EN 15634-2:2019

SLOVENSKI STANDARD
01-december-2019
Nadomešča:
SIST-TS CEN/TS 15634-2:2012
Živila - Odkrivanje prisotnosti alergenov v živilih z molekularno biološkimi
metodami - 2. del: Zelena (Apium graveolens) - Odkrivanje specifičnega niza DNK
v obarjenih klobasah s PCR v realnem času
Foodstuffs - Detection of food allergens by molecular biological methods - Part 2: Celery
(Apium graveolens) - Detection of a specific DNA sequence in cooked sausages by real-
time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 2: Sellerie (Apium graveolens) - 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 2 : Céleri (Apium graveolens) - Détection d'une
séquence d'ADN spécifique dans des saucisses cuites par PCR en temps réel
Ta slovenski standard je istoveten z: EN 15634-2:2019
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.120.10 Meso in mesni proizvodi Meat and meat products
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN 15634-2
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 07.100.30; 67.120.10 Supersedes CEN/TS 15634-2:2012
English Version
Foodstuffs - Detection of food allergens by molecular
biological methods - Part 2: Celery (Apium graveolens) -
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 2: Sellerie
moléculaire - Partie 2 : Céleri (Apium graveolens) - (Apium graveolens) - Nachweis einer spezifischen
Détection d'une séquence d'ADN spécifique dans des DNA-Sequenz in Brühwürsten mittels Real-time-PCR
saucisses cuites par PCR en temps réel
This European Standard was approved by CEN on 12 August 2019.

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, Turkey 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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 15634-2:2019 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
6 Apparatus and equipment . 7
7 Procedure. 8
8 Expression of results . 13
9 Validation . 13
10 Test report . 19
Bibliography . 20

European foreword
This document (EN 15634-2:2019) 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 April 2020, and conflicting national standards shall be
withdrawn at the latest by April 2020.
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 15634-2:2012.
Significant technical changes between this standard and CEN/TS 15634-2:2012 are as follows:
a) the document was converted from a Technical