oSIST prEN 15634-8:2026

SLOVENSKI STANDARD
01-marec-2026
Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 8. del:
Arašidi (Arachis hypogaea), lešniki (Corylus spp.), orehi (Juglans regia) in indijski
oreščki (Anacardium occidentale) - Kvalitativno določanje specifičnega zaporedja
DNK v živilih s PCR v realnem času
Foodstuffs - Detection of food allergens by molecular biological methods - Part 8: Peanut
(Arachis hypogaea), hazelnut (Corylus spp.), walnut (Juglans regia) and cashew
(Anacardium occidentale) - Qualitative detection of specific DNA sequences in food by
real-time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 8: Erdnuss (Arachis hypogaea), Haselnuss (Corylus spp.), Walnuss
(Juglans regia) und Cashew (Anacardium occidentale) - Qualitativer Nachweis
spezifischer DNA-Sequenzen in Lebensmitteln mittels Real-time PCR
Produits alimentaires - Détection des allergènes alimentaires par des méthodes
d’analyse de biologie moléculaire - Partie 8 : Arachide (Arachis hypogaea), noisette
(Corylus spp.), noix (Juglans regia) et noix de cajou (Anacardium occidentale) -
Détection qualitative de séquences d’ADN spécifiques dans les produits alimentaires par
PCR en temps réel
Ta slovenski standard je istoveten z: prEN 15634-8
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 8: Peanut (Arachis hypogaea),
hazelnut (Corylus spp.), walnut (Juglans regia) and cashew
(Anacardium occidentale) - Qualitative detection of
specific DNA sequences 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 8: Erdnuss
moléculaire - Partie 8 : Arachide (Arachis hypogaea), (Arachis hypogaea), Haselnuss (Corylus spp.), Walnuss
noisette (Corylus spp.), noix (Juglans regia) et noix de (Juglans regia) und Cashew (Anacardium occidentale) -
cajou (Anacardium occidentale) - Détection qualitative Qualitativer Nachweis spezifischer DNA-Sequenzen in
de séquences d'ADN spécifiques dans les produits Lebensmitteln mittels Real-time PCR
alimentaires par PCR en temps réel
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 15634-8:2026 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 . 6
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, MgCl ), as
a dilutable concentrate. . 7
5.3.2 Oligonucleotides . 7
6 Apparatus and equipment . 8
6.1 General . 8
6.2 DNA extraction . 8
6.3 PCR . 9
7 Procedure . 9
7.1 General . 9
7.2 Sample preparation . 9
7.3 Preparation of DNA extracts . 9
7.3.1 DNA extraction with CTAB and DNA purification . 9
7.3.2 Optional quantification of DNA concentration . 10
7.4 Real-time PCR set-up . 11
7.4.1 Reaction mix for real-time PCR . 11
7.4.2 Positive control for DNA targets . 12
7.4.3 Negative control for DNA targets . 12
7.4.4 Amplification reagent control . 12
7.4.5 Extraction blank control . 12
7.4.6 Positive extraction control . 12
7.4.7 Temperature/time program (real-time PCR) . 12
7.4.8 Accept/Reject criteria . 13
7.4.9 Identification . 13
8 Validation . 13
8.1 General . 13
8.2 Specificity . 13
8.3 Sensitivity . 14
8.4 Method validating interlaboratory study (ring trial) . 15
8.4.1 Setup of the ring trial . 15
8.4.2 Deviations from the ring trial protocol . 16
8.4.3 Ring trial validation results . 16
9 Test report . 24
Bibliography . 25
European foreword
This document (prEN 15634-8:2026) 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
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 the species-specific DNA of peanut
(Arachis hypogaea), hazelnut (Corylus spp.), walnut (Juglans regia) and cashew (Anacardium
occidentale) in food of animal and plant origin, using real-time PCR, 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, hazelnut, walnut and cashew in
rice biscuits, cooked sausage, sauce powder, vegan cookie and veggie burger (powder).
The limit of detection of each real-time PCR has been determined experimentally to be about 5 mg/kg
(10 mg/kg for roasted peanuts).
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 15634-1and 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.
From the extracted sample DNA, nut-specific DNA sequences are amplified using real-time PCR.
For the different genera the following specific DNA target sequences are used:
— Peanut: multicopy sequence from mitochondrial DNA close to the coding region of the ATPase
subunit 6 (atp6) of Arachis hypogaea, 104 base pairs (bp) long (Genbank : MW448460.1 [2][3]
— Hazelnut: multicopy sequence from the internal transcribed spacer 2 (ITS2) region from the 5.8S
ribosomal RNA gene of Corylus spp., 62 bp long (GenBank: MG237811.1 [4])
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.
