oSIST prEN ISO 20813:2022

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)

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.

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)

Analiza molekulskih biomarkerjev - Analitske metode za odkrivanje in prepoznavanje živalskih vrst v živilih in živilskih proizvodih (metoda, ki temelji na nukleinskih kislinah) - Splošne zahteve in definicije (ISO 20813:2019)

General Information

• Status: Not Published
• Public Enquiry End Date: 30-Oct-2022
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 4020 - Public enquire (PE) (Adopted Project)
• Start Date: 31-Aug-2022
• Due Date: 18-Jan-2023
• Ref Project: prEN ISO 20813 - 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)

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 20813:2022
01-oktober-2022
Analiza molekulskih biomarkerjev - Analitske metode za odkrivanje in

prepoznavanje živalskih vrst v živilih in živilskih proizvodih (metoda, ki temelji na

nukleinskih kislinah) - 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: prEN ISO 20813
ICS:
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
oSIST prEN ISO 20813:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 20813:2022
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
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oSIST prEN ISO 20813:2022
ISO 20813:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© 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: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved
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oSIST prEN ISO 20813:2022
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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

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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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oSIST prEN ISO 20813:2022
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.
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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;
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— 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

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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
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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;
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— 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 o
...