EN 15634-4:2023

Foodstuffs - Detection of food allergens by molecular biological methods - Part 4: Peanut (Arachis hypogaea) - Qualitative detection of a specific DNA sequence in chocolate by real-time PCR

This document specifies a method for the detection of peanut (Arachis hypogaea) in chocolate.
Real-time PCR (Polymerase Chain Reaction) detection of peanut is based on an 86 bp (base pair) sequence from the Ara h 2 gene of peanut.

Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 4: Erdnuss (Arachis hypogaea) - Qualitativer Nachweis einer spezifischen DNA-Sequenz in Schokolade mittels Real-time PCR

Dieses Dokument legt ein Verfahren zum Nachweis von Erdnuss (Arachis hypogaea) in Schokolade fest.
Die Real time-PCR (Polymerase-Kettenreaktion) zum Nachweis von Erdnuss beruht auf einer 86 bp (Basenpaar) großen Sequenz des Ara h 2 Gens der Erdnuss.

Produits alimentaires - Détection des allergènes alimentaires par des méthodes d’analyse de biologie moléculaire - Partie 4 : Arachide (Arachis hypogaea) - Détection qualitative d’une séquence d’ADN spécifique dans du chocolat, par PCR en temps réel

Le présent document décrit une méthode de détection de l’arachide (Arachis hypogaea) dans du chocolat.
La détection de l’arachide par PCR (réaction de polymérisation en chaîne) en temps réel repose sur une séquence de 86 pb (paires de bases) provenant du gène Ara h 2 de l’arachide.

Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 4. del: Arašidi (Arachis hypogaea) - Kvalitativno določanje specifičnega zaporedja DNK v čokoladi s PCR v realnem času

Ta metoda opisuje postopek za kvalitativno odkrivanje arašidov (Arachis hypogaea) v
v čokoladi z verižno reakcijo s polimerazo (PCR) v realnem času na podlagi gena za alergen iz arašida Ara h 2 [4, 5].

General Information

• Status: Published
• Publication Date: 21-Feb-2023
• ICS: 07.100.30 - Food microbiology
• ICS: 67.190 - Chocolate
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 12 - Food allergens
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 22-Feb-2023
• Due Date: 15-Aug-2022
• Completion Date: 22-Feb-2023
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: SIST EN 15634-4:2023 - Foodstuffs - Detection of food allergens by molecular biological methods - Part 4: Peanut (Arachis hypogaea) - Qualitative detection of a specific DNA sequence in chocolate by real-time PCR

Relations

• Replaces: CEN/TS 15634-4:2016 - Foodstuffs - Detection of food allergens by molecular biological methods - Part 4: Peanut (Arachis hypogaea) - Qualitative detection of a specific DNA sequence in chocolate by real-time PCR
• Effective Date: 19-Jan-2023

Standards Content (Sample)

SLOVENSKI STANDARD
01-maj-2023
Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 4. del:
Arašidi (Arachis hypogaea) - Kvalitativno določanje specifičnega zaporedja DNK v
čokoladi s PCR v realnem času
Foodstuffs - Detection of food allergens by molecular biological methods - Part 4: Peanut
(Arachis hypogaea) - Qualitative detection of a specific DNA sequence in chocolate by
real-time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 4: Erdnuss (Arachis hypogaea) - Qualitativer Nachweis einer
spezifischen DNA-Sequenz in Schokolade mittels Real-time PCR
Produits alimentaires - Détection des allergènes alimentaires par des méthodes
d’analyse de biologie moléculaire - Partie 4 : Arachide (Arachis hypogaea) - Détection
qualitative d’une séquence d’ADN spécifique dans du chocolat, par PCR en temps réel
Ta slovenski standard je istoveten z: EN 15634-4:2023
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
67.190 Čokolada Chocolate
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 15634-4
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2023
EUROPÄISCHE NORM
ICS 07.100.30; 67.190 Supersedes CEN/TS 15634-4:2016
English Version
Foodstuffs - Detection of food allergens by molecular
biological methods - Part 4: Peanut (Arachis hypogaea) -
Qualitative detection of a specific DNA sequence in
chocolate 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 4:
moléculaire - Partie 4 : Arachide (Arachis hypogaea) - Erdnuss (Arachis hypogaea) - Qualitativer Nachweis
Détection qualitative d'une séquence d'ADN spécifique einer spezifischen DNA-Sequenz in Schokolade
dans du chocolat, par PCR en temps réel mittels Real-time PCR
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 15634-4:2023 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 . 6
6 Apparatus and equipment . 7
6.1 General . 7
6.2 DNA extraction . 7
6.3 PCR . 7
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 of DNA concentration and purity/quality check . 9
7.4 Real-time PCR set-up . 9
7.4.1 Reaction mix for real-time PCR . 9
7.4.2 Positive control for DNA targets . 10
7.4.3 Negative control for DNA targets . 10
7.4.4 PCR inhibition control . 11
7.4.5 Amplification reagent control . 11
7.4.6 Extraction blank control . 11
7.4.7 Positive extraction control . 11
7.4.8 Temperature/time programme (real-time PCR) . 11
7.4.9 Accept/Reject criteria . 11
7.4.10 Identification . 12
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
9 Test report . 14
Bibliography . 16
European foreword
This document (EN 15634-4:2023) 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 15634-4:2016.
In comparison with CEN/TS 15634-4:2016, 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 7.4.9 “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 method for the detection of peanut (Arachis hypogaea) in chocolate.
Real-time PCR (Polymerase Chain Reaction) detection of peanut is based on an 86 bp (base pair) sequence
from the Ara h 2 gene of peanut.
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 chocolate 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 measured by photospectrometry. Real-time PCR is used to detect a
peanut specific DNA sequence. The real-time PCR method involves a fluorescence detection with a
sequence specific hydrolysis probe [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 %.
5.2.3 Ethylenediaminetetraacetic acid disodium salt (Na EDTA).
5.2.4 Cetyltrimethylammoniumbromide (CTAB).
5.2.5 Hydrochloric acid, mass fraction w = 37 %.
...