ISO/FDIS 17174

Molecular biomarker analysis — DNA barcoding of fish and fish products using defined mitochondrial cytochrome b and cytochrome c oxidase I gene segments

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.

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

General Information

• Status: Not Published
• ICS: 67.120.30 - Fish and fishery products
• Technical Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Drafting Committee: ISO/TC 34/SC 16 - Horizontal methods for molecular biomarker analysis
• Current Stage: 5020 - FDIS ballot initiated: 2 months. Proof sent to secretariat
• Start Date: 11-Jun-2024
• Completion Date: 11-Jun-2024

Relations

• Consolidates: ISO/IEC 23126:2021 - Information technology for learning, education and training — Ubiquitous learning resource organization and description framework
• Effective Date: 28-Jan-2023

Standards Content (Sample)

FINAL DRAFT
International
Standard
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
DNA barcoding of fish and
Voting begins on:
fish products using defined
2024-06-11
mitochondrial cytochrome b
Voting terminates on:
and cytochrome c oxidase I gene
2024-08-06
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
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/CEN PARALLEL PROCESSING LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
FINAL DRAFT
International
Standard
ISO/TC 34/SC 16
Molecular biomarker analysis —
Secretariat: ANSI
DNA barcoding of fish and
Voting begins on:
fish products using defined
mitochondrial cytochrome b
Voting terminates on:
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
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2024
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
ISO/CEN PARALLEL PROCESSING
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
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 Reference number
ii
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
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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 34, Food products, Subcommittee SC 16, Horizontal
methods for molecular biomarker analysis, in collaboration with the European Committee for Standardization
(CEN) Technical Committee CEN/TC 460, Food authenticity, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
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
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 reliable,
harmonized and standardized protocols for the authentication of fish products is necessary to ensure
consumer protection and the detection of potential food fraud.

v
FINAL DRAFT International Standard ISO/FDIS 17174:2024(en)
Molecular biomarker analysis — DNA barcoding of fish and
fish products using defined mitochondrial cytochrome b and
cytochrome c oxidase I gene segments
1 Scope
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.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content c
...

ISO/DIS FDIS 17174.2:2024(E)
ISO/TC 34/SC 16/WG 8
2024-03-27
Secretariat: ANSI
Date: 2024-05-28
Molecular biomarker analysis — 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
FDIS stage
ISO/DISFDIS 17174.2:2024(Een)
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: [phone]
EmailE-mail: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2024 – All rights reserved
ii
ISO/DISFDIS 17174.2:2024(Een)
Contents
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 . 4
7 Apparatus . 5
8 Procedure . 5
8.1 Sample preparation . 5
8.2 DNA extraction . 6
8.3 PCR . 6
8.3.1 General . 6
8.3.2 PCR setup . 6
8.3.3 PCR controls . 7
8.3.4 Thermal cycling . 7
8.4 Evaluation of PCR products . 8
8.5 Evaluation of the PCR results . 8
9 Sequencing . 9
9.1 Sequencing of PCR products . 9
9.2 Evaluation of sequence data . 9
9.3 Comparison of the sequence with public databases . 10
9.3.1 General . 10 ®
9.3.2 Sequence comparison of cytb and/or cox1 DNA sequences with GenBank . 11
9.3.3 Sequence comparison of cox1 DNA sequences with BOLD . 11
10 Interpretation of database query results . 12
11 Validation status and performance criteria . 13
11.1 Collaborative study for the identification of fish species based on cytb sequence analysis13
11.2 Collaborative study for the identification of fish species based on cox1 sequence analysis14
12 Test report . 15
Annex A (informative) Practical laboratory experiences with the amplifiability of cytb and cox1
segments from tested fish species . 17
Bibliography . 21

iii
ISO/DISFDIS 17174.2:2024(Een)
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent rights
in respect thereof. As of the date of publication of this document, ISO [had/had not] received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that this
may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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 34, Food products, Subcommittee SC 16, Horizontal
methods for molecular biomarker analysis, in collaboration with the European Committee for Standardization
(CEN) Technical Committee CEN/TC 460, Food authenticity, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
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 2024 – All rights reserved
iv
ISO/DISFDIS 17174.2:2024(Een)
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 reliable,
harmonized and standardized protocols for the authentication of fish products is necessary to to ensure
consumer protection and the detection of potential food fraud.
v
DRAFT INTERNATIONAL STANDARD ISO/DIS 17174.2:2024(E)

Molecular biomarker analysis — DNA barcoding of fish and fish
products using defined mitochondrial cytochrome b and cytochrome
c oxidase I gene segments
1 Scope
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.
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 — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
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
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— — ISO Online browsing platform: available at https://www.iso.org/obp
— — IEC Electropedia: available at https://www.electropedia.org/
3.1
alignment
sequence alignment
arrangement of nucleic acid sequences or protein sequences according to regions of similarity
ISO/DISFDIS 17174.2:2024(Een)
Note 1 to entry: Alignment is a process or result of matching up the nucleotide residues of two or more biological
sequences to achieve maximal levels of identity (3.3(3.3).).
[SOURCE: ISO 16577:2022, 3.7.18, modified — " — “alignment"” was added as the preferred term.; Note 1 to
entry was added.]
3.2
FASTA format
text-based format for representing either nucleotide sequences or amino acid (protein) sequences, in which
nucleotides or amino acids are represented using single-letter codes
Note 1 to entry: A sequence in FASTA format begins with a single-line description, followed by lines of sequence data.
The description line (defline) is distinguished from the sequence data by a greater-than (“>”) symbol at the beginning. It
is recommended that all lines of text be shorter than 80 characters in length.
Note 2 to entry: An example sequence in FASTA format is:
>Sample_04_cytb
ATGGCCAGCCTCCGAAAAACTCATCCCCTTCTAAAGATTGCTAATGATGCATTAGTAGACCTTCCTGCCCCCTCT
AACCTCTCAACATTATGAAACTTCGGGTCTCTCCTAGGCCTCTGCTTAGCCGCCCAAATCTTAACAGGACTATTT
CTAGCGATACATTATACCGCAAACGTCGAGATAGCTTTCTCATCCGTCGTACACATCTGCCGCGACGTAAATTAC
GGATGACTAATCCGCAACATACACGCCAACGGCGCTTCTTTCTTCTTCATCTGCCTCTACCTACACATTGCACGA
GGCCTATATTACGGCTCCTACTTATTCATAGAGACCTGAAACATTGGAGTTGTACTATTCCTTTTAGTAATAATG
ACCGCCTTCGTAGGCTACGTCCTCCCT
Note 3 to entry: Blank lines are not allowed in the middle of FASTA input. Sequences are represented in the standard
IUB/IUPAC amino acid and nucleic acid codes, with these exceptions:
— — lower-case letters are accepted and are mapped into upper-case;
— — a single hyphen or dash can be used to represent a gap of indeterminate length.
It is common to end the sequence with an “*” (asterisk) character and to leave a blank line between the description and
the sequenc
...