EN ISO 20813:2022

SLOVENSKI STANDARD
01-maj-2023
Analiza molekularnih biomarkerjev - Analitske metode za odkrivanje in
prepoznavanje živalskih vrst v živilih in živilskih proizvodih (metodana osnovi
nukleinskih kislin) - Splošne zahteve in definicije (ISO 20813:2019)
Molecular biomarker analysis - Methods of analysis for the detection and identification of
animal species in foods and food products (nucleic acid-based methods) - General
requirements and definitions (ISO 20813:2019)
Untersuchung auf molekulare Biomarker - Verfahren zur Identifizierung und zum
Nachweis von Tierarten in Lebensmitteln (Nukleinsäureverfahren) - Allgemeine
Anforderungen und Definitionen (ISO 20813:2019)
Analyse moléculaire de biomarqueurs - Méthodes d'analyse pour la détection et
l'identification des espèces animales dans les aliments et les produits alimentaires
(méthodes basées sur l'utilisation des acides nucléiques) - Exigences générales et
définitions (ISO 20813:2019)
Ta slovenski standard je istoveten z: EN ISO 20813:2022
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 20813
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2022
EUROPÄISCHE NORM
ICS 67.050
English Version
Molecular biomarker analysis - Methods of analysis for the
detection and identification of animal species in foods and
food products (nucleic acid-based methods) - General
requirements and definitions (ISO 20813:2019)
Analyse moléculaire de biomarqueurs - Méthodes Untersuchung auf molekulare Biomarker - Verfahren
d'analyse pour la détection et l'identification des zur Identifizierung und zum Nachweis von Tierarten in
espèces animales dans les aliments et les produits Lebensmitteln (Nukleinsäureverfahren) - Allgemeine
alimentaires (méthodes basées sur l'utilisation des Anforderungen und Definitionen (ISO 20813:2019)
acides nucléiques) - Exigences générales et définitions
(ISO 20813:2019)
This European Standard was approved by CEN on 18 December 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 20813:2022 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
The text of ISO 20813:2019 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 20813:2022
by Technical Committee CEN/TC 460 “Food Authenticity” 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 June 2023, and conflicting national standards shall be
withdrawn at the latest by June 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 20813:2019 has been approved by CEN as EN ISO 20813:2022 without any modification.

INTERNATIONAL ISO
STANDARD 20813
First edition
2019-05
Molecular biomarker analysis —
Methods of analysis for the detection
and identification of animal species
in foods and food products (nucleic
acid-based methods) — General
requirements and definitions
Analyse moléculaire de biomarqueurs — Méthodes d'analyse pour la
détection et l'identification des espèces animales dans les aliments et
les produits alimentaires (méthodes basées sur l'utilisation des acides
nucléiques) — Exigences générales et définitions
Reference number
ISO 20813:2019(E)
©
ISO 2019
ISO 20813:2019(E)
© ISO 2019
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
Fax: +41 22 749 09 47
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

ISO 20813:2019(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Performance characteristics of the methods . 2
4.1 General . 2
4.2 Scope of the method . 2
4.3 Scientific basis . 2
4.4 Units of measurement . . 2
4.5 Applicability . 2
4.6 Specificity . 3
4.6.1 General. 3
4.6.2 Requirements for inclusivity testing . 3
4.6.3 Requirements for exclusivity testing . 3
4.7 Sensitivity . 4
4.7.1 General. 4
4.7.2 Limit of detection (LOD) . 4
4.8 Specific requirements for quantitative methods . 5
4.8.1 General. 5
4.8.2 Limit of quantification (LOQ) . 5
4.8.3 Dynamic range . 5
4.8.4 Determination of precision and trueness for quantitative methods . 6
4.9 Robustness . 6
4.9.1 General. 6
4.9.2 Robustness determination by interlaboratory study . 6
4.9.3 Robustness determination by a multifactorial orthogonal test design . 6
5 Single-laboratory validation . 6
6 Interlaboratory study (collaborative study) . 7
6.1 General . 7
6.2 Qualitative methods. 7
6.3 Quantitative methods . 7
7 General laboratory and procedural requirements . 7
7.1 General . 7
7.2 Facilities, materials and equipment . 8
7.3 Sample preparation and DNA extraction . 8
7.4 Use of controls . 9
7.5 Data analysis . 9
7.5.1 Control . 9
7.5.2 Conventional PCR .10
7.5.3 Real-time PCR amplification curves .10
7.6 Expression of results .10
7.6.1 Expression of positive results .10
7.6.2 Expression of negative results .11
7.6.3 Expression of quantitative results .11
8 Test report .11
Annex A (informative) List of typical species used for inclusivity and exclusivity testing .12
Annex B (informative) Examples of unit conversion methods from DNA copy numbers to
the ratio of masses .17
Bibliography .26
ISO 20813:2019(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 2019 – All rights reserved

INTERNATIONAL STANDARD ISO 20813:2019(E)
Molecular biomarker analysis — Methods of analysis
for the detection and identification of animal species in
foods and food products (nucleic acid-based methods) —
General requirements and definitions
1 Scope
This document specifies minimum requirements of performance characteristics for the detection of
nucleic acid sequences (DNA) by molecular methods, such as the polymerase chain reaction (PCR),
including different post-PCR detection methods, real-time PCR, single and/or multiple probe-based
detection techniques as well as the combination of such methods.