— Walnut: multicopy sequence from a non-coding segment of the large single copy (LSC) region of
the chloroplast genome of Juglans regia, 67 bp long (GenBank : MF167463.1 [4])
— Cashew: multicopy sequence from the internal transcribed spacer 2 (ITS2) region from the 5.8S
ribosomal RNA gene of Anacardium occidentale, 69 bp long (GenBank : AB071690.1 [4]).
The real-time PCR method involves a fluorescence detection with sequence-specific hydrolysis
probes, see [2].
5 Reagents
5.1 General
The following general conditions for analysis should be followed, unless specified otherwise. 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 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), molar concentration c = 1,4 mol/l,
— TRIS (5.2.12), c = 0,1 mol/l,
— Na EDTA (5.2.3), 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 should 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 with 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 down to room temperature slowly.
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, MgCl ), as a dilutable
concentrate.
Ready to use reagents or single components may be used as a PCR master mix.
5.3.2 Oligonucleotides
The oligonucleotides are shown in Table 1.
Table 1 — Primers and probes for the real-time PCR
Name DNA sequence of the oligonucleotide
Peanut
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′ – HEX - AAg gCg AAg AAg ggT CAg AT - BHQ1 – 3′
Hazelnut
H-nuss-F 5′ - gCg gCT ggC CTA AAA gC - 3′
H-nuss-R 5′ - ggT TTg TCA ACC ACC gAT TgT - 3′
5′ - 6-FAM - AgT CCT Cgg CgA CgA g - MGB – BMN-Q535
H-nuss-MGB
b
- 3′
Walnut
WalN F 5‘ - TTT CCA TAT CgA TCC ATg Cg - 3‘
WalN R 5‘ - TCT CTT TgT CAC ATC CgT TCT CC - 3‘
c
WalN ROX LNA 5’ - ROX - TCC CAT TgC Cgg A+AC A - BMN-Q620 - 3’
Cashew
CashITS-F 5‘ - gAA CgA ACC CgA TgA TCC - 3‘
CashITS-R 5‘ - CCA TCg Agg gTC AAg gAg - 3‘
d
CashITS-CY5 5‘ - CY5 - ggA CgC GCT CTC TCT gTg - BHQ2 - 3′
a HEX: hexachlorofluorescein; BHQ1: black hole quencher 1
b FAM: 6-carboxyfluorescein; MGB: minor groove binder; BMN-Q535: quencher by Fa. Biomers.net (Ulm)
c ROX: carboxy-X-rhodamine +A = A-LNA: locked nucleic acid BMN-Q620: quencher by Fa. Biomers.net (Ulm)
; ;
d CY5: cyanin 5; BHQ2: black hole quencher 2
Equivalent reporter and/or quencher dyes may be used
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 PCR analytics and the risk for 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.
2 –2
g = 9,81 m⋅s
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 [5]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.
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.
The analyses should be carried out twice in accordance with the following scheme:
— Weigh 2 g of the homogenized sample into a 50 ml centrifuge tube (tube A).
— Add 10 ml of CTAB extraction buffer solution (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 of Proteinase K solution (5.2.15) and mix.
— Incubate and shake for 90 min at 65 °C and mix.
— 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 sodium chloride solution (5.2.20) for at least 30 min at 37 °C while
gently shaking to dissolve the pellet.
— Add 10 μl RNase A solution (5.2.18) and incubate for 10 min at 37 °C.
— Add 900 μl chloroform and mix for 30 s (vortex mixer).
— Centrifuge for 10 min at 14 500 g at room temperature.
— Add the supernatant (the upper, aqueous phase, approx. 800 µl) from tube C to a new 1,5 ml reaction
vial (tube D).
— Add 0,6 parts by volume (approx. 480 μl) of isopropyl alcohol (5.2.6) and mix carefully by turning
upside down several times.
— Incubate at room temperature for at least 20 min (if necessary, overnight).
— Centrifuge for 10 min at 14 500 g at room temperature.
— Carefully remove and discard the supernatant.
— Wash pellet with 500 μl ethanol (70 %) (5.2.2).
— Centrifuge for 10 min at 14 500 g at room temperature.
— Carefully pour off the supernatant completely and discard it.
— Dry extracted DNA at room temperature or under vacuum.
— Dissolve dried DNA in 50 μl 0,2 × TE buffer (5.2.21): agitate gently for 1 hour at 37 °C or for several
hours at room temperature, possibly keeping it refrigerated overnight.
The DNA extracts can be stored refrigerated for a short period or should be stored frozen. Repeated
freezing and thawing should be avoided.
