EN 15634-3:2023

Foodstuffs - Detection of food allergens by molecular biological methods - Part 3: Hazelnut (Corylus avellana) - Qualitative detection of a specific DNA sequence in chocolate by real-time PCR

This document specifies a method for the detection of hazelnut (Corylus avellana) in chocolate.
Real-time PCR (Polymerase chain reaction) detection of hazelnut is based on an152 bp (base pair) sequence from the corA 1 gene of hazelnut.

Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 3: Haselnuss (Corylus avellana) - Qualitativer Nachweis einer spezifischen DNA-Sequenz in Schokolade mittels Real-time PCR

Dieses Dokument legt ein Verfahren zum Nachweis von Haselnuss (Corylus avellana) in Schokolade fest.
Die Real time-PCR (Polymerase-Kettenreaktion) zum Nachweis von Haselnuss beruht auf einer 152 bp (Basenpaar) großen Sequenz des corA 1 Gens der Haselnuss.

Produits alimentaires - Détection des allergènes alimentaires par des méthodes d’analyse de biologie moléculaire - Partie 3 : Noisette (Corylus avellana) - 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 la noisette (Corylus avellana) dans du chocolat.
La détection de la noisette par PCR (réaction de polymérisation en chaîne) en temps réel repose sur une séquence de 152 pb (paires de base) provenant du gène corA 1 de la noisette.

Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 3. del: Lešnik (Corylus avellana) - Kvalitativno določanje specifičnega zaporedja DNK v čokoladi s PCR v realnem času

Ta metoda opisuje postopek za kvalitativno odkrivanje lešnikov (Corylus avellana) v
čokoladi. DNK se izvleče iz čokolade in specifičen niz DNK za lešnik
se ugotovi iz gena za corA 1.

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 15634-3:2023 - Foodstuffs - Detection of food allergens by molecular biological methods - Part 3: Hazelnut (Corylus avellana) - Qualitative detection of a specific DNA sequence in chocolate by real-time PCR

Relations

• Replaces: CEN/TS 15634-3:2016 - Foodstuffs - Detection of food allergens by molecular biological methods - Part 3: Hazelnut (Corylus avellana) - Qualitative detection of a specific DNA sequence in chocolate by real-time PCR
• Effective Date: 19-Jan-2023

Overview

EN 15634-3:2023 (CEN) specifies a qualitative real-time PCR method for the detection of hazelnut (Corylus avellana) in chocolate. The assay targets a 152 bp sequence from the corA1 gene of hazelnut and uses a sequence-specific hydrolysis probe for fluorescence-based detection. The standard defines sample preparation, CTAB-based DNA extraction, DNA purification, PCR setup, controls and acceptance criteria to support reliable food allergen detection by molecular biological methods.

Key topics and technical requirements

• Scope: Qualitative detection of hazelnut DNA in chocolate matrices using real-time PCR.
• Molecular target: 152 bp fragment of the corA1 gene (Corylus avellana).
• Oligonucleotides:
  • Hazelnut iF: 5´– TAC AgC ATC ATC gAg ggA ggT C –3´
  • Hazelnut iR: 5´– CTC CTC ATT gAT TgA AgC gTT g –3´
  • Hazelnut probe: 5´– FAM–AgA Tgg Cgg CAg CCC CTC AT–TAMRA–3´
• Sample extraction: CTAB extraction buffer with Proteinase K, chloroform:isoamyl alcohol partitioning, isopropanol precipitation, ethanol wash and re-suspension in TE buffer; solid-phase column purification to remove PCR inhibitors.
• PCR reagents & equipment: PCR master mix (HotStart Taq, dNTPs, MgCl2), real-time PCR instrument capable of FAM/TAMRA detection, aerosol-protected filter tips, centrifuges and spectrophotometer for DNA quantification.
• Controls and validation:
  • Positive and negative controls for DNA targets
  • Extraction blank and positive extraction control
  • PCR inhibition control and amplification reagent control
  • Defined accept/reject criteria and identification procedures
  • Validation and ring-trial requirements described for specificity and method performance
• Quality practice: Use DNA-free reagents and sterilized consumables; follow EN ISO 21571 and EN 15634-1 general considerations.

Applications and users

• Food testing laboratories performing allergen screening and compliance testing for chocolate and chocolate-containing products.
• Food manufacturers and ingredient suppliers for internal quality control, supplier verification and allergen management.
• Regulatory and certification bodies verifying labeling compliance and food safety requirements.
• Research and method development teams working on molecular detection of tree-nut allergens.

This standard supports accurate hazelnut detection in chocolate, helping reduce allergic risk, ensure regulatory compliance and improve consumer safety through robust real-time PCR workflows.

Related standards

• EN 15634-1:2019 - Foodstuffs: Detection of food allergens by molecular biological methods - General considerations
• EN 15842 - Foodstuffs: Detection of food allergens - General considerations and validation of methods

Keywords: EN 15634-3:2023, hazelnut detection, real-time PCR, Corylus avellana, corA1 gene, CTAB extraction, chocolate allergen testing, food allergen detection, molecular biological methods.

