SIST EN ISO 17174:2024

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.

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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
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Email: copyright@iso.org
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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 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
ISO 17174:2024(en)
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
International Standard ISO 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 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
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).
[SOURCE: ISO 16577:2022, 3.7.18, modified — “alignment” was added as the preferred term; Note 1 to entry
was added.]
ISO 17174:2024(en)
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:
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 sequence.
[SOURCE: ISO 16577:2022, 3.1.2, modified — Example in Note 2 to entry was replaced; the third list item in
Note 3 to entry was deleted.]
3.3
identity
extent to which two (nucleotide or amino acid) sequences have the same residues at the same positions in an
alignment (3.1)
Note 1 to entry: Identity is often expressed as a percentage.
Note 2 to entry: In the sequence database of the Barcode of Life (BOLD), the term "similarity" is used instead of identity.
3.4
introgressed DNA
allele from one species incorporated in the gene pool of another, divergent species
Note 1 to entry: Introgression has usually happened via hybridization and backcrossing of individuals belonging to
different species.
3.5
query
sequence (or other type of search term) that is compared to entries in a database
3.6
query coverage
percentage of the query (3.5) covered by alignment (3.1) to the database sequence

ISO 17174:2024(en)
4 Symbols and abbreviated terms
bp base pairs
Glu glutamic acid, glutamate
tRNA transfer RNA
cox1, COI cytochrome c oxidase I gene
cytb cytochrome b gene
DNA deoxyribonucleic acid
RNA ribonucleic acid
dNTP deoxyribonucleotide triphosphate
A adenine
C cytosine
G guanine
I inosine
R purine base (adenine or guanine)
T thymine
Y pyrimidine base (cytosine or thymine)
IUU illegal, unreported and unregulated
PCR polymerase chain reaction
5 Principle
DNA is extracted from fish and fish products applying a suitable method. Segments of approximately 460
base pairs of cytb and/or approximately 650 base pairs of cox1 are amplified by PCR. For the amplification
of the cytb segment a set of two primers is used. Amplification of cox1 uses a set of four primers to ensure
the amplification of DNA of as many fish species as possible. Some of the used primers include ambiguous
bases to increase the number of species detected by the two methods. The nucleotide sequences of the PCR
products are determined by a suitable DNA sequencing method (e. g. Sanger sequencing). The two PCR
primers used to generate the cytb amplicon are also used for sequencing. The cox1 primers have M13 tails
that enable sequencing of the cox1 amplicons using M13 sequencing primers. The determined sequence is
evaluated by comparison to sequence entries in databases, thus allowing the assignment to a fish species or
genus according to the degree of identity with available sequences.
The decision whether the cytb or cox1 segment or both are used for fish identification depends on the
declared fish species, especially the applicability of the PCR method for the fish species and the availability
of comparative sequences in the public databases.
6 Reagents and materials
During the analysis, unless otherwise stated, use only reagents of recognized molecular biology grade and
distilled, demineralized water or water of equivalent purity, in accordance with ISO 20813. Laboratory
organization shall follow ISO 20813.