7.3.2 Optional quantification of DNA concentration
The concentration of a DNA aliquot can be determined by UV spectrophotometry at a wavelength of
260 nm with the following Formula (1):
C   =  50  ×   OD  ×   d
(1)
DNA
where
C is the concentration of the DNA in ng/μl;
DNA
OD is the optical density;
d is the dilution factor of the measured aliquot.
In order to check its purity, the sample may in addition be measured at 280 nm. The ratio of the values
for optical density at wavelengths of 260 nm and 280 nm should be approximately 1,8.
The DNA mass concentration may also be estimated using other suitable procedures.
NOTE The DNA concentration determined by this method is influenced by the matrix and the processing state
of the extracted sample. The DNA concentration determined this way does not allow any conclusion on the content
of nut DNA in the sample.
7.4 Real-time PCR set-up
7.4.1 Reaction mix for real-time PCR
As an example, the procedure is described below for a total reaction volume of 25 µl (comprising 20 µl
of PCR mix and 5 µl of DNA extract), using the reagents listed in Table 2. The final reagent concentrations
shown in Table 2 have been proven suitable. The PCR can also be performed with different volumes,
provided the reagent concentrations are adjusted accordingly.
Prior to use, reagents should be gently thawed, for example on ice or a cooling block, and briefly
centrifuged. During PCR mix preparation, reagents should be kept on ice or in a cooling block, if
necessary. Care should be taken to thoroughly mix each reagent immediately before pipetting.
A PCR mix should be prepared containing all components except the DNA extract. The required volume
of PCR mix should be calculated based on the number of reactions to be performed, including a 10 %
safety margin. Each DNA extract should be analysed in duplicate. For each reaction, 5 µl of DNA extract
should be added. Alongside the test samples, the controls specified in 7.4.2 to 7.4.6 should be included.
Table 2 — Reaction mix for real-time PCR
Reagent Final concentration Volume per reaction
[µl]
PCR master mix (2×), see 5.3.1 1-fold 12,5
a
0,6 µmol 0,5
H-nuss f (30 µmol )
a
0,6 µmol 0,5
H-nuss r (30 µmol )
a
0,04 µmol 0,5
H-nuss p FAM (2 µmol )
a
0,3 µmol 0,5
atp-6 F (15 µmol )
a
0,3 µmol 0,5
atp-6 R (15 µmol )
a
0,04 µmol 0,5
atp-6 pr HEX (2 µmol )
a
0,3 µmol 0,5
WalN F (15 µmol )
a
0,3 µmol 0,5
WalN R (15 µmol )
a
0,01 µmol 0,1
WalN ROX (2,5 µmol )
a
0,2 µmol 0,5
Cash ITS F (10 µmol )
a
0,2 µmol 0,5
Cash ITS R (10 µmol )
a
0,04 µmol 0,5
Cash ITS CY5 (2 µmol )
Water 1,9
DNA extract (undiluted) 5,0
a
Other working concentrations can be used. The volumes shall then be adjusted accordingly.
— Mix the PCR reagents (see Table 2), centrifuge shortly and pipette 20 µl per PCR in each reaction vial.
— For each of the sample reactions, pipette 5 µl of sample DNA extract into the PCR master mix.
— For the positive control for DNA targets (7.4.2), pipette 5 µl of the target containing DNA into the
PCR master mix.
— For the negative control for DNA targets (7.4.3), pipette 5 µl of the peanut free sample DNA extract
into the PCR master mix.
— For the amplification reagent control (7.4.4), pipette 5 µl of water into the PCR master mix.
— For the extraction blank control (7.4.5), pipette 5 µl of extract from the negative extraction control
sample into the PCR master mix.
— For the positive extraction control (7.4.6), pipette 5 µl of the peanut-containing sample DNA into
the PCR master mix.
— Place the reaction vials in the PCR device and start the temperature/time program.
7.4.2 Positive control for DNA targets
The positive control is a control reaction containing the target DNA in a specified quantity or number
of copies. DNA for the positive PCR control is extracted from pure nut samples,, as described in 7.3.1.
7.4.3 Negative control for DNA targets
The negative control is a sample of the food matrix without target sequence, which passes through all
steps of the analytical process.
7.4.4 Amplification reagent control
The amplification reagent control (No template control) is a control containing all reagents, except
extracted test sample template DNA. Instead of the template DNA, a corresponding volume of nucleic
acid free water or buffer is added to the reaction.
7.4.5 Extraction blank control
The extraction blank control is a control performing all steps of the DNA extraction procedure, except
addition of the test portion, e.g. by substitution of a corresponding amount of water for the test portion.
7.4.6 Positive extraction control
The positive extraction control is a sample of the food matrix with known quantity of nuts, which passes
through all steps of the analytical proce
...