SIST EN 18033:2025
(Main)Food authenticity - Quantitation of equine DNA relative to mammalian DNA in raw beef (meat)
Food authenticity - Quantitation of equine DNA relative to mammalian DNA in raw beef (meat)
This document specifies a real-time PCR procedure for the quantitation of the amount of equine DNA relative to total mammalian DNA in a raw meat sample.
Results of this equine assay are expressed in terms of equine (Equus genus) haploid genome copy numbers relative to total mammalian haploid genome copy numbers. This assay is specific for representatives of the genus Equus and therefore detects horse, mule, donkey and zebra DNA.
The method has been previously validated in a collaborative trial and applied to DNA extracted from samples that consist of raw horse meat in a raw beef (meat) background.
The limit of detection has been determined experimentally to be at least 9 genomic equivalent copies (~ 17 haploid target gene copies) for both the horse genome (E. caballus) and mammalian genome (raw meat samples) based on the lowest dilution on the respective calibration curves through single laboratory validation. The lowest relative horse content of the target sequence included in the collaborative trial was a mass fraction of 0,1 % based on gravimetrically prepared raw horse muscle tissue in a raw beef muscle tissue background.
The compliance assessment process is not part of this document.
Lebensmittelauthentizität - Quantifizierung von Equiden-DNA im Verhältnis zu Säugetier-DNA in rohem Rindfleisch
Dieses Dokument legt ein Real-time-PCR-Verfahren für die Quantifizierung der Menge an Equiden-DNA im Verhältnis zur gesamten Säugetier-DNA in einer Probe von rohem Fleisch fest.
Die Ergebnisse dieser Untersuchung auf Equiden (Equiden-Assay) werden in Form von Kopienzahlen des haploiden Genoms von Equiden (Gattung Equus) im Verhältnis zu den Kopienzahlen des haploiden Genoms von Säugetieren insgesamt ausgedrückt. Diese Untersuchung ist spezifisch für Vertreter der Gattung Equus und weist daher DNA von Pferden, Maultieren, Eseln und Zebras nach.
Das Verfahren wurde zuvor in einem Ringversuch validiert und auf DNA angewendet, die aus Proben extrahiert wurde, die aus rohem Pferdefleisch in rohem Rindfleisch als Hintergrund bestehen.
Die Nachweisgrenze wurde experimentell auf mindestens 9 Kopien von Genomäquivalenten (~ 17 haploide Zielgenkopien) sowohl für das Pferdegenom (E. caballus) als auch für das Säugetiergenom (Proben von rohem Fleisch) auf der Grundlage der niedrigsten Verdünnung auf den jeweiligen Kalibrierkurven durch Einzellaborvalidierung bestimmt. Der niedrigste relative Pferdeanteil der in den Ringversuch einbezogenen Zielsequenz war ein Massenanteil von 0,1 % auf der Grundlage von gravimetrisch vorbereitetem rohem Pferde-Muskelgewebe in rohem Rinder-Muskelgewebe als Hintergrund.
Der Prozess der Konformitätsbewertung ist nicht Teil dieses Dokuments.
Authenticité des aliments - Quantification de l’ADN équin par rapport à l’ADN mammalien dans la viande de bœuf crue
Le présent document spécifie un protocole de PCR en temps réel pour quantifier l’ADN équin par rapport à l’ADN mammalien total dans un échantillon de viande crue.
Les résultats de cet essai équin sont exprimés comme le rapport du nombre de copies de génomes haploïdes équins (genre Equus) sur le nombre total de copies de génomes haploïdes mammaliens. Cet essai est spécifique des représentants du genre Equus et détecte donc l’ADN de cheval, de mulet, d’âne et de zèbre.
La méthode a déjà été validée par un essai interlaboratoires et appliquée à de l’ADN extrait d’échantillons constitués de viande crue de cheval sur une base de viande crue de bœuf.
La limite de détection a été déterminée par expérimentation comme étant égale à au moins 9 copies d’équivalents génome (environ 17 copies de gène cible haploïde), à la fois pour le génome du cheval (E. caballus) et pour le génome mammalien (échantillons de viande crue), sur la base de la dilution la plus faible des courbes d’étalonnage respectives et par validation intralaboratoire. La plus petite quantité relative de viande de cheval de la séquence cible de l’essai interlaboratoires était une fraction massique de 0,1 % correspondant à des tissus musculaires crus de cheval préparés par gravimétrie sur une base de tissus musculaires crus de bœuf.
Le présent document ne traite pas du processus d’évaluation de la conformité.
