ISO/TS 20224-6:2020
(Main)Molecular biomarker analysis — Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR — Part 6: Horse DNA detection method
Molecular biomarker analysis — Detection of animal-derived materials in foodstuffs and feedstuffs by real-time PCR — Part 6: Horse DNA detection method
This document specifies a real-time polymerase chain reaction (real-time PCR) method for the qualitative detection of horse-specific DNA derived from food and feed. It requires the extraction of an adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection of horse material derived from domestic horse (Equus caballus), mule (Equus caballus ♀ × Equus asinus ♂), hinny (Equus caballus ♂ × Equus asinus ♀) and zebroid (Equus caballus × Equus simplicidens). The assay also detects the species Przewalski's horse (Equus przewalskii) and zebra (Equus burchellii). The target sequence is an Equus caballus isolate Twilight breed thoroughbred chromosome 28, EquCab3.0, whole genome shotgun sequence (i.e. GenBank accession number NC_009171.3)[1], which is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD95 %).
Analyse de biomarqueurs moléculaires — Détection de matériaux d'origine animale dans les denrées alimentaires et les aliments pour animaux par PCR en temps réel — Partie 6: Méthode de détection de l'ADN de cheval
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TECHNICAL ISO/TS
SPECIFICATION 20224-6
First edition
2020-07
Molecular biomarker analysis —
Detection of animal-derived materials
in foodstuffs and feedstuffs by real-
time PCR —
Part 6:
Horse DNA detection method
Analyse de biomarqueurs moléculaires — Détection de matériaux
d'origine animale dans les denrées alimentaires et les aliments pour
animaux par PCR en temps réel —
Partie 6: Méthode de détection de l'ADN de cheval
Reference number
©
ISO 2020
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Scientific basis . 2
5 Reagents and materials . 2
5.1 General . 2
5.2 PCR reagents . 2
6 Apparatus . 3
7 Procedure. 3
7.1 Preparation of the test portion/sample . 3
7.2 Preparation of DNA extracts . 3
7.3 PCR setup . 3
7.3.1 Reaction mixes . 3
7.3.2 PCR controls . . 4
7.3.3 Real-time PCR thermocycler plate set-up . 4
7.4 Temperature-time programme . 4
8 Accept/reject criteria . 4
8.1 General . 4
8.2 Identification . 5
9 Validation status and performance criteria . 5
9.1 General . 5
9.2 Robustness . 5
9.3 Reproducibility . 6
9.4 Sensitivity . 6
9.5 Specificity . 9
10 Test report .11
Annex A (informative) BlastN 2.9.0 results for query of GenBank refseq_genomes (452
databases) .12
Bibliography .15
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.
A list of all parts in the ISO 20224 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 © ISO 2020 – All rights reserved
Introduction
Fraudulent adulteration of meat in food and feed threatens both public safety and commerce.
Adulteration can affect those adhering to ethnological dietary rules, economic development and social
stability. This document provides a real-time polymerase chain reaction (real-time PCR) analytical
method for the identification of meat animal species from nucleic acid present in the ingredients of food
and feed.
Animal-derived biological materials in food and feed are detected and identified in the laboratory with
the following successive (or simultaneous) steps: preparation of the test portion/sample, nucleic acid
extraction and purification, PCR amplification and interpretation of results. This document provides
guidance for PCR amplification and interpretation of results, specific to horse DNA detection.
The ISO 20224 series consists of technical specifications that describe specific applications. New
species DNA detection methods established in the future will be added as independent parts.
TECHNICAL SPECIFICATION ISO/TS 20224-6:2020(E)
Molecular biomarker analysis — Detection of animal-
derived materials in foodstuffs and feedstuffs by real-
time PCR —
Part 6:
Horse DNA detection method
1 Scope
This document specifies a real-time polymerase chain reaction (real-time PCR) method for the
qualitative detection of horse-specific DNA derived from food and feed. It requires the extraction of an
adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection
of horse material derived from domestic horse (Equus caballus), mule (Equus caballus ♀ × Equus asinus
♂), hinny (Equus caballus ♂ × Equus asinus ♀) and zebroid (Equus caballus × Equus simplicidens). The
assay also detects the species Przewalski’s horse (Equus przewalskii) and zebra (Equus burchellii).
The target sequence is an Equus caballus isolate Twilight breed thoroughbred chromosome 28,
[1]
EquCab3.0, whole genome shotgun sequence (i.e. GenBank accession number NC_009171.3) , which
is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute
limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD ).
95 %
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 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 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
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 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/
4 Scientific basis
DNA is extracted from the test portion by applying a suitable method (see ISO 21571:2005, A.1). The
DNA analysis consists of two parts:
— verification of the quality and amplifiability of the extracted DNA using a PCR assay specific for
eukaryotes (e.g. 18S rRNA gene) or mammals and poultry (e.g. myostatin gene);
— detection of the horse species-specific DNA sequence of the single-copy Equus caballus isolate
Twilight breed thoroughbred chromosome 28, EquCab3.0, whole genome shotgun sequence.
