIST EN ISO 24276:2006
ISO 24276:2006(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
ISO 24276 was prepared by the European Committee for Standardization (CEN) Technical Committee
CEN/TC 275, Food analysis — Horizontal methods, in collaboration with Technical Committee ISO/TC 34,
Food products, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna
Agreement).
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SIST EN ISO 24276:2006
ISO 24276:2006(E)
Introduction
The purpose of such an analysis is to identify and quantify genetic elements or proteins common to genetically
modified organisms (GMOs) and their derived products in a given matrix.
The main focus of this International Standard is polymerase chain reaction (PCR) based methodologies.
However, because of the rapid rate of technological change in this area, other technologies may be
considered in the future.
The search for ingredients of genetically modified origin is performed by means of the following successive (or
simultaneous) steps. After sample collection, nucleic acids or proteins are extracted from the test portion.
Extracted analytes 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 PCR or
Enzyme-Linked Immunosorbent Assay (ELISA). These steps are detailed in this International Standard and in
the following documents:
EN/TS 21568, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — Sampling strategies
ISO 21569, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Qualitative nucleic acid based methods
ISO 21570, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Quantitative nucleic acid based methods
ISO 21571, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Nucleic acid extraction
ISO 21572, Foodstuffs — Methods for the detection of genetically modified organisms and derived
products — Protein based methods
Specific information pertaining to protein detection methods is found in ISO 21572.

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SIST EN ISO 24276:2006

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SIST EN ISO 24276:2006
INTERNATIONAL STANDARD ISO 24276:2006(E)

Foodstuffs — Methods of analysis for the detection of
genetically modified organisms and derived products —
General requirements and definitions
1 Scope
This International Standard specifies how to use the standards for sampling strategies (EN/TS 21568), nucleic
acid extraction (ISO 21571), qualitative nucleic acid analysis (ISO 21569), quantitative nucleic acid analysis
(ISO 21570) and protein-based methods (ISO 21572), and explains their relationship in the analysis of
genetically modified organisms in foodstuffs.
It contains general definitions, requirements and guidelines for laboratory set-up, method validation
requirements, description of methods and test reports.
It has been established for food matrices, but could also be applied to other matrices (e.g. seeds, feed and
plant samples from the environment).
2 Normative references
The following referenced documents are indispensable for the application 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 5725-1, Accuracy (trueness and precision) of measurement methods and results — Part 1: General
principles and definitions
3 Terms and definitions
For the purpose of this document, the terms and definitions given in ISO 5725-1 concerning validation, those
in Reference [1] and the following apply.
3.1 General definitions
3.1.1
target taxon
taxon to which the genetically modified organism belongs
NOTE In this context, taxon usually means species but it could be of lower or higher taxonomic rank.
3.1.2
laboratory sample
sample as prepared for sending to the laboratory and intended for inspection or testing
[ISO 7002:1986]
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SIST EN ISO 24276:2006
ISO 24276:2006(E)
3.1.3
test sample
test portion
sample, as prepared for testing or analysis, the whole quantity being used for analyte extraction at one time
3.1.4
specificity
property of a method to respond exclusively to the characteristic or analyte under investigation
3.1.5
sensitivity
change in the response divided by the corresponding change in the concentration of a standard (calibration)
curve
NOTE This is the slope of the analytical calibration curve.
3.1.6
limit of detection
LOD
minimum amount or concentration of the analyte in a test sample which can be detected reliably but not
necessarily quantified, as demonstrated by a collaborative trial or other appropriate validation
NOTE See Reference [2] for collaborative trial and Reference [3] for validation.
3.1.7
limit of quantitation
LOQ
〈analytical procedure〉 lowest concentration or amount of the analyte in a test sample which can be
quantitatively determined with an acceptable level of precision and accuracy, as demonstrated by a
collaborative trial or other appropriate validation