The document is applicable to the detection, identification and quantification of DNA from animal
species of higher and lower taxonomic groups in foodstuffs, and the validation of applicable methods.
It is applicable to mammals, birds, reptiles, amphibians, fishes, molluscs, crustaceans and insects.
Typical examples for each are listed in Annex A.
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 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, ISO 24276 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 http: //www .electropedia .org/
3.1
basic local alignment search tool
BLAST
sequence comparison algorithm optimized for speed that is used to search sequence databases for
optimal local alignments to a query
Note 1 to entry: This algorithm directly approximates alignments that optimize a measure of local similarity, the
maximum signal pair (MST) score or high-scoring segment pair (HSP) score.
Note 2 to entry: See Reference [2].
Note 3 to entry: BLASTn is applicable to nucleotide sequence comparison.
ISO 20813:2019(E)
3.2
conventional polymerase chain reaction
conventional PCR
PCR method that requires a post-PCR step such as gel electrophoresis for detection or visualization of
amplification products to provide a qualitative result
4 Performance characteristics of the methods
4.1 General
The methods to be used for animal species analysis shall meet the performance characteristics in
accordance with this document. The results of all interlaboratory and/or single-laboratory validations
and the performance characteristics shall be described.
NOTE Some guidelines are available for implementation of methods, see Reference [10].
4.2 Scope of the method
Information regarding the intended use and the limitations of the methods shall be provided. In
particular, information shall indicate that the criteria set out in this document have been fulfilled.
4.3 Scientific basis
An overview of the principles and references to relevant scientific publications should be provided.
4.4 Units of measurement
Qualitative analyses indicate the presence or absence (lack of detection) of a certain target.
In quantitative analyses, the measured value is calculated as ratio of DNA copy numbers (c/c). The use
of this ratio should examine possible influences, including the number of DNA copies with regard to the
target in the genome. Other units (e.g. ratio of masses) can be employed. The principles of calculation of
the ratio shall be reported.
If a quantitative method is intended to judge the mass/mass ratio of different animal species ingredients
in a sample, it should be indicated that the values measured for the DNA copy number ratio cannot
reflect in all cases the mass/mass ratio of animal constituents in the sample.
4.5 Applicability
When assessing if a method is fit for purpose, the following aspects regarding the nature of the target
should be considered:
— the location of the target (nuclear or mitochondrial);
— the copy number per cell;
— the length of the target sequence.
For quantitative species-specific methods, a nuclear gene, excluding mitochondrial DNA shall be
targeted. The target sequence shall be present as a single copy per haploid genome or the copy number
shall be determined/known.
When assessing if a method is fit for purpose, the following aspects regarding the matrix should be
considered:
— the nature of the potential sample matrices;
— the degree of processing of the sample constituents;
2 © ISO 2019 – All rights reserved

ISO 20813:2019(E)
— the different species and animal tissue types involved;
— the preparation of the sample matrix.
The applicability of the method shall be tested by extracting DNA from test samples reflecting the
matrices and analytical scope.
