prEN 15634-6

SLOVENSKI STANDARD
01-marec-2026
Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 6. del:
Pšenica (Triticum L.) in rž (Secale cereale) - Kvalitativno določanje specifičnega
zaporedja DNK v živilih s PCR v realnem času
Foodstuffs - Detection of food allergens by molecular biological methods - Part 6: Wheat
(Triticum L.) and Rye (Secale cereale) - Qualitative detection of a specific DNA
sequence in cooked sausages by real-time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 6: Weizen (Triticum L.) and Roggen (Secale cereale) - Qualitativer
Nachweis einer spezifischen DNA-Sequenz in Brühwürsten mittels Real-time PCR
Produits alimentaires - Détection des allergènes alimentaires par des méthodes
d’analyse de biologie moléculaire - Partie 6 : Blé (Triticum L.) et seigle (Secale cereale) -
Détection qualitative d’une séquence d’ADN spécifique dans des saucisses cuites, par
PCR en temps réel
Ta slovenski standard je istoveten z: prEN 15634-6
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.060 Žita, stročnice in proizvodi iz Cereals, pulses and derived
njih 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; 67.060
English Version
Foodstuffs - Detection of food allergens by molecular
biological methods - Part 6: Wheat (Triticum L.) and Rye
(Secale cereale) - Qualitative detection of a specific DNA
sequence in cooked sausages 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 6: Weizen
moléculaire - Partie 6 : Blé (Triticum L.) et seigle (Triticum L.) and Roggen (Secale cereale) - Qualitativer
(Secale cereale) - Détection qualitative d'une séquence Nachweis einer spezifischen DNA-Sequenz in
d'ADN spécifique dans des saucisses cuites, par PCR en Brühwürsten mittels Real-time PCR
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 15634-6:2026 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 . 5
5.2 Extraction reagents . 6
5.2.13 CTAB extraction buffer solution, containing: . 6
5.2.14 Proteinase K solution, ρ = 20 mg/ml. . 6
5.3 DNA purification by means of solid phase extraction . 6
5.4 Real-time PCR reagents . 7
5.4.1 Master mix for real-time PCR, containing thermostable DNA polymerase (for hot-
start PCR) and PCR buffer solution (containing reaction buffer, dNTPs, and MgCl2),
dilutable concentrate. . 7
5.4.2 Oligonucleotides . 7
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 DNA extracts . 8
7.3.1 DNA extraction with CTAB and DNA purification . 8
7.3.2 Optional quantification 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 . 10
7.4.4 Amplification reagent control . 10
7.4.5 Extraction blank control . 10
7.4.6 Positive extraction control . 10
7.4.7 Temperature/time programme (real-time PCR) . 11
7.4.8 Accept/Reject criteria . 11
7.4.9 Identification . 11
8 Validation . 11
8.1 General . 11
8.2 Specificity . 12
8.3 Sensitivity . 12
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 protocol . 14
8.4.3 Ring trial validation results . 14
9 Test report . 17
Bibliography . 18
European foreword
This document (prEN 15634-6:2026) 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 DNA of the general wheat and rye in
cooked sausages using real-time PCR based on the glutenin gene, in the context of allergen analyses.
This document does not apply to differentiating between wheat (Triticum L.) and rye (Secale cereale).
The method was previously validated in an interlaboratory study (ring trial).
The limit of detection of the wheat and rye real-time PCR has been determined experimentally to be
around 80 mg wheat or rye per kg for the matrix ‘cooked sausage’. For autoclaved material the detection
limit can increase significantly.
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 15842 and EN 15634-1and
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 by pürification with solid phase
columns and the DNA content can be measured. From the extracted sample DNA, a wheat- and rye-
specific DNA sequence from the glutenin genes B1-1 of wheat or 1-R of rye, respectively, is amplified
using real-time PCR. The amplicons have a lengths of 85 base pairs (bp). The real-time PCR method
involves flüorescence detection with a seqüence-specific hydrolysis probe, see [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
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 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).
5.2.13 CTAB extraction buffer solution, containing:
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,
— Na2EDTA (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.
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 0,2 × TE buffer solution, containing:
— TRIS (5.2.11), c = 0,002 mol/l,
— Na2EDTA (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 (5.2.10).
NOTE For all solutions commercially available alternatives can be used.
5.3 DNA purification by means of solid phase extraction
For the DNA pürification, different methods may be used.
NOTE Several formats are commercially available, among them spin filter columns or plates. Commercially
available kits can be used if appropriate.
5.4 Real-time PCR reagents
5.4.1 Master mix for real-time PCR, containing thermostable DNA polymerase (for hot-start
PCR) and PCR buffer solution (containing reaction buffer, dNTPs, and MgCl2), dilutable
concentrate.
Ready to use reagents or single components may be used as a master mix.
5.4.2 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
tritprglut 3 F 5´ – CCT CTT TgC ggC AgT AgT YgT – 3´
tritprglut 3 R 5´ – CTC gCR CTC ACA BTg TAg TTg – 3´
a
tritglut 5´ – FAM – CCT CgT ggC TCT CAC CRY CgC T – BHQ1 – 3′
a FAM: 6-carboxyflüorescein; 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 8 000 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.
1 –2
g = 9,81 m⋅s
6.3.2 Microcentrifuge for PCR tubes.
6.3.3 Real-time PCR equipment, suitable for excitation and for emission measurement of
flüorescence-labeled oligonucleotides.
7 Procedure
7.1 General
General aspects are described inEN 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.
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 50 ml centrifuge tubes (tube A).
— Add 10 ml of CTAB extraction buffer solution (5.2.13).
— Add 30 µl of Proteinase K solution (5.2.14) and mix.
— Incubate and shake for 90 min at 65 °C.
— Centrifuge for 5 min at 6 000 g at room temperature.
— Place 500 µl of chloroform isoamyl alcohol mixture (5.2.12) in a 2 ml reaction vial (tube B).
— Add 700 µl of supernatant from tube A to tube B and mix thoroughly for 30 s.
— Centrifuge for 15 min at about 14 500 g at room temperature, to separate phases.
— Place 500 µl of cold isopropanol (5.2.7) in a 1,5 ml reaction vial (tube C).
— Add 500 µl of supernatant (aqueous phase) from tube B to tube C and mix carefully.
— Incubate for 30 min at room temperature.
— Centrifuge for 15 min at about 14 500 g at room temperature.
— Carefully remove and discard the supernatant.
— Wash pellet with 500 μl ethanol (70 %) (5.2.2).
— Centrifuge for 5 min at about 14 500 g at room temperature.
— Carefully remove and discard the supernatant.
— Dry extracted DNA at room temperature or under vacuum.
— Dissolve the dried DNA extract in 100 µl of 0,2 × TE buffer solution (5.2.15).
— Purify the DNA extract using e.g. solid phase extraction. For commercial kits the instructions given
by the manufacturer should be followed.
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 inflüenced by the
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