SIST EN ISO 17174:2024

Molecular biomarker analysis - DNA barcoding of fish and fish products using defined mitochondrial cytochrome b and cytochrome c oxidase I gene segments (ISO 17174:2024)

This document describes a procedure for the identification of single fish and fish fillets to the
level of genus or species.
The identification of fish species is carried out by PCR amplification of either a segment of the
mitochondrial cytochrome b gene (cytb) [1] or the cytochrome c oxidase I gene (cox1, syn COI)
[2], [3] or both, followed by sequencing of the PCR products and subsequent sequence
comparison with entries in databases [4], [5]. The methodology allows the identification of a large
number of commercially important fish species.
The decision whether the cytb or cox1 gene segment or both are used for fish identification
depends on the declared fish species, the applicability of the PCR method for the fish species
and the availability of comparative sequences in the public databases.
This method has been successfully validated on raw fish fillets, however, laboratory experience
is available that it can also be applied to processed, e.g. cold smoked, hot smoked, salted,
frozen, cooked, fried, deep fried
samples.
This document is usually unsuitable for the analysis of highly processed foods, e.g. tins of fish,
with highly degraded DNA where the fragment lengths are not sufficient for amplification of the
targets. Furthermore, it is not applicable for complex fish products containing mixtures of two or
more fish species.

Untersuchung auf molekulare Biomarker - DNA-Barcoding von Fisch und Fischprodukten anhand definierter mitochondrialer Cytochrom b- und Cytochrom c-Oxidase I-Genabschnitte (ISO 17174:2024)

Dieses Dokument legt ein Verfahren für die Identifizierung von einzelnen Fischen und Fischfilets bis zur Ebene der Gattung oder Art fest. Es ermöglicht die Identifizierung einer großen Anzahl kommerziell wichtiger Fischarten mittels DNA-Barcoding.
Dieses Verfahren wurde an rohem Fisch validiert. Laborerfahrungen deuten auf eine zusätzliche Anwendbarkeit auf verarbeitete Fischerzeugnisse hin, z. B. kalt geräucherte, heiß geräucherte, gesalzene, tiefgefrorene, gekochte, gebratene und frittierte Proben.
Das beschriebene Verfahren ist in der Regel ungeeignet für die Analyse stark verarbeiteter Lebensmittel, z. B. Fischkonserven, mit stark degradierter DNA, bei denen die Fragmentlängen nicht für eine Amplifikation der Zielsequenzen (Targets) ausreichen. Außerdem ist es nicht anwendbar auf zusammengesetzte Fischerzeugnisse, die Mischungen aus zwei oder mehr Fischarten enthalten.
Die Identifizierung der Fischarten erfolgt durch PCR-Amplifikation entweder eines Segments des mitochondrialen Cytochrom-b-Gens (cytb) oder des Cytochrom-c-Oxidase-I-Gens (cox1, syn COI) oder von beiden, gefolgt von der Sequenzierung der PCR-Produkte und einem anschließenden Sequenzvergleich mit Einträgen in Datenbanken.

Analyse de biomarqueurs moléculaires - Codes-barres d’ADN de poissons et de produits à base de poisson à l’aide de segments de gènes mitochondriaux de cytochrome b et cytochrome c oxydase I (ISO 17174:2024)

Le présent document spécifie une méthode d’identification au niveau du genre ou de l’espèce des poissons individuels et des filets de poisson. Il permet l’identification d’un grand nombre d’espèces de poissons à forte importance commerciale au moyen de codes-barres d’ADN.
Cette méthode a été validée sur des poissons crus. L’expérience menée en laboratoire indique une applicabilité supplémentaire aux produits à base de poisson après transformation (fumage à froid ou à chaud, salage, congélation, cuisson, friture, etc.).
De manière générale, la méthode décrite est inadaptée à l’analyse d’aliments hautement transformés (comme les poissons en conserve dont l’ADN est fortement dégradé et dont les longueurs des fragments se révèlent insuffisantes pour l’amplification des cibles). En outre, la méthode présentée ici ne s’applique pas aux produits complexes à base de poisson qui mélangent deux espèces de poissons et plus.
L’identification des espèces de poisson s’effectue par amplification par réaction de polymérisation en chaîne (PCR : Polymerase Chain Reaction) d’un segment du gène du cytochrome b mitochondrial (cytb), du gène du cytochrome c oxydase I (cox1 ou COI) ou des deux, suivie d’un séquençage des produits de la PCR et d’une comparaison des séquences aux entrées de bases de données.