DNA should be extracted from a minimum of three matrices of the most relevant types, including those
types reflecting the method scope, containing a known mass/mass content of the target(s) species
materials (evenly distributed over the percentage dynamic range of the method) and tissues relevant
for the application.
NOTE 1 Mitochondrial PCR targets cannot be used for reliable quantification of haploid genome copy number
ratios of different species, because the number of mitochondrial targets differs with tissue type.
NOTE 2 Different animal tissue types can have variable DNA contents per mass equivalent.
NOTE 3 The practical limit of detection (LOD) (see ISO 21569) can differ significantly for different matrices.
Furthermore, different processing grades of animal constituents in the same product will further contribute to
DNA degradation and a possible asymmetric DNA distribution between ingredients. For example, a product can
be composed of different types of animal tissue containing different amounts of DNA. This imbalance can be
further intensified if some ingredients underwent pre-processing, like cooking or acid treatment, lowering DNA
quality, whereas other ingredients were added for example raw or processed differently.
4.6 Specificity
4.6.1 General
The specificity should be assessed in a two-step procedure: theoretical and experimental evaluation of
the inclusivity and exclusivity.
In silico testing of the specificity of primers and probes with available bioinformatics tools shall be
performed.
[1] [2]
NOTE 1 Examples are testing primer-dimer formation with primer3 and BLAST searches in nucleic acid
sequence databases.
If sequence data are used for verification of animal speciation results, they should be based on
appropriate databases with due consideration of the timing of submission of individual entries and any
subsequent changes in taxonomic classification or naming.
NOTE 2 In cases of unexpected results, further investigation can be carried out with appropriate techniques,
such as sequencing, gel electrophoresis or hybridization techniques in order to confirm reference material
identity.
4.6.2 Requirements for inclusivity testing
Experimental results from testing the method with the target animal species should be provided.
This testing should include relevant breeds of the animal species according to the scope of the
method (see 4.2).
[6]
Material for experimental inclusivity testing should contain approximately 100 target DNA copies .
Each sample material shall be at a minimum tested in duplicate. Sequence variants of the target animal
species should be detected with comparable amplification efficiency, if they occur.
NOTE The target animal species for inclusivity testing are normally more than five breeds.
4.6.3 Requirements for exclusivity testing
Experimental results from testing the method with non-target animal species shall be provided. This
testing should include both taxonomically close and not closely related animal species. Animal species
ISO 20813:2019(E)
or taxonomic groups relevant with regard to the scope of the method shall be tested, e.g. species
commonly used in food in general and particularly in matrices considered in the scope of the method.
The method should clearly distinguish between target and non-target animal species.
[6]
Sufficient DNA should be used for experimental exclusivity testing. A number of 2 500 target copies
ensures that cross reactivity can be identified.
Select a minimum of 10 species that could cause interference with the target animal species present in
the food test material. Examples of suitable organisms are listed in Annex A.
Other species should be included if relevant, e.g. if there are sequence homologies of oligonucleotides to
nucleic acid sequences.
Cross-reactivity of matrix should be characterized.
The suitability of the DNA used for amplification should be confirmed by an amplification control, e.g.
by a single copy (chromosomal) DNA consensus PCR system (e.g. myostatin or actin).
4.7 Sensitivity
4.7.1 General
Experimental results from testing the method at different concentrations in order to test the range of
use of the method shall be available. They shall be described in the validation report.
If applicable, detailed information about how a cut-off value can be established and used in the
laboratory should be provided.
Animal species that require qualitative testing should be detected at levels relevant for the interested
party, e.g. the consumer.
4.7.2 Limit of detection (LOD)
4.7.2.1 Absolute LOD
The absolute LOD (LOD ) shall be indicated in copy numbers of the target sequence per reaction with
abs
the confidence level (typically 95 %) specified.
NOTE 1 Twenty copies or less can be applied for single copy genes and an appropriate number of haploid
genome equivalents for high copy number genes.
NOTE 2 If for the LOD determination a DNA with known copy number of target sequence is not available,
plasmid DNA can be used.