(GenBank accession number NC_009171.3) in a real-time PCR.
NOTE The copy number of the eukaryotic ribosomal 18S RNA (18S rRNA) gene in a cell varies from several
hundred to several thousand, while the specific target sequence in the horse genome and myostatin gene in
mammals and poultry genome are single copy. The copy number of the specific target sequence in the horse
genome was confirmed by bioinformatics analysis at the whole genome scale (see Annex A) and digital PCR for
absolute quantification
...
TECHNICAL ISO/TS
SPECIFICATION 20224-6
First edition
2020-07
Molecular biomarker analysis —
Detection of animal-derived materials
in foodstuffs and feedstuffs by real-
time PCR —
Part 6:
Horse DNA detection method
Analyse de biomarqueurs moléculaires — Détection de matériaux
d'origine animale dans les denrées alimentaires et les aliments pour
animaux par PCR en temps réel —
Partie 6: Méthode de détection de l'ADN de cheval
Reference number
©
ISO 2020
© ISO 2020
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Scientific basis . 2
5 Reagents and materials . 2
5.1 General . 2
5.2 PCR reagents . 2
6 Apparatus . 3
7 Procedure. 3
7.1 Preparation of the test portion/sample . 3
7.2 Preparation of DNA extracts . 3
7.3 PCR setup . 3
7.3.1 Reaction mixes . 3
7.3.2 PCR controls . . 4
7.3.3 Real-time PCR thermocycler plate set-up . 4
7.4 Temperature-time programme . 4
8 Accept/reject criteria . 4
8.1 General . 4
8.2 Identification . 5
9 Validation status and performance criteria . 5
9.1 General . 5
9.2 Robustness . 5
9.3 Reproducibility . 6
9.4 Sensitivity . 6
9.5 Specificity . 9
10 Test report .11
Annex A (informative) BlastN 2.9.0 results for query of GenBank refseq_genomes (452
databases) .12
Bibliography .15
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.
A list of all parts in the ISO 20224 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 © ISO 2020 – All rights reserved
Introduction
Fraudulent adulteration of meat in food and feed threatens both public safety and commerce.
Adulteration can affect those adhering to ethnological dietary rules, economic development and social
stability. This document provides a real-time polymerase chain reaction (real-time PCR) analytical
method for the identification of meat animal species from nucleic acid present in the ingredients of food
and feed.
Animal-derived biological materials in food and feed are detected and identified in the laboratory with
the following successive (or simultaneous) steps: preparation of the test portion/sample, nucleic acid
extraction and purification, PCR amplification and interpretation of results. This document provides
guidance for PCR amplification and interpretation of results, specific to horse DNA detection.
The ISO 20224 series consists of technical specifications that describe specific applications. New
species DNA detection methods established in the future will be added as independent parts.
TECHNICAL SPECIFICATION ISO/TS 20224-6:2020(E)
Molecular biomarker analysis — Detection of animal-
derived materials in foodstuffs and feedstuffs by real-
time PCR —
Part 6:
Horse DNA detection method
1 Scope
This document specifies a real-time polymerase chain reaction (real-time PCR) method for the
qualitative detection of horse-specific DNA derived from food and feed. It requires the extraction of an
adequate amount of PCR amplifiable DNA from the relevant matrix and can be applied to the detection
of horse material derived from domestic horse (Equus caballus), mule (Equus caballus ♀ × Equus asinus
♂), hinny (Equus caballus ♂ × Equus asinus ♀) and zebroid (Equus caballus × Equus simplicidens). The
assay also detects the species Przewalski’s horse (Equus przewalskii) and zebra (Equus burchellii).
The target sequence is an Equus caballus isolate Twilight breed thoroughbred chromosome 28,
[1]
EquCab3.0, whole genome shotgun sequence (i.e. GenBank accession number NC_009171.3) , which
is present as a single copy per haploid genome. The provided PCR assay for this target has an absolute
limit of detection of five copies per reaction, with ≥ 95 % replicability at this concentration (LOD ).
95 %
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 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 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
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 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/
4 Scientific basis
DNA is extracted from the test portion by applying a suitable method (see ISO 21571:2005, A.1). The
DNA analysis consists of two parts:
— verification of the quality and amplifiability of the extracted DNA using a PCR assay specific for
eukaryotes (e.g. 18S rRNA gene) or mammals and poultry (e.g. myostatin gene);
— detection of the horse species-specific DNA sequence of the single-copy Equus caballus isolate
Twilight breed thoroughbred chromosome 28, EquCab3.0, whole genome shotgun sequence.
(GenBank accession number NC_009171.3) in a real-time PCR.
NOTE The copy number of the eukaryotic ribosomal 18S RNA (18S rRNA) gene in a cell varies from several
hundred to several thousand, while the specific target sequence in the horse genome and myostatin gene in
mammals and poultry genome are single copy. The copy number of the specific target sequence in the horse
genome was confirmed by bioinformatics analysis at the whole genome scale (see Annex A) and digital PCR for
absolute quantification
...
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