prEN 15634-7

SLOVENSKI STANDARD
01-marec-2026
Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 7.
del:Arašidi (Arachis hypogaea) - Kvalitativno določanje specifičnega zaporedja
DNK v živilih s PCR v realnem času
Foodstuffs - Detection of food allergens by molecular biological methods - Part 7: Peanut
(Arachis hypogaea) - Qualitative detection of a specific DNA sequence in food by real-
time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 7: Erdnuss (Arachis hypogaea) - Qualitativer Nachweis einer
spezifischen DNA-Sequenz in Lebensmitteln mittels Real-time PCR
Produits alimentaires - Détection des allergènes alimentaires par des méthodes
d’analyse de biologie moléculaire - Partie 7 : Arachide (Arachis hypogaea) - Détection
qualitative d’une séquence d’ADN spécifique dans des produits alimentaires, par PCR en
temps réel
Ta slovenski standard je istoveten z: prEN 15634-7
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.080.10 Sadje in sadni proizvodi Fruits and derived products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
EUROPEAN STANDARD
NORME EUROPÉENNE
EUROPÄISCHE NORM
January 2026
ICS 07.100.30; 67.080.10
English Version
Foodstuffs - Detection of food allergens by molecular
biological methods - Part 7: Peanut (Arachis hypogaea) -
Qualitative detection of a specific DNA sequence in food 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 7: Erdnuss
moléculaire - Partie 7 : Arachide (Arachis hypogaea) - (Arachis hypogaea) - Qualitativer Nachweis einer
Détection qualitative d'une séquence d'ADN spécifique spezifischen DNA-Sequenz in Lebensmitteln mittels
dans des produits alimentaires, par PCR en temps réel Real-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, Türkiye 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
© 2026 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 15634-7:2026 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 . 6
5.2 Extraction reagents . 6
5.3 Real-time PCR reagents . 7
5.3.1 Master mix for real-time PCR, containing thermostable DNA polymerase (for hot-
start PCR) and PCR buffer solution (containing reaction buffer, dNTPs, and MgCl2),
as a dilutable concentrate. . 7
5.3.2 Oligonucleotides . 7
6 Apparatus and equipment . 7
6.1 General . 7
6.2 DNA extraction . 8
6.2.7 Equipment for DNA quantity estimation (optional), e.g. UV-photometer. . 8
6.3 PCR . 8
7 Procedure . 8
7.1 General . 8
7.2 Sample preparation . 8
7.3 Preparation of DNA extracts . 8
7.3.1 DNA extraction with CTAB and DNA purification . 8
7.3.2 Optional quantification of DNA concentration . 10
7.4 Real-time PCR set-up . 10
7.4.1 Reaction mix for real-time PCR . 10
7.4.2 Positive control for DNA targets . 11
7.4.3 Negative control for DNA targets . 11
7.4.4 Amplification reagent control . 11
7.4.5 Extraction blank control . 11
7.4.6 Positive extraction control . 11
7.4.7 Temperature/time program (real-time PCR) . 11
7.4.8 Accept/Reject criteria . 12
7.4.9 Identification . 12
8 Validation . 12
8.1 General . 12
8.2 Specificity . 12
8.3 Sensitivity . 13
8.4 Method validating interlaboratory study (ring trial) . 13
8.4.1 Setup of the ring trial . 13
8.4.2 Deviations from the ring trial study protocol . 14
8.4.3 Ring trial validation results . 14
9 Test report . 17
Bibliography . 18
European foreword
This document (prEN 15634-7:2026) has been prepared by Technical Committee CEN/TC 275 "Food
analysis - Horizontal method", the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
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.
1 Scope
This document specifies a method for the qualitative detection of peanut (Arachis hypogaea) DNA in
food using real-time PCR and targeting a multicopy mitochondrial sequence, in the context of allergen
analyses.
The method was previously validated in an interlaboratory study (ring trial) and applied to DNA
extracted from samples that consist of defined proportions of peanut in rice biscuits, wheat biscuits,
cooked sausage and milk powder.