The LOD of the method is determined experimentally by preparing a dilution series of target material
abs
with dilutions in the range of the expected/targeted limit of detection. Guidance for assessment of the
LOD is described in Reference [6].
abs
4.7.2.2 Relative LOD
The relative LOD (LOD ) shall be determined in relevant non-target animal species DNA as
rel
background. Depending on test requirements, the LOD is adjusted to this value. The LOD expresses
rel rel
the relative c/c % of the target animal species DNA in other animal species DNA which is detected with
95 % confidence.
The LOD should be determined experimentally by preparing one or more defined reference samples
rel
with defined percentage content of the target DNA in the range of the limit of detection. Each reference
sample is analysed in at least 10 replicates. The percentage of the reference sample where at least 95 %
of the replicates give positive results is considered the LOD .
rel
4 © ISO 2019 – All rights reserved

ISO 20813:2019(E)
4.7.2.3 Asymmetric LOD (for multiplex methods only)
In the case of multiplex methods where the detection of different targets is restricted by competitive
effects, as in the case of multiplex real-time PCR methods, the LOD for the single targets in an
asymmetric target situation expressed as target ratio needs to be validated. Different contents of
the specific animal target sequence are mixed to obtain defined copy ratios (i.e. ratios of 1:1 000 and
1 000:1; 1:100 and 100:1). The ratio where each target animal is detected with 95 % confidence is
determined experimentally with an appropriate number of replicates for the defined reference sample.
4.8 Specific requirements for quantitative methods
4.8.1 General
The upper and lower limit of the linear range of the method shall be determined. The assessment of
these limits and the linear range shall be carried out on samples containing animal non-target DNA
relevant to the food item.
4.8.2 Limit of quantification (LOQ)
The absolute LOQ (LOQ ) shall be indicated as copy numbers of the target sequence. It shall be equal
abs
to the smallest amount included in the dynamic range.
The relative LOQ (LOQ ) shall be determined in DNA of other relevant animal species. Depending on
rel
the test requirements, the LOQ should be adjusted to this value. The LOQ expresses the ratio of the
rel rel
target animal species DNA copy number to other animal species DNA copies or to the DNA copies of a
reference gene representative for the whole taxonomic rank. The LOQ should be equal to the smallest
rel
concentration included in the dynamic range.
If, for the LOQ determination, a DNA with known copy number of target sequence is not available,
plasmid DNA should be used. This plasmid can also serve as a calibrator.
A minimum of 15 replicates with a target concentration of the expected LOQ shall be tested. The criteria
for precision and trueness shall be fulfilled for the results.
NOTE The LOQ values reported from collaborative study data generally refer to the lowest level of analyte
that was observed to have a relative reproducibility standard deviation of 25 % or less.
4.8.3 Dynamic range
The dynamic range should cover the percentage values as well as the copy numbers according to the
expected use and scope of the method.
In order to define the relevant minimum copy number, the desired dynamic range in terms of target
copy percentages shall be determined. It should be considered that the genome size of the species in the
expected sample material restricts the maximum copy number that can be used for the analysis (e.g.
100 ng to 200 ng, depending on the method).
NOTE 1 For example, for cattle, a genome size of 4 pg can be assumed, which results in a maximum copy
[18][22]
number of 25 000 in 100 ng of sample DNA material. See Table B.2 .
The copy numbers of the dynamic range for both, target and reference sequence, shall be then
determined as follows:
— for the reference sequence, the maximum number of copies can be calculated considering genome
sizes and amount of sample DNA used for analysis as described above;
— for the target, the lowest copy number should be the absolute LOQ; as a prerequisite, the lowest
possible value considering the ratio compared to the maximum number of copies of total/reference
DNA should be taken into consideration;
ISO 20813:2019(E)
— the minimum copy number of the reference sequence and the maximum copy number for the target
sequence should be given by the ratio of the minimum and maximum, respectively, percentage value.
NOTE 2 The dynamic range is established on the basis of a standard curve with a minimum of four
concentration levels evenly distributed at least in duplicate.
NOTE 3 For a desired upper limit of the percentage dynamic range of 100 %, the minimum copy number of the
reference can be equal to the lower limit of the copy number range of the target sequence, and for a desired LOQ
of 0,1 % at an absolute LOQ of 30 copies, the upper limit of the reference target is 30,000 copies.