NOTE See Reference [2] for collaborative trial and Reference [3] for validation.
3.1.8
accuracy
closeness of agreement between a test result and the accepted reference value
3.1.9
trueness
closeness of agreement between the average value obtained from a large series of test results and an
accepted reference value
NOTE The measure of trueness is usually expressed in terms of bias. Trueness has been referred to as “accuracy of
the mean”.
3.1.10
precision
closeness of agreement between independent test results obtained under stipulated conditions
NOTE 1 Precision depends only on the distribution of random errors and does not relate to the true value or to the
specified value.
NOTE 2 The measure of precision usually is expressed in terms of imprecision and computed as a standard deviation
of the test results. Lower precision is reflected by a larger standard deviation.
NOTE 3 “Independent test results” means results obtained in a manner not influenced by any previous result on the
same or similar test object. Quantitative measures of precision depend critically on the stipulated conditions. Repeatability
and reproducibility conditions are particular sets of extreme conditions.
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SIST EN ISO 24276:2006
ISO 24276:2006(E)
3.1.11
repeatability
precision under repeatability conditions
3.1.12
reproducibility
precision under reproducibility conditions
3.1.13
repeatability conditions
conditions where independent test results are obtained with the same method on identical test items in the
same laboratory by the same operator using the same equipment within short intervals of time
3.1.14
reproducibility conditions
conditions where test results are obtained with the same method on identical test items in different
laboratories with different operators using different equipment
NOTE When different methods give test results that do not differ significantly, or when different methods are
permitted by the design of the experiment (as in a proficiency study or a material-certification study for the establishment
of a consensus value of a reference material), the term “reproducibility” may be applied to the resulting parameters. The
conditions must be explicitly stated.
3.1.15
repeatability standard deviation
standard deviation of test results obtained under repeatability conditions
NOTE Repeatability standard deviation is a measure of the dispersion of the distribution of test results under
repeatability conditions. Similarly “repeatability variance” and “repeatability coefficient of variation” could be defined and
used as measures of the dispersion of test results under repeatability conditions.
3.1.16
reproducibility standard deviation
the standard deviation of test results obtained under reproducibility conditions
NOTE Reproducibility standard deviation is a measure of the dispersion of the distribution of test results under
repeatability conditions. Similarly “reproducibility variance” and “reproducibility coefficient of variation” could be defined
and used as measures of the dispersion of test results under reproducibility conditions.
3.1.17
repeatability limit
value less than or equal to which the absolute difference between two test results obtained under repeatability
conditions may be expected to be with a probability of 95 %
NOTE 1 The symbol used is r.
NOTE 2 When examining two single test results obtained under repeatability conditions, the comparison should be
made with the repeatability limit r = 2,8 s .

r
3.1.18
reproducibility limit
value less than or equal to which the absolute difference between two test results obtained under
reproducibility conditions may be expected to be with a probability of 95 %
NOTE 1 The symbol used is R.
NOTE 2 When examining two single test results obtained under reproducibility conditions, the comparison should be
made with the reproducibility limit R = 2,8 s .
R
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SIST EN ISO 24276:2006
ISO 24276:2006(E)
3.1.19
collaborative trial
interlaboratory study
study in which several laboratories detect and/or determine an analyte in one or more “identical” portions of
homogeneous, stable materials under documented conditions
NOTE Guidelines for performing collaborative trials are elaborated in ISO 5725-2 and ISO/AOAC/IUPAC harmonized
[6]
protocol .
3.1.20
fitness for purpose
applicability
scope of application of the method which identifies the matrix, analyte or species being measured, its
concentration range and the type of study/monitoring effort for which the procedure, as judged from its
performance characteristics, is suited
[3]
NOTE It also describes the known limitations of the method.
3.1.21
practicability
ease of operations, in terms of sample throughput and costs, to achieve the required performance criteria and
thereby meet the specified purpose
3.1.22
applicability range
range of quantitation/linearity/dynamic range
quantity interval within which the analytical procedure has been demonstrated by a collaborative trial or other
appropriate validation to have a suitable level of precision and accuracy

NOTE See Reference [2] for collaborative trial and Reference [3] for validation.
3.1.23
measurement uncertainty
parameter associated with the result of a measurement, which characterizes the dispersion of the values that
could reasonably be attributed to the analyte
3.1.24
screening method
method that will rapidly and reliably eliminate (screen) a large number of negative (or positive) test samples
and restrict the number of test samples requiring the application of a rigorous method