The limit of detection of the peanut real-time PCR has been determined experimentally to be at least
0,5 mg peanut/kg.
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, 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 15634-1 and EN 15842and
the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at http://www.iso.org/obp
— IEC Electropedia: available at http://www.electropedia.org/
4 Principle
Total DNA is extracted from the sample using a cetyltrimethylammonium bromide (CTAB) method EN
ISO 21571 [1]. In general, CTAB functions as a cationic surfactant, leading to the formation of complexes
with DNA which are precipitated in the presence of low salt concentration leaving other impurities in
solution.
In short, potential PCR inhibitors are removed from the DNA extract through chloroform extraction and
subsequent CTAB precipitation followed by an alcoholic precipitation of the DNA. The DNA precipitate
is washed, dissolved in buffer solution and the DNA content can be measured. Real-time PCR is used to
detect a 104 base pair (bp) long sequence targeting a multicopy sequence from mitochondrial peanut
DNA close to the coding region of the ATPase subunit 6 (atp6) (Genbank : MW448460. The real-time
PCR method involves a fluorescence detection with sequence-specific hydrolysis probes, see [2].
5 Reagents
NCBI-GenBank® is an example of a suitable search tool for free use. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN.
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
relevant reagents in water and autoclaving, unless otherwise specified.
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 Cetyltrimethylammonium bromide (CTAB).
5.2.5 Hydrochloric acid, mass fraction w = 37 %.
5.2.6 Isopropyl alcohol.
5.2.7 Proteinase K.
5.2.8 RNase A.
5.2.9 α-Amylase (>1 500 U/mg Protein).
5.2.10 Sodium chloride.
5.2.11 Sodium hydroxide solution.
5.2.12 Tris(hydroxymethyl)-aminomethane (TRIS).
5.2.13 Tris(hydroxymethyl)-aminomethane Hydrochloride (Tris-HCl).
5.2.14 CTAB extraction buffer solution, containing:
— CTAB (5.2.4 ), mass concentration ρ = 20 g/l,
— sodium chloride (5.2.10), substance concentration c = 1,4 mol/l,
— TRIS (5.2.12), 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).
5.2.15 Proteinase K solution, ρ = 20 mg/ml.
The freshly produced Proteinase K solution should be stored in the form of aliquots at −20 °C.
5.2.16 Amylase solution, ρ = 10 mg/ml.
Weigh 100 mg of α-amylase (5.2.9) into a sterile glass container and add 10 ml of double distilled, sterile
water.
The solution should be kept refrigerated after preparation until analysis. Frozen storage is possible,
whereby repeated freezing and thawing is to be avoided.
Any sediment in the solution has no negative influence on the test.
5.2.17 Solution buffer for RNase A, containing:
— Tris-HCl (5.2.13 ), (c = 0,01 mol/l);
— sodium chloride (5.2.10), (c = 0,015 mol/l).
The pH is adjusted to 7,5 by adding hydrochloric acid (5.2.5) or sodium hydroxide solution (5.2.11).
5.2.18 RNase A solution, ρ = 10 mg/ml.
Weigh 100 mg RNase A (5.2.8) into a sterile glass jar and add 10 ml RNase A solution buffer (5.2.17).
Heat for 15 min to 100 °C and cool slowly to room temperature.
The aliquoted solution can be stored frozen at -20 °C for at least 12 months.
5.2.19 CTAB precipitation buffer solution, containing:
— CTAB (5.2.4 ) (ρ = 5 g/l),
— sodium chloride (5.2.10) (c = 0,04 mol/l).
5.2.20 Sodium chloride solution, c = 1,2 mol/l.
5.2.21 0,2 × TE buffer solution, containing:
— TRIS (5.2.12), c = 0,002 mol/l,
— Na EDTA (5.2.3), c = 0,000 2 mol/l.