4.8.4 Determination of precision and trueness for quantitative methods
The precision should be determined and expressed as relative repeatability standard deviation (S ).
r
A sufficient number of replicates (at least 15) for at least three DNA materials with different target
percentages covering the whole dynamic range should be analysed.
NOTE Mitochondrial DNA cannot be used for the targets of quantitative methods.
The S for all replicates shall be ≤ 25 % over the whole dynamic range of the method.
r
The trueness shall be within 25 % of the accepted reference value for all replicates over the whole
dynamic range of the method.
4.9 Robustness
4.9.1 General
Results from the empirical testing of the method against small but deliberate variations in method
parameters (e.g. variation in concentration of kit components, variation in apparatus) should be
provided, if available.
4.9.2 Robustness determination by interlaboratory study
An interlaboratory study introduces a deliberate change in the laboratory performing the method and
meets the criteria for an evaluation of robustness. Empirically, a robust method shall be selected by
considering that the results from different laboratories do not vary significantly.
4.9.3 Robustness determination by a multifactorial orthogonal test design
The test should be carried out in a multifactorial approach where several alterations, including, but
not limited to, mastermix concentration, reaction volume, primer and probe concentration, annealing
temperature and thermocycler platform are assessed.
NOTE 1 A detailed procedure is described in Reference [6].
For qualitative methods, at least three replicates should be tested. The target animal species copy number
used in the test should be in a concentration threefold the LOD (95 % confidence) of the method.
abs
For quantitative methods, three defined target concentrations over the whole dynamic range of the
method should be tested in three replicates each.
NOTE 2 The method is considered to be robust if all reactions give the expected results.
5 Single-laboratory validation
An analytical method should have been sufficiently tested within a laboratory to disclose the required
specification prior to the interlaboratory study, see ISO 13495.
6 © ISO 2019 – All rights reserved

ISO 20813:2019(E)
Reference materials or certified reference materials (CRMs) should be considered for use in the
validation of detection and quantification methods of nucleic acids.
6 Interlaboratory study (collaborative study)
6.1 General
Information about the collaborative study (organizer, protocol, number of participating laboratories,
etc.) and the performance data obtained by the study shall be reported with appropriate references
to the relevant documents. Collaborative studies for the validation of PCR methods for detection,
identification and quantification of specific DNA sequences can be performed according to other
[7]
relevant documents (e.g. Codex Alimentarius CAC/GL 74-2010 ).
NOTE A small-scale collaborative study (pre-validation study involving, for example, two to four
laboratories) can be performed to test the general transferability of the method before the expense of organizing
a large-scale study is incurred.
For validation of the precision of detection and identification methods, data are collected from multiple
laboratories having facilities and proficiency in molecular biology testing. In ISO 13495:2013, the
required number of laboratories is eight and four for the international and national levels of validation,
[23]
respectively. According to AOAC International (2002) , the required number is eight laboratories.
Statistical analysis is calculated based on ISO 5725-1:1994, 6.3.
6.2 Qualitative methods
A collaborative validation study of a qualitative PCR method shall be designed by considering the
probability of detection (POD) (see ISO/TS 16393) within the range of the method.
NOTE Traditional nonparametric 5 % false positive and 5 % false negative rates reflect PODs of 5 % and 95 %.
6.3 Quantitative methods
The relative reproducibility standard deviation (S ) should be ≤ 25 % over the whole dynamic range of
R
the method.
NOTE At levels of 0,1 % (copy/copy) an S of 50 % can be acceptable.
R
7 General laboratory and procedural requirements
7.1 General
The procedure shall be documented to include the following steps:
— sample representability shall be addressed;
— preparation of the test sample (optional: if the test sample is not the whole laboratory sample,
homogenize the laboratory sample and obtain test samples in accordance with the relevant
International Standards);
— grinding and homogenization of the test sample;
— preparation of test portions;
— extraction of DNA;
— testing, interpretation and reporting of the results.
Manufacturers' safety recommendations shall be followed.
ISO 20813:2019(E)
7.2 Facilities, materials and equipment
The work area in the laboratory should be designed for preventing accidental DNA contamination
originating from, for example, dust, human material and spreading aerosols, including consideration of:
— systematic containment of the methodological steps involved in the production of the results;
— a forward flow principle for sample handling.