NOTE 1 See Reference [4].
NOTE 2 In this International Standard, a screening method is a method to detect gene products (such as proteins)
and/or genetic elements common to several GMOs (such as promoters, terminators, or other genetic elements of interest).
3.1.25
construct-specific method
method that targets a combination of inserted DNA sequences that are only found in GMO-derived material
3.1.26
event-specific method
method that detects a specific sequence that is only present in that event
NOTE This is commonly targeted at the integration-border region.
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SIST EN ISO 24276:2006
ISO 24276:2006(E)
3.2 Terms relative to extraction and purification of DNA
3.2.1
DNA extraction
separation of DNA from the other components in a test sample
3.2.2
DNA purification
method resulting in a more purified DNA
NOTE In this context, purity refers to the reduction of observable and measurable effects of PCR inhibitors.
3.2.3
PCR quality DNA
DNA template of sufficient length, chemical purity and structural integrity to be amplified by PCR.
3.3 Terms referring to DNA amplification and PCR
3.3.1
identification of nucleic acid sequences
identity of nucleic acid sequences
establishment of identity by comparison with a reference nucleic acid fragment/sequence
NOTE For example, specific hybridization with a probe, matching restriction digest profiles or matching nucleic acid
sequences.
3.3.2
junction region
DNA sequence encompassing two consecutive sequence elements, such as a promoter and the coding
region of a gene
3.3.3
integration-border region
junction region where one element originates from the host organism and the other originates from the DNA
introduced during transformation
3.3.4
taxon-specific (endogenous) target sequence
sequence known to be specific for the target taxon
NOTE 1 That is consistently present in the target taxon and absent in other taxa.
NOTE 2 There are at least two types of target taxon-specific sequences:
⎯ variable number or multicopy sequences that can be used, for example, to assess the presence of nucleic acid from
the target taxon;
⎯ low copy number or single copy sequences that can also be used, for example, as a reference sequence to establish
the background of target taxon genome equivalents in a quantitative analysis.
3.3.5
forward flow
principle of material/sample handling applied to ensure that the laboratory sample, raw and processed test
portion (including amplified DNA) remain physically segregated during the whole procedure
3.4 Definitions referring to DNA and PCR controls
NOTE Controls applicable to protein-based methods are described in ISO 21572. The following definitions apply to
DNA-based methods.
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SIST EN ISO 24276:2006
ISO 24276:2006(E)
3.4.1
positive DNA target control
reference DNA, or DNA extracted from a certified reference material, or known positive sample representative
of the sequence or organism under study
NOTE This control is used to demonstrate that the PCR reagents are working as intended.
3.4.2
negative DNA target control
reference DNA, or DNA extracted from a certified reference material, or known negative sample not containing
the sequence under study
NOTE This control demonstrates that the results of analyses of test samples not containing the target sequence will
be negative.
3.4.3
PCR inhibition control
reaction mixture that provides the means to monitor PCR inhibition of the assay for the specific sample of the
target analyte
NOTE 1 This control allows the determination of the presence of soluble PCR inhibitors, which is particularly necessary
in the case of negative amplification and of quantitative PCR.
NOTE 2 Generally, a known amount of target DNA is added to the reaction that is to be controlled. This could be the
original target or a spike, for example a slightly modified target such as a competitor plasmid.
3.4.4
PCR reagent control
control containing all the amplification reagents except the extracted test sample template DNA
NOTE This control is used to demonstrate the absence of contaminating nucleic acids in the reagents. Instead of the
template DNA, for example, a corresponding volume of nucleic acid free water is added to the reaction.
3.4.5
extraction blank control
control generated by performing all steps of the extraction procedure except the addition of the test portion
NOTE 1 For example by substitution of water for the test portion.
NOTE 2 This control is used to demonstrate the absence of contaminating nucleic acid during extraction.
3.4.6
positive extraction control
control used to demonstrate that the DNA extraction procedure has been performed in a way that will allow for extraction
of a target DNA
NOTE For example by using a sample material known to contain the target DNA.
3.4.7
environment control
control used to determine that there is no nucleic acid contamination from, for example, the air in the laboratory
NOTE The control is a tube containing a suitable volume of nucleic acid free water that is left open to the air
throughout the entire process.
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SIST EN ISO 24276:2006
ISO 24276:2006(E)
3.5 Terms relative to reference materials
3.5.1
reference material
material or substance, one or more of whose property values are sufficiently homogeneous and well
established to be used for the calibration of an apparatus, the assessment of a measurement method, or for
assigning values to materials
[ISO Guide 30]
3.5.2
certified reference material
reference material accompanied by a certificate, one or more of whose property values are certified by a
technically valid procedure, accompanied by or traceable to a certificate or other documentation which is
issued by a certifying body
[ISO Guide 30]
3.6 Terms relative to quantitation
NOTE Controls applicable to protein-based methods are described in ISO 21572. The following definitions apply to
DNA-based methods.
3.6.1
endogenous DNA sequence
defined reference DNA sequence native to the corresponding taxon
NOTE The endogenous DNA sequence can be used to determine the quantity of genome equivalents of the target
taxon if the sequence is present in a constant copy number and does not show allelic variation among cultivars of the
target tax
...