The pH is adjusted to 8,0 by adding hydrochloric acid (5.2.5) or sodium hydroxide solution (5.2.11).
NOTE For all solutions commercially available alternatives can be used.
5.3 Real-time PCR reagents
5.3.1 Master mix for real-time PCR, containing thermostable DNA polymerase (for hot-start
PCR) and PCR buffer solution (containing reaction buffer, dNTPs, and MgCl2), as a dilutable
concentrate.
Ready to use reagents or single components may be used as a master mix.
5.3.2 Oligonucleotides
An overview of the oligonucleotides is provided in Table 1 .
Table 1 — Primers and probes for the real-time PCR
Name DNA sequence of the oligonucleotide
atp6-F 5′ – CAg ggC ATC CTT AAC Tgg Ag – 3′
atp6-R 5′ – ggA AAg ACg ggT Tgg TgA TA – 3′
a
atp6-P
5′ – FAM – AAg gCg AAg AAg ggT CAg AT – BHQ1 – 3′
a FAM: 6-carboxyfluorescein; BHQ1: black hole quencher 1
6 Apparatus and equipment
6.1 General
In addition to the typical laboratory facilities, the following equipment shall be used.
Due to the high sensitivity of the PCR analytics and the risk of DNA contaminations, the use of aerosol-
protected filter-tips in the DNA extraction procedure is mandatory. Plastic and glass materials shall be
sterilized and free of DNA before use.
Further general requirements are given in EN ISO 21571 [1].
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 6 000 g.
If no centrifuge with a correspondingly high g-force is available, it is also possible to centrifuge at a
speed of about 4 000 g for a correspondingly extended time until a supernatant is obtained.
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 Equipment for DNA quantity estimation (optional), e.g. UV-photometer.
6.2.8 Vacuum dryer (optional).
6.2.9 Mechanical quick-shaker, e.g. vortex mixer.
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.
7 Procedure
7.1 General
General aspects are described in EN 15634-1 [3]and EN ISO 21571 [1].
7.2 Sample preparation
It should be ensured that the test sample taken after milling or homogenizing is representative of the
laboratory sample.
7.3 Preparation of DNA extracts
7.3.1 DNA extraction with CTAB and DNA purification
The sample DNA analysed in the ring trial were obtained using the DNA extraction method described
below.
2 –2
g = 9,81 m⋅s
The use of a commercially available kit is acceptable in place of the DNA extraction procedure described
below, provided it yields comparable or better results.
In parallel to the test samples, the controls listed in 7.4.5 and 7.4.6 should be performed adequately.
— Weigh 2 g of the homogenized sample into a 50 ml centrifuge tube (tube A).
— Add 10 ml of CTAB extraction buffer (5.2.14) and 20 μl of amylase solution (5.2.16). Suspend well
by rotating the centrifuge tube around the longitudinal axis. Incubate for 30 min at 65 °C while
stirring or gently shaking; alternatively, use a hybridization oven.
— Add 20 μl Proteinase K solution (5.2.15) and mix.
— Incubate and shake for a further 90 min at 65 °C.
— Centrifuge for 10 min at 6 000 g at room temperature.
— Place 900 µl of chloroform (5.2.1) in a 2 ml reaction vial (tube B).
— Add 900 µl of supernatant from tube A to tube B and mix thoroughly for 30 s.
— Centrifuge for 10 min at about 14 500 g at room temperature for phase separation.
— The interphase should be as small as possible, if necessary, mix briefly again and centrifuge for
another 20 min.
— Add 600-650 µl of supernatant from tube B to a new 2 ml reaction vial (tube C).
— Add 2 parts by volume of CTAB precipitation buffer solution (5.2.19) and mix.
— Incubate for 60 min at room temperature.
— Centrifuge for 10 min at about 14 500 g at room temperature.
— Carefully remove and discard the supernatant.
— Incubate the precipitate in 900 μl NaCl solution (5.2.20) for at least 30 min at 37 °C while gently
shaking to dissolve the pellet
...