Analiza molekularnih biomarkerjev - Črtno kodiranje DNK rib in ribjih proizvodov z uporabo definiranih mitohondrijskih segmentov genov citokroma b in citokroma c oksidaze I (ISO 17174:2024)

General Information

• Status: Published
• Public Enquiry End Date: 01-Aug-2023
• Publication Date: 08-Oct-2024
• ICS: 67.120.30 - Fish and fishery products
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 04-Oct-2024
• Due Date: 09-Dec-2024
• Completion Date: 09-Oct-2024
• Directive: 2017/625 - Regulation (EU) 2017/625 of the European Parliament and of the Council of 15 March 2017 on official controls and other official activities performed to ensure the application of food and feed law, rules on animal health and welfare, plant health and plant protection products
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: EN ISO 17174:2024 - Molecular biomarker analysis - DNA barcoding of fish and fish products using defined mitochondrial cytochrome b and cytochrome c oxidase I gene segments (ISO 17174:2024)

Relations

• Replaces: SIST-TS CEN/TS 17303:2019 - Foodstuffs - DNA barcoding of fish and fish products using defined mitochondrial cytochrome b and cytochrome c oxidase I gene segments
• Effective Date: 01-Dec-2024

Standards Content (Sample)

SLOVENSKI STANDARD
01-december-2024
Nadomešča:
SIST-TS CEN/TS 17303:2019
Analiza molekularnih biomarkerjev - Črtno kodiranje DNK rib in ribjih proizvodov z
uporabo definiranih mitohondrijskih segmentov genov citokroma b in citokroma c
oksidaze I (ISO 17174:2024)
Molecular biomarker analysis - DNA barcoding of fish and fish products using defined
mitochondrial cytochrome b and cytochrome c oxidase I gene segments (ISO
17174:2024)
Untersuchung auf molekulare Biomarker - DNA-Barcoding von Fisch und Fischprodukten
anhand definierter mitochondrialer Cytochrom b- und Cytochrom c-Oxidase I-
Genabschnitte (ISO 17174:2024)
Analyse de biomarqueurs moléculaires - Codes-barres d’ADN de poissons et de produits
à base de poisson à l’aide de segments de gènes mitochondriaux de cytochrome b et
cytochrome c oxydase I (ISO 17174:2024)
Ta slovenski standard je istoveten z: EN ISO 17174:2024
ICS:
67.120.30 Ribe in ribji proizvodi Fish and fishery products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 17174
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2024
EUROPÄISCHE NORM
ICS 67.120.30 Supersedes CEN/TS 17303:2019
English Version
Molecular biomarker analysis - DNA barcoding of fish and
fish products using defined mitochondrial cytochrome b
and cytochrome c oxidase I gene segments (ISO
17174:2024)
Analyse de biomarqueurs moléculaires - Codes-barres Untersuchung auf molekulare Biomarker - DNA-
d'ADN de poissons et de produits à base de poisson à Barcoding von Fisch und Fischprodukten anhand
l'aide de segments de gènes mitochondriaux de definierter mitochondrialer Cytochrom b- und
cytochrome b et cytochrome c oxydase I (ISO Cytochrom c-Oxidase I-Genabschnitte (ISO
17174:2024) 17174:2024)
This European Standard was approved by CEN on 7 September 2024.

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
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 17174:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 17174:2024) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with 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 March 2025, and conflicting national standards shall
be withdrawn at the latest by March 2025.
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.
This document supersedes CEN/TS 17303:2019.
This document has been prepared under a standardization request addressed to CEN by the European
Commission. The Standing Committee of the EFTA States subsequently approves these requests for its
Member States.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. 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 17174:2024 has been approved by CEN as EN ISO 17174:2024 without any modification.

International
Standard
ISO 17174
First edition
Molecular biomarker analysis —
2024-09
DNA barcoding of fish and
fish products using defined
mitochondrial cytochrome b
and cytochrome c oxidase I gene
segments
Analyse de biomarqueurs moléculaires — Codes-barres d’ADN de
poissons et de produits à base de poisson à l’aide de segments de
gènes mitochondriaux de cytochrome b et cytochrome c oxydase I
Reference number
ISO 17174:2024(en) © ISO 2024
ISO 17174:2024(en)
© ISO 2024
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]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii
ISO 17174:2024(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Symbols and abbreviated terms. 3
5 Principle . 3
6 Reagents and materials . 3
7 Apparatus . 5
8 Procedure . 5
8.1 Sample preparation .5
8.2 DNA extraction .5
8.3 PCR .5
8.3.1 General .5
8.3.2 PCR setup .5
8.3.3 PCR controls .6
8.3.4 Thermal cycling .7
8.4 Evaluation of PCR products . .7
8.5 Evaluation of the PCR results .7
9 Sequencing . 8
9.1 Sequencing of PCR products .8
9.2 Evaluation of sequence data .8
9.3 Comparison of the sequence with public databases .9
9.3.1 General .9 ®
9.3.2 Sequence comparison of cytb and/or cox1 DNA sequences with GenBank .9
9.3.3 Sequence comparison of cox1 DNA sequences with BOLD.10
10 Interpretation of database query results . 10
11 Validation status and performance criteria .11
11.1 Collaborative study for the identification of fish species based on cytb sequence analysis .11
11.2 Collaborative study for the identification of fish species based on cox1 sequence analysis . 12
12 Test report .13
Annex A (informative) Practical laboratory experiences with the amplifiability of cytb and
cox1 segments from tested fish species. 14
Bibliography . 17

iii
ISO 17174:2024(en)
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 dif
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