For DNA-based methods, separation (temporal and/or physical) of work is required to prevent
contamination. Designated contained/dedicated work areas with their own apparatus are
recommended, as follows:
a) a work area for grinding and homogenization;
b) a work area for extraction of the nucleic acid from the test material;
c) a work area dedicated to the setup of PCR/amplification reactions;
d) a work area dedicated to subsequent processing, including analysis and characterization of the
amplified DNA sequences, if applicable.
If human DNA is detected by the method, appropriate contamination prevention measures (e.g. use
of masks, gloves and disposable coats) should be taken in order to prevent false-positive results by
contamination with the operator’s or other human DNA during analysis.
Physical separation through the use of different rooms is the most effective and preferable way of
ensuring separate work areas, but other methods can be used as a protection against contamination,
provided their effectiveness is comparable. The air flow system should be set up and directed in a way
that prevents intrusion of dust/amplicons from work areas with higher contamination risk to work
areas with lower contamination risk.
For the analysis, unless otherwise stated, only analytical grade reagents suitable for molecular
biology, free from DNA and DNases should be used. Reagents and solutions should be stored at room
temperature, unless otherwise specified. PCR reagents should be stored in small aliquots to minimize
the risk of contamination. The water used shall be double-distilled, deionized or of comparable
quality. Solutions should be prepared by dissolving the appropriate reagents in water and autoclaved,
unless specified differently. Sterile filtration devices (possibly 0,22 μm pore size) may be used when
autoclaving is not possible.
In order to avoid contamination, sterile technique should be adopted in the PCR set-up area, e.g. powder-
free gloves, sterilized plastic ware, autoclaved reagents, disposable plasticware and aerosol-protected,
DNA/RNA free and DNase/RNase free filtered pipette tips should be used.
Materials and all containers and disposables containing reagents shall be preserved from any
contaminating agent (e.g. dust).
Manufacturers’ recommendations for the use of reagents should be followed. Appropriate controls can
be used to assess the integrity of reagents and the absence of DNase.
No unintended enzyme activities (e.g. exonuclease) that might interfere with PCR shall be present in the
preparation. The reaction buffer shall be suitable for the polymerase used.
7.3 Sample preparation and DNA extraction
A representative sample should be tested. It shall be ensured that the test samples used for DNA
extraction are representative of the laboratory sample, such as by homogenizing the sample or
appropriate portions thereof. At least two aliquots should be taken from the homogenized laboratory
sample as test portions for DNA extraction and subsequent analysis.
If possible, the sample material should not be taken from the surface of the laboratory sample in order
to minimize the risk of the amplification of adhering contaminants.
8 © ISO 2019 – All rights reserved

ISO 20813:2019(E)
If the analytical method to be used for the sample detects human DNA, special contamination prevention
measures should be taken.
Concerning the preparation of DNA from the test portion, the general instructions and measures
described in ISO 21571 should be followed. One of the DNA extraction methods described in
ISO 21571:2005, Annex A, should be considered. Alternatively, commercial kits can be used for the
extraction and purification of DNA.
7.4 Use of controls
Controls should be carried out according to Table 1 (see also ISO 24276).
Negative DNA target control should be prepared from DNA extracted from non-target species prevalent
in the sample (e.g. for a horse assay in cattle meat, the non-target species is cattle).
Table 1 — Flow diagram showing intersection of successive steps and inclusion of controls
Control step Environment Extraction Positive Positive Negative DNA PCR reagent PCR inhibition
b c f g h
control blank control extraction DNA target target control control control
d e
control control
Homogenization Recommended — — — — — —
Nucleic acid Mandatory at
a
↓ One per series — — — —
extraction regular intervals
Assessment
of nucleic acid ↓ ↓ ↓ — — — —
quality
Recommended,
Nucleic acid
↓ ↓ ↓ Mandatory Recommended Mandatory but mandatory in
amplification
i
certain cases
Assessment
of results of
↓ ↓ ↓ ↓ ↓ ↓ ↓
nucleic acid
amplification
Interpretation — ↓ ↓ ↓ ↓ ↓ ↓
Test report — ↓ ↓ ↓ ↓ ↓ ↓
a
The arrows indicate that this control should be applie
...