CEN/TS 17303:2019
(Main)Foodstuffs - DNA barcoding of fish and fish products using defined mitochondrial cytochrome b and cytochrome c oxidase I gene segments
Foodstuffs - 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) 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. 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.
Lebensmittel - DNA-Barcoding von Fisch und Fischprodukten anhand definierter mitochondrialer Cytochrom-b- und Cytochrom-c-Oxidase-I-Genabschnitte
Dieses Dokument legt ein Verfahren für die Identifizierung von Einzelfischen oder Fischfilets auf Gattungs- oder Speziesebene fest.
Die Identifizierung der Fischspezies erfolgt durch PCR-Amplifikation entweder eines Segments des mitochondrialen Cytochrom-b-Gens (cytb) [1] oder des Cytochrom-c-Oxidase-I-Gens (cox1, syn COI) [2], [3] - oder von beiden, gefolgt von der Sequenzierung der PCR-Produkte und einem anschließenden Datenbankabgleich der Sequenzen [4], [5]. Diese Methodik erlaubt die Identifizierung einer großen Zahl kommerziell wichtiger Fischspezies.
Die Entscheidung, ob der cytb- oder der cox1-Genabschnitt oder beide für die Fischidentifizierung herangezogen werden, hängt von der deklarierten Fischspezies, der Anwendbarkeit des PCR-Verfahrens für die Fischspezies und der Verfügbarkeit vergleichbarer Sequenzen in den öffentlichen Datenbanken ab.
Dieses Verfahren wurde erfolgreich an rohen Fischfilets validiert, Laborerfahrungen zeigen jedoch, dass dieses Verfahren auch an verarbeiteten Proben wie z. B. kalt- und heißgeräucherten, gesalzenen, tiefgefrorenen, gekochten, gebratenen und frittierten Proben angewendet werden kann.
Dieses Dokument ist in der Regel ungeeignet für die Untersuchung stark verarbeiteter Lebensmittel - z. B. Fischkonserven mit stark degradierter DNA, bei denen die Fragmentlängen nicht für eine Amplifizierung der Zielsequenzen ausreichen. Außerdem ist es nicht anwendbar auf zusammengesetzte Fischprodukte, die aus mehr als einer Fischspezies bestehen.
Produits alimentaires - Codes-barres d’ADN de poissons et de produits à base de poissons à l’aide de segments de gènes mitochondriaux du cytochrome b et cyctochrome c oxydase I
Le présent document décrit un mode opératoire d’identification au niveau du gène ou de l’espèce des poissons individuels et des filets de poisson.
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) 1], du gène du cytochrome c oxydase I (cox1 ou COI) [2], [3] ou des deux, suivie d’un séquençage des produits de la PCR et d’une comparaison des séquences aux entrées de bases de données [4], [5]. Cette méthodologie permet l’identification d’un grand nombre d’espèces de poisson à forte importance commerciale.
Le choix de l’utilisation de segments de gènes de cytb ou de cox1 ou des deux pour l’identification du poisson dépend de l’espèce de poisson déclarée, de la facilité d’application de la méthode de la PCR à l’espèce de poisson considérée et de la disponibilité des séquences de comparaison dans les bases de données publiques.
Cette méthode a été validée avec succès sur des filets de poisson cru mais des expériences menées en laboratoire montrent qu’elle est également applicable à des échantillons après transformation (fumage à froid ou à chaud, salage, congélation, cuisson, friture, etc.).
Le présent document est de manière générale inadapté 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 n’est pas applicable aux produits complexes à base de poisson qui mélangent deux espèces de poissons et plus.
Živila - Črtno kodiranje DNK rib in ribjih proizvodov z uporabo segmentov genov, ki nosijo zapis za mitohondrijski citokrom b in citokrom c oksidaze I
Ta metoda določa postopek za identifikacijo posameznih surovih, hladno prekajenih, soljenih, zamrznjenih ali kuhanih (prekuhanih, pečenih, ocvrtih, vroče dimljenih) rib in ribjih filejev glede na rod ali vrsto.
Identifikacija vrst rib se izvaja s povečanjem polimerazne verižne reakcije (PCR) segmenta mitohondrijskega gena za citokrom b (cytb) ali gena za citokrom c oksidaze I (cox1, syn COI) ali obojega, čemur sledi sekvenciranje produktov polimerazne verižne reakcije in nadaljnja primerjava sekvenc z vnosi v zbirkah podatkov. Metodologija omogoča identifikacijo velikega števila komercialno pomembnih vrst rib.
Odločitev o tem, ali se za identifikacijo rib uporablja segment gena cytb ali cox1 ali oba, je odvisna od navedene vrste rib, uporabnosti metode polimerazne verižne reakcije za vrsto rib in razpoložljivosti primerjalnih sekvenc v javnih zbirkah podatkov.