Pristnost živil - Kvantitativno določanje DNK kopitarjev glede na DNK sesalcev v surovi govedini (meso)
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-februar-2025
Pristnost živil - Kvantitativno določanje DNK kopitarjev glede na DNK sesalcev v
surovi govedini (meso)
Food authenticity - Quantitation of equine DNA relative to mammalian DNA in raw beef
(meat)
Lebensmittelauthentizität - Quantifizierung von Equiden-DNA im Verhältnis zu Säugetier-
DNA in rohem Rindfleisch
Authenticité des aliments - Quantification de l’ADN équin par rapport à l’ADN mammalien
dans la viande de bœuf crue
Ta slovenski standard je istoveten z: EN 18033:2024
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.120.10 Meso in mesni proizvodi Meat and meat products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 18033
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2024
EUROPÄISCHE NORM
ICS 07.100.30; 67.120.10
English Version
Food authenticity - Quantitation of equine DNA relative to
mammalian DNA in raw beef (meat)
Authenticité des aliments - Quantification de l'ADN Lebensmittelauthentizität - Quantifizierung von
équin par rapport à l'ADN mammalien dans la viande Equiden-DNA im Verhältnis zu Säugetier-DNA in
de bœuf crue rohem Rindfleisch
This European Standard was approved by CEN on 18 November 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 18033:2024 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 Symbols and abbreviations . 5
5 Principle . 6
6 Reagents and materials . 6
7 Apparatus . 8
8 Procedure . 8
8.1 Preparation of the test sample and test portion . 8
8.2 Preparation of DNA extracts . 8
8.3 Preparation of horse calibration standards. 8
8.4 PCR setup . 9
8.4.1 Samples and controls . 9
8.4.2 Reaction mixes . 9
8.4.3 Thermal cycling . 9
9 Accept/Reject criteria . 10
9.1 General. 10
9.2 Data analysis . 10
10 Validation status and performance criteria . 11
10.1 General. 11
10.2 Repeatability . 11
10.3 Reproducibility . 12
10.4 Limit of Detection (LOD) . 12
10.5 Limit of Quantitation (LOQ). 12
10.6 Specificity . 12
10.7 Measurement uncertainty . 12
11 Test report . 12
Bibliography . 13
European foreword
This document (EN 18033:2024) has been prepared by 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 June 2025, and conflicting national standards shall be
withdrawn at the latest by June 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.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations 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.
Introduction
Food authenticity and integrity are key aspects in terms of consumer protection. Since the late 1980s,
globalization has taken place in the trade of food. During the last decades, many methods applying PCR
and particularly real-time PCR protocols have been established for the identification of animal species
used for food consumption (e.g. pig, cattle, sheep, horse, chicken, and turkey).
The European Union (EU) and United Kingdom (UK) horse meat issue in 2013, where a significant amount
of horse DNA was found in a beef burger intended for sale to the public at a supermarket, brought into
perspective that the development of harmonized and standardized protocols for the authentication of
meat products is necessary to establish reliable methods for the detection of potential food fraud.
This document contains public sector information licensed under the UK Open Government Licence
[1]
v3.0.
1 Scope
This document specifies a real-time PCR procedure for the quantitation of the amount of equine DNA
relative to total mammalian DNA in a raw meat sample.
Results of this equine assay are expressed in terms of equine (Equus genus) haploid genome copy
numbers relative to total mammalian haploid genome copy numbers. This assay is specific for
representatives of the genus Equus and therefore detects horse, mule, donkey and zebra DNA.
The method has been previously validated in a collaborative study and applied to DNA extracted from
samples that consist of raw horse meat in a raw beef (meat) background.
The limit of detection has been determined experimentally to be at least 17 horse haploid genome
equivalents (HGE) for both the equine PCR and the mammalian PCR based on the lowest dilution on the
respective calibration curves through single laboratory validation. The lowest relative horse content of
the target sequence included in the collaborative study was a mass fraction of 0,1 % based on
gravimetrically prepared raw horse muscle tissue in a raw beef muscle tissue background.
The compliance assessment process is not part of this document.
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 11781 , Molecular biomarker analysis - General guidelines for single-laboratory validation of
qualitative real-time PCR methods (ISO 11781)
EN 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 (ISO 20813)
EN ISO 21571:2005 , Foodstuffs — Methods of analysis for the detection of genetically modified organisms
and derived products — Nucleic acid extraction (ISO 21571:2005)
ISO 16577, Molecular biomarker analysis — Vocabulary for molecular biomarker analytical methods in
agriculture and food production
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16577 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/
4 Symbols and abbreviations
bp base pairs
dNTP deoxyribonucleotide triphosphate
Under preparation. Stage at the time of preparation: prEN ISO 11781.
As impacted by EN ISO 21571:2005/A1:2013.
DNA deoxyribonucleic acid
PCR polymerase chain reaction
GHR growth hormone receptor
HGE haploid genome equivalents
UNG uracil-N glycosylase
A adenine
C cytosine
G guanine
T thymine
5 Principle
Test samples containing equine DNA in a background of beef DNA are analysed by a relative quantitation
approach utilizing singleplex real-time PCR assays targeting the equine growth hormone receptor
[2] [3]
(GHR) gene and the myostatin gene present in mammals and poultry. DNA sample concentration is
quantified prior to the real-time PCR to normalize test input levels. 100 % horse (E. caballus) DNA derived
from authenticated raw horse meat materials that have been extracted and treated in the same manner
as the test samples are used to generate separate calibration curves for both the equine-specific target
and the mammalian target based on estimated haploid genome equivalents.
Test samples are evaluated using the same equine-specific and universal mammalian qPCR assays.
Estimated haploid genome equivalents are determined for the test samples using the equine and
mammalian calibration curves.
The percentage equine DNA content of the test sample is expressed as a ratio of the number of equine
haploid genome equivalents relative to the total mammalian haploid genome equivalents present in the
sample.
6 Reagents and materials
During the analysis, unless otherwise stated, use only reagents of recognized molecular biology grade,
and distilled or demineralized water or water of equivalent purity, according to EN ISO 20813. Regarding
laboratory organization, see EN ISO 20813.
Although it is not obligatory to use the rea
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