EN 15634-5:2023

SLOVENSKI STANDARD
01-maj-2023
Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 5. del:
Gorčica (Sinapis alba) in soja (Glycine max) - Kvalitativno določanje specifičnega
zaporedja DNK v obarjenih klobasah s PCR v realnem času
Foodstuffs - Detection of food allergens by molecular biological methods - Part 5:
Mustard (Sinapis alba) and soya (Glycine max) - Qualitative detection of a specific DNA
sequence in cooked sausages by real-time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 5: Senf (Sinapis alba) sowie Soja (Glycine max) - Qualitativer Nachweis
einer spezifischen DNA-Sequenz in Brühwürsten mittels Real-time PCR
Produits alimentaires - Détection des allergènes alimentaires par des méthodes
d’analyse de biologie moléculaire - Partie 5 : Moutarde (Sinapis alba) et soja (Glycine
max) - Détection qualitative d’une séquence d’ADN spécifique dans des saucisses
cuites, par PCR en temps réel
Ta slovenski standard je istoveten z: EN 15634-5:2023
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.050 Splošne preskusne in General methods of tests and
analizne metode za živilske analysis for food products
proizvode
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 15634-5
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2023
EUROPÄISCHE NORM
ICS 07.100.30; 67.120.10 Supersedes CEN/TS 15634-5:2016
English Version
Foodstuffs - Detection of food allergens by molecular
biological methods - Part 5: Mustard (Sinapis alba) and
soya (Glycine max) - Qualitative detection of a specific
DNA sequence in cooked sausages by real-time PCR
Produits alimentaires - Détection des allergènes Lebensmittel - Nachweis von Lebensmittelallergenen
alimentaires par des méthodes d'analyse de biologie mit molekularbiologischen Verfahren - Teil 5: Senf
moléculaire - Partie 5 : Moutarde (Sinapis alba) et soja (Sinapis alba) sowie Soja (Glycine max) - Qualitativer
(Glycine max) - Détection qualitative d'une séquence Nachweis einer spezifischen DNA-Sequenz in
d'ADN spécifique dans des saucisses cuites, par PCR en Brühwürsten mittels Real-time PCR
temps réel
This European Standard was approved by CEN on 16 January 2023.
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
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 15634-5:2023 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 . 5
5 Reagents . 5
5.1 General . 5
5.2 Extraction reagents . 5
5.3 DNA purification by means of solid phase extraction . 6
5.4 Real-time PCR reagents . 7
6 Apparatus and equipment . 7
6.1 General . 7
6.2 DNA extraction . 7
6.3 PCR . 8
7 Procedure. 8
7.1 General . 8
7.2 Sample preparation . 8
7.3 Preparation of extracts . 8
7.3.1 DNA extraction with CTAB and DNA purification . 8
7.3.2 Quantification and normalization of DNA concentration . 9
7.4 Real-time PCR set-up . 10
7.4.1 Reaction mix for real-time PCR . 10
7.4.2 Amplification reagent control . 11
7.4.3 Extraction blank control . 11
7.4.4 Positive extraction control . 11
7.4.5 Temperature/time programme (real-time PCR) . 11
7.4.6 Accept/Reject criteria . 11
7.4.7 Identification . 11
8 Validation . 12
8.1 General . 12
8.2 Specificity . 12
8.3 Ring trial validation study . 12
8.3.1 Setup of the ring trial study . 12
8.3.2 Ring trial validation results . 13
8.3.3 Qualitative interpretation. 14
9 Test report . 16
Bibliography . 17
European foreword
This document (EN 15634-5:2023) has been prepared by Technical Committee CEN/TC 275 “Food
analysis – Horizontal methods”, 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 August 2023, and conflicting national standards shall be
withdrawn at the latest by August 2023.
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 15634-5:2016.
In comparison with CEN/TS 15634-5:2016, the following technical modifications have been made:
a) the document was converted from a Technical Specification into a European standard;
b) normative references and terms and definitions clause added;
c) PCR controls moved from Clause 3 “Reagents” to Clause 7 “Procedure”;
d) new subclause 7.4.6 “Accept/Reject criteria” added;
e) restructured clauses in alignment with EN 15634-2:2019.
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
For the use of this document the term:
— ‘shall’ indicates a requirement;
— ‘should’ indicates a recommendation;
— ‘may’ indicates a permission;
— ‘can’ indicates a possibility and/or a capability.
1 Scope
This document specifies a procedure for the qualitative detection of species specific DNA from white
mustard (Sinapis alba) and soya (Glycine max) in cooked sausages using singleplex real-time PCR based
on the genes MADS-D (mustard) and lectin (soya).
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 15634-1:2019, Foodstuffs - Detection of food allergens by molecular biological methods - Part 1: General
considerations
EN 15842, Foodstuffs - Detection of food allergens - General considerations and validation of methods
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 15842 and EN 15634-1 apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
• IEC Electropedia: available at https://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp/ui
4 Principle
Total DNA from cooked sausages is extracted from the sample using a cetyltrimethylammoniumbromide
(CTAB) method. Potential PCR inhibitors are removed from the DNA extracted by purification with solid
phase columns and the DNA content is estimated. Real-time PCR is used to detect a 74 base pair (bp) long
sequence of the DNA for the MADS-D protein of Sinapis alba (NCBI accession no. Y08626 ) or a 81 bp
long sequence from the soya lectin gene. The real-time PCR method involves a fluorescence detection
with sequence specific hydrolysis probes, see [1] or EN ISO 21570:2005, C.1 [2].
5 Reagents
5.1 General
The following general conditions for analysis should be followed, unless specified differently. Use only
analytical grade reagents suitable for molecular biology. All water shall be free from DNA and nucleases,
e.g. double distilled or equivalent (molecular grade). Solutions shall be prepared by dissolving the
appropriate reagents in water and autoclaving, unless specified differently.
5.2 Extraction reagents
5.2.1 Chloroform.
5.2.2 Ethanol, volume fraction φ = 70 %.

NCBI-GenBank® is an example of a suitable search tool for
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