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 specifies a method for the identification of single fish and fish fillets to the level of genus or species. It allows the identification of a large number of commercially important fish species using DNA barcoding.
This method was validated on raw fish. Laboratory experience indicates additional applicability to processed fish products (e.g. cold smoked, hot smoked, salted, frozen, cooked, fried and deep-fried samples).
The described method 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 does not apply to complex fish products containing mixtures of two or more fish species.
The identification of fish species is carried out by PCR amplification of either a segment of the mitochondrial cytochrome b gene (cytb) or the cytochrome c oxidase I gene (cox1, syn COI), or both, followed by sequencing of the PCR products and subsequent sequence comparison with entries in databases.

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 gilt es nicht für 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
Publication Date
17-Sep-2024
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Start Date
18-Sep-2024
Due Date
08-Dec-2023
Completion Date
18-Sep-2024

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SLOVENSKI STANDARD
oSIST prEN ISO 17174:2023
01-julij-2023
Analiza molekularnih biomarkerjev - Črtno kodiranje DNK rib in ribjih proizvodov z
uporabo segmentov genov, ki nosijo zapis za mitohondrijski citokrom b in
citokrom c oksidaze I (ISO/DIS 17174:2023)
Molecular biomarker analysis - DNA barcoding of fish and fish products using defined
mitochondrial cytochrome b and cytochrome c oxidase I gene segments (ISO/DIS
17174:2023)
Untersuchung auf molekulare Biomarker - DNA-Barcoding von Fisch und Fischprodukten
anhand definierter mitochondrialer Cytochrom b- und Cytochrom c-Oxidase I-
Genabschnitte (ISO/DIS 17174:2023)
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/DIS 17174:2023)
Ta slovenski standard je istoveten z: prEN ISO 17174
ICS:
67.120.30 Ribe in ribji proizvodi Fish and fishery products
oSIST prEN ISO 17174:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

oSIST prEN ISO 17174:2023
oSIST prEN ISO 17174:2023
DRAFT INTERNATIONAL STANDARD
ISO/DIS 17174
ISO/TC 34/SC 16 Secretariat: ANSI
Voting begins on: Voting terminates on:
2023-05-15 2023-08-07
Molecular biomarker analysis — DNA barcoding of fish and
fish products using defined mitochondrial cytochrome b
and cytochrome c oxidase I gene segments
ICS: 67.120.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
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NATIONAL REGULATIONS.
ISO/DIS 17174:2023(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
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PROVIDE SUPPORTING DOCUMENTATION. © ISO 2023

oSIST prEN ISO 17174:2023
DRAFT INTERNATIONAL STANDARD
ISO/DIS 17174
ISO/TC 34/SC 16 Secretariat: ANSI
Voting begins on: Voting terminates on:

Molecular biomarker analysis — DNA barcoding of fish and
fish products using defined mitochondrial cytochrome b
and cytochrome c oxidase I gene segments
ICS: 67.120.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
© ISO 2023
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
NOT BE REFERRED TO AS AN INTERNATIONAL
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on STANDARD UNTIL PUBLISHED AS SUCH.
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
IN ADDITION TO THEIR EVALUATION AS
or ISO’s member body in the country of the requester. BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
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CH-1214 Vernier, Geneva
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NATIONAL REGULATIONS.
Website: www.iso.org ISO/DIS 17174:2023(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
Published in Switzerland
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
ii
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2023

oSIST prEN ISO 17174:2023
ISO/DIS 17174:2023(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 4
5 Reagents and materials . 4
5.1 General . 4
5.2 PCR reagents . 4
6 Apparatus . 5
7 Procedure .5
7.1 Sample preparation . 5
7.2 DNA extraction . 6
7.3 PCR . 6
7.3.1 General . 6
7.3.2 PCR setup . 6
7.3.3 Temperature-time program . 7
7.3.4 PCR controls . 8
8 Evaluation . 8
8.1 Evaluation of PCR products . . 8
8.2 Evaluation of the PCR results . 8
8.3 Sequencing of PCR products . 9
8.4 Evaluation of sequence data . 9
8.5 Comparison of the sequence with public databases . 9
8.5.1 General . 9
8.5.2 Sequence comparison of cytb and/or cox1 DNA sequences with GenBank . 10
8.5.3 Sequence comparison of cox1 DNA sequences with BOLD. 10
9 Interpretation of database query results .11
10 Validation status and performance criteria .12
10.1 Collaborative study for the identification of fish species based on cytb sequence
analysis .12
10.2 Collaborative study for the identification of fish species based on cox1 sequence
analysis .13
11 Test report .14
Annex A (informative) Practical laboratory experiences with the amplifiability of cytb and
cox1 segments from tested fish species.15
Bibliography .18
iii
oSIST prEN ISO 17174:2023
ISO/DIS 17174:2023(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
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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
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www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee 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
oSIST prEN ISO 17174:2023
ISO/DIS 17174:2023(E)
Introduction
Food safety is a key aspect in terms of consumer protection. In the last three decades, globalization has
taken place in the trade of food. Fish trade channels are becoming steadily longer and more complicated
so that sophisticated traceability tools are needed to ensure food safety. Correct food labelling is
a prerequisite to ensure safe fish products and fair trade as well as to minimize illegal, unreported
and unregulated (IUU) fishing. In particular, the fact that fish is increasingly being processed in
export countries makes the identification of species by morphological characteristics impossible.
The development of harmonized and standardized protocols for the authentication of fish products is
necessary to establish reliable methods for the detection of potential food fraud.
v
oSIST prEN ISO 17174:2023
oSIST prEN ISO 17174:2023
DRAFT INTERNATIONAL STANDARD ISO/DIS 17174:2023(E)
Molecular biomarker analysis
...

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