ISO 17174:2024(en)
1)
6.1 Thermostable DNA polymerase .
1)
6.2 PCR reaction buffer (including MgCl or with separate MgCl solution) .
2 2
6.3 dNTP mix (dATP, dCTP, dGTP and dTTP).
NOTE dNTP can also be part of a commercial PCR master mix.
6.4 Oligonucleotides (see Tables 1 and 2).
NOTE 1 The abbreviations of the DNA bases in Tables 1 and 2 are based on the recommendations for unambiguous,
[4]
uniform, and consistent nomenclature, published by the International Union of Pure and Applied Chemistry (IUPAC).
[1]
Table 1 — Oligonucleotides for amplification of the cytb gene region
Name DNA sequence of oligonucleotide
L14735 5’-AAA AAC CAC CGT TGT TAT TCA ACT A-3’
H15149ad 5’-GCI CCT CAR AAT GAY ATT TGT CCT CA-3’
NOTE 2 Laboratory experience has shown that M13 tails can also be used with cytb primers.
[2][3]
Table 2 — Oligonucleotides for amplification of the cox1 gene region
a
Name DNA sequence of oligonucleotide
VF2_t1 5’-TGT AAA ACG ACG GCC AGT CAA CCA ACC ACA AAG ACA TTG GCA C-3’
FishF2_t1 5’-TGT AAA ACG ACG GCC AGT CGA CTA ATC ATA AAG ATA TCG GCA C-3’
FishR2_t1 5’-CAG GAA ACA GCT ATG ACA CTT CAG GGT GAC CGA AGA ATC AGA A-3’
FR1d_t1 5’-CAG GAA ACA GCT ATG ACA CCT CAG GGT GTC CGA ARA AYC ARA A-3’
a
M13 tails of the primers are highlighted in bold and italic.
6.5 Trehalose.
6.6 Agarose.
6.7 DNA size standard.
6.8 Sequencing primers (see Table 3).
[3]
Table 3 — Sequencing primers for cox1 PCR products
Name DNA sequence of oligonucleotide
M13F (−21) 5’-TGT AAA ACG ACG GCC AGT-3’
M13R (−27) 5’-CAG GAA ACA GCT ATG AC-3’ ®
1) During the collaborative studies, the Maxima Hot Start PCR Master Mix (2 ×) of Fermentas GmbH (ready-to -use
PCR buffer solution including thermostable DNA polymerase) was used for the cytb amplification and the BIOTAQ™ DNA
polymerase of Bioline with 10 × reaction buffer and separate MgCl solution for the cox1 amplification. In addition to ®
the recommended BIOTAQ™ DNA polymerase, other mastermixes and polymerases were successfully used. Maxima
Hot Start PCR Master Mix (2 ×) of Fermentas GmbH (ready-to-use PCR buffer solution including thermostable DNA
polymerase) and BIOTAQ™ DNA polymerase of Bioline are examples of suitable products available commercially. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of
these products.
ISO 17174:2024(en)
7 Apparatus
In addition to standard laboratory equipment, the following apparatus should be used.
7.1 UV-spectrophotometer or fluorometer, to determine the concentration of DNA.
7.2 Thermocycler.
7.3 Gel electrophoresis device.
7.4 Gel documentation system.
7.5 DNA sequencer.
8 Procedure
8.1 Sample preparation
The test portion used for DNA extraction shall be representative of the laboratory sample, for example, using
the following guidance. In samples that consist of processed materials (e.g. convenience foods), single fish
pieces shall be analysed separately. For the analysis of samples composed of several pieces (e.g. bags with
different fillets), test portions for every putative fish species are taken and analysed separately. To minimize
the risk of amplifying adhering contaminants, test sample material shall not be taken from the surface of the
[7]
laboratory sample, see also ISO 20813 and ISO 22949-1 .
8.2 DNA extraction
[5]
General instructions and measures specified in ISO 21571:2005 should be followed for the extraction of
[5]
DNA from the test sample. For example, the DNA extraction methods specified in ISO 21571:2005, Annex A,
can be used. Commercial kits can be used for the extraction and purification of DNA, if their applicability for
the extraction of DNA from fish has been experimentally confirmed.
8.3 PCR
8.3.1 General
Universal primers are used for amplification of segments of mitochondrial cytb and cox1. The primer
H15149ad is a universal primer with fish-specific adaptations that binds to the cytb section. L14735 binds to
[1]
a section in the neighbouring highly conserved tRNA-Glu gene. The primers used for amplification of the
[2][3]
mitochondrial cox1 fragment were designed to the 5’ region of the cox1 of fish.
Both primer sets have been tested against a very broad taxonomic range of fish species. It is currently
known that, with some exceptions, the primer pair L14735/H15149ad did not react with samples labelled as
[6]
barramundi (Lates calcarifer) or Nile perch (Lates niloticus). The cox1 primer set has only failed in a small
[2]
minority of cases (< 5 % of species tested).
8.3.2 PCR setup
The method was validated for a total volume of 25 µl (cytb) or 20 µl (cox1) per PCR. The reagents given in
Tables 4 and 5 shall be used for the cytb and cox1 PCR, respectively.
Reagents shall be completely thawed and should be centrifuged briefly before usage. A PCR reagent mixture
is prepared containing all PCR components in the given concentrations except for the DNA extract or the
controls. The amount of PCR mixture prepared depends on the total volume per PCR and the total number of
reactions including a sufficient pipetting reserve.

ISO 17174:2024(en)
Positive PCR results are expected when using a DNA concentration of approximately 1 ng DNA per μl of the
final solution [25 ng (cytb) or 20 ng (cox1)].
To improve the PCR result, the DNA quantity can be increased (e.g. to increase the yield of PCR product) or
decreased (e.g. to avoid PCR inhibition).
Table 4 — Components for the cytb PCR
Reagent (stock solution) Final composition in the reaction solution
PCR buffer 1 ×
a
MgCl 1,5 mmol/l
a
dNTP mix 0,2 mmol/l for each dNTP
Primer L14735 500 nmol/l
Primer H15149ad 500 nmol/l
Thermostable DNA polymerase 0,5 units to 1 unit
Water Add to obtain final volume
Sample DNA Approximately 1 ng/µl
a
Use a reagent only if one is not already included in the PCR buffer.
Table 5 — Components for the cox1 PCR
Reagent (stock solution) Final composition in the reaction solution
a
Trehalose 5 %
PCR buffer 1 ×
b
MgCl 2,5 mmol/l
b
dNTP mix 0,2 mmol/l for each dNTP
Primer VF2_t1 100 nmol/l
Primer FishF2_t1 100 nmol/l
Primer FishR2_t1 100 nmol/l
Primer FR1d_t1 100 nmol/l
Thermostable DNA polymerase 0,5 units
Water Add to obtain final volume
Sample DNA Approximately 1 ng/µl
a
Optional.
b
Use a reagent only if one is not already included in the PCR buffer.
Mix the PCR reagent mixture, centrifuge briefly and split into the individual reactions. Pipette the DNA
extracts to be examined or the PCR controls (see 8.3.3) into the different reaction solutions.
For further information on PCR controls, see also ISO 20813.
8.3.3 PCR controls
In addition to the reaction setups for the samples to be analysed, an amplification reagent control and an
extraction blank control in accordance with ISO 20813 shall be included.
A positive DNA target control (see ISO 20813) can be used to dem
...