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 method describes a procedure for the qualitative detection of peanut (Arachis hypogaea) in
chocolate using real-time PCR based on the gene for the peanut allergen Ara h 2 [4, 5].

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 reel

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

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SLOVENSKI STANDARD
oSIST prEN 15634-4:2022
01-januar-2022

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 reel

Ta slovenski standard je istoveten z: prEN 15634-4
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
oSIST prEN 15634-4:2022 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 15634-4:2022
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oSIST prEN 15634-4:2022
DRAFT
EUROPEAN STANDARD
prEN 15634-4
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2021
ICS 07.100.30; 67.190 Will supersede 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: Erdnuss

moléculaire - Partie 4 : Arachide (Arachis hypogaea) - (Arachis hypogaea) - Qualitativer Nachweis einer

Détection qualitative d'une séquence d'ADN spécifique spezifischen DNA-Sequenz in Schokolade mittels Real-

dans du chocolat, par PCR en temps reel time PCR

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 275.

If this draft becomes a European Standard, 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.

This draft European Standard was established by CEN 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, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 15634-4:2021 E

worldwide for CEN national Members.
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prEN 15634-4:2021 (E)
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........................................................................................................................................................... 7

7.1 General ................................................................................................................................................................ 7

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 ................................................................................................................................ 10

7.4.5 Amplification reagent control ................................................................................................................. 10

7.4.6 Extraction blank control ............................................................................................................................ 10

7.4.7 Positive extraction control ....................................................................................................................... 10

7.4.8 Temperature/time programme (real-time PCR) ............................................................................. 11

7.4.9 Accept/Reject criteria ................................................................................................................................ 11

7.4.10 Identification ................................................................................................................................................. 11

8 Validation ........................................................................................................................................................ 12

8.1 General ............................................................................................................................................................. 12

8.2 Specificity ........................................................................................................................................................ 12

8.3 Inter-laboratory validation ...................................................................................................................... 12

8.3.1 Setup of the inter-laboratory study ....................................................................................................... 12

8.3.2 Inter-laboratory validation results ....................................................................................................... 12

9 Test report ...................................................................................................................................................... 14

Bibliography ................................................................................................................................................................. 15

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prEN 15634-4:2021 (E)
European foreword

This document (prEN 15634-4:2021) has been prepared by Technical Committee CEN/TC 275 “Food

analysis – Horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document will supersede CEN/TS 15634-4:2016.

In comparison with the previous edition, 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) added new subclause 7.4.9 “Accept/Reject criteria”;
e) restructured clauses in alignment with EN 15634-2:2019.
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prEN 15634-4:2021 (E)
Introduction
For the use of this document the term:
— ‘shall’ indicates a requirement;
— ‘should’ indicates a recommendation;
— ‘may’ indicates a permission; and
— ‘can’ indicates a possibility and/or a capability.
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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 http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
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 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 %.
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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 data for this (see also 8.3.1).

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, 10 µmol each.
5.4.2.1 Peanut (AR-58 F), gCA gCA gTg ggA ACT CCA Agg AgA CA.
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5.4.2.2 Peanut (AR-143 R), gCA TgA gAT gTT gCT CgC Ag.

5.4.2.3 Peanut probe (AR-103 T), FAM – CgA gAg ggC gAA CCT gAg gCC – TAMRA– or BHQ1 .

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.
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 spectrometer or other detection instruments,
...

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