Standards Content (Sample)

SLOVENSKI STANDARD
01-maj-2023
Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 3. del:
Lešnik (Corylus avellana) - Kvalitativno določanje specifičnega zaporedja DNK v
čokoladi s PCR v realnem času
Foodstuffs - Detection of food allergens by molecular biological methods - Part 3:
Hazelnut (Corylus avellana) - Qualitative detection of a specific DNA sequence in
chocolate by real-time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 3: Haselnuss (Corylus avellana) - 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 3 : Noisette (Corylus avellana) - Détection
qualitative d’une séquence d’ADN spécifique dans du chocolat, par PCR en temps réel
Ta slovenski standard je istoveten z: EN 15634-3:2023
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 15634-3
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2023
EUROPÄISCHE NORM
ICS 07.100.30; 67.190 Supersedes CEN/TS 15634-3:2016
English Version
Foodstuffs - Detection of food allergens by molecular
biological methods - Part 3: Hazelnut (Corylus avellana) -
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 3:
moléculaire - Partie 3 : Noisette (Corylus avellana) - Haselnuss (Corylus avellana) - Qualitativer Nachweis
Détection qualitative d'une séquence d'ADN spécifique einer spezifischen DNA-Sequenz in Schokolade mittels
dans du chocolat, par PCR en temps réel 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 15634-3:2023 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 and normalization of DNA concentration .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 . 11
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 . 12
7.4.10 Identification . 12
8 Validation . 12
8.1 General . 12
8.2 Specificity . 12
8.3 Ring trial validation study . 13
8.3.1 Setup of the ring trial study . 13
8.3.2 Ring trial validation results . 13
9 Test report . 15
Bibliography . 16
European foreword
This document (EN 15634-3:2023) 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 15634-3:2016.
In comparison with CEN/TS 15634-3:2016, 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 hazelnut (Corylus avellana) in chocolate.
Real-time PCR (Polymerase chain reaction) detection of hazelnut is based on a 152 bp (base pair)
sequence from the corA 1 gene of hazelnut.
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 estimated. Real-time PCR is used to detect a hazelnut
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 %.
5.2.6 Isoamyl alcohol.
5.2.7 Isopropanol.
5.2.8 Proteinase K.
5.2.9 Sodium chloride.
5.2.10 Sodium hydroxide solution.
5.2.11 Tris(hydroxymethyl)aminomethane (TRIS).
5.2.12 Chloroform isoamyl alcohol mixture, 24 parts by volume of chloroform (5.2.1) are mixed
with one part by volume of isoamyl alcohol (5.2.6).
Similar mixtures commercially available may be used.
5.2.13 CTAB extraction buffer solution, containing:
— CTAB (5.2.4), mass concentration ρ = 20 g/l;
— sodium chloride (5.2.9), substance concentration c = 1,4 mol/l;
— TRIS (5.2.11), substance concentration c = 0,1 mol/l;
— Na EDTA (5.2.3), substance concentration c = 0,02 mol/l.
The pH is adjusted to 8,0 by adding hydrochloric acid (5.2.5).
5.2.14 Proteinase K solution, ρ = 20 mg/ml.
The freshly produced Proteinase K solution should be stored in the form of aliquots at −20 °C.
5.2.15 TE buffer solution, containing:
— TRIS (5.2.11), c = 0,001 mol/l;
— Na EDTA (5.2.3), c = 0,000 1 mol/l.
The pH is adjusted to 8,0 by adding hydrochloric acid (5.2.5) or sodium hydroxide solution (5.2.10).
5.3 DNA purification by means of solid phase extraction
For the DNA purification, different methods may be used.
Several formats are commercially available, among them spin filter columns or plates. Commercially
available kits may be used if appropriate. Follow the manufacturer's instructions for this.
5.4 Real-time PCR reagents
5.4.1 PCR master mix, containing reaction buffer, dNTPs, MgCl and Hotstart Taq polymerase.
Ready to use reagents or single components may be used as a PCR master mix, insofar as they provide
comparable or better results.
5.4.2 Oligonucleotides, 5 µmol each.
5.4.2.1 Hazelnut iF, 5´– TAC AgC ATC ATC gAg ggA ggT C – 3´.
5.4.2.2 Hazelnut iR, 5´– CTC CTC ATT gAT TgA AgC gTT g – 3´.
5.4.2.3 Hazelnut probe, 5´– FAM – AgA Tgg Cgg CAg CCC CTC AT – TAMRA – 3´.
Equivalent reporter dyes and/or quencher dyes may be used if they are shown to give comparable or
better results.
6 Apparatus and equipment
6.1 General
In addition to the usual laboratory facilities, the following equipment shall be used.
Due to the high sensitivity of the PCR analytics and the risk of DNA contaminations resulting from it,
the use of aerosol protected filter tips in the DNA extraction procedure is obligatory. Plastic and glass
materials shall be sterilized and free of DNA before use.
Further general requirements are given in EN ISO 21571.
6.2 DNA extraction
6.2.1 Suitable reaction vials, 1,5 ml and 2 ml, DNA-free.
6.2.2 50 ml centrifuge tubes, sterile.
6.2.3 Thermostat or water bath, preferably with shaker function.
6.2.4 Centrifuge, suitable for centrifuging 50 ml centrifuge tubes at 8 000 g .
6.2.5 Centrifuge, suitable for centrifuging 1,5 ml and 2 ml reaction vials at 14 500 g.
6.2.6 Equipment and/or material for grinding the sample, e.g. blender or mill.
6.2.7 UV spectrophotometer or other detection instruments, suitable for estimating the amount
of DNA.
6.3 PCR
6.3.1 Suitable PCR tubes.
6.3.2 Microcentrifuge for PCR tubes.
6.3.3 Real-time PCR equipment, suitable for excitation and for emission measurement of
fluorescence-marked oligonucleotides.
...