Ta standard običajno ni primeren za analizo živil z visoko stopnjo predelave, npr. ribjih konzerv, z zelo razgrajeno DNK, pri katerih dolžine delcev ne zadostujejo za povečanje ciljev. Poleg tega se ne uporablja za kompleksne ribje proizvode, ki vsebujejo mešanico dveh ali več vrst rib.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
01-maj-2019
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Foodstuffs - DNA barcoding of fish and fish products using defined mitochondrial
cytochrome b and cytochrome c oxidase I gene segments
Lebensmittel - DNA-Barcoding von Fisch und Fischprodukten anhand definierter
mitochondrialer Cytochrom b- und Cytochrom c-Oxidase I-Genabschnitte
Produits alimentaires - Codes-barres d’ADN de poissons et de produits à base de
poissons à l'aide de segments de gènes mitochondriaux du cytochrome b et
cyctochrome c oxydase I
Ta slovenski standard je istoveten z: CEN/TS 17303:2019
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.
CEN/TS 17303
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2019
TECHNISCHE SPEZIFIKATION
ICS 67.120.30
English Version
Foodstuffs - DNA barcoding of fish and fish products using
defined mitochondrial cytochrome b and cytochrome c
oxidase I gene segments
Produits alimentaires - Codes-barres d'ADN de Lebensmittel - DNA-Barcoding von Fisch und
poissons et de produits à base de poissons à l'aide de Fischprodukten anhand definierter mitochondrialer
segments de gènes mitochondriaux du cytochrome b et Cytochrom-b- und Cytochrom-c-Oxidase-I-
cyctochrome c oxydase I Genabschnitte
This Technical Specification (CEN/TS) was approved by CEN on 14 January 2019 for provisional application.
The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to
submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.
CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS
available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in
parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey 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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 17303:2019 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 5
4 Principle . 7
5 Reagents and materials . 7
5.1 General . 7
5.2 PCR reagents . 8
6 Apparatus . 9
7 Procedure. 9
7.1 Sample preparation . 9
7.2 DNA extraction . 9
7.3 PCR . 9
7.3.1 General . 9
7.3.2 PCR setup . 10
7.3.3 Temperature-time program . 11
7.3.4 PCR controls . 11
8 Evaluation . 12
8.1 Evaluation of PCR products . 12
8.2 Evaluation of the PCR results . 12
8.3 Sequencing of PCR products . 12
8.4 Evaluation of sequence data . 13
8.5 Comparison of the sequence with public databases. 13
8.5.1 General . 13
8.5.2 Sequence comparison of cytb and/or cox1 DNA sequences with GenBank . 13
8.5.3 Sequence comparison of cox1 DNA sequences with BOLD . 14
9 Interpretation of database query results . 15
10 Validation status and performance criteria . 15
10.1 Collaborative study for the identification of fish species based on cytb sequence
analysis . 15
10.2 Collaborative study for the identification of fish species based on cox1 sequence
analysis . 16
11 Test report . 18
Annex A (informative) Practical laboratory experiences with the amplificability of cytb or
cox1 segments from tested fish species . 19
Bibliography . 24
European foreword
This document (CEN/TS 17303:2019) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
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.
According to the CEN/CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
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.
1 Scope
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.
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.
EN ISO 24276, Foodstuffs — Methods of analysis for the detection of genetically modified organisms and
derived products — General requirements and definitions (ISO 24276)
ISO 16577, Molecular biomarker analysis — Terms 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 terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http://www.electropedia.org/
— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
alignment
process or result of matching up the nucleotide residues of two or more biological sequences to achieve
maximal levels of identity
[SOURCE: BLAST Glossary]
3.2
BLAST
(The Basic Local Alignment Search Tool) [4]
sequence comparison algorithm optimized for speed used to search sequence databases for optimal
local alignments to a query
Note 1 to entry: It directly approximates alignments that optimize a measure of local similarity, the maximum
signal pair (MSP) score or high scoring signal pair (HSP) score.
3.3
BOLD
(Barcode of Life Data Systems) [5]
informatics workbench aiding the acquisition, storage, analysis, and publication of DNA barcode
records
Note 1 to entry: By assembling molecular, morphological, and distributional data, it bridges a traditional
bioinformatics chasm. BOLD is freely available to any researcher with interests in DNA barcoding. By providing
specialized services, it aids the assembly of records that meet the standards needed to gain BARCODE designation
in the global sequence databases. Because of its web-based delivery and flexible data security model, it is also well
positioned to support projects that involve broad research alliances.
[SOURCE: BOLDSYSTEMS About Us]
3.4
FASTA format
text-based format for representing either nucleotide sequences or amino acid sequences, which begins
with a single-line description, followed by lines of sequence data
Note 1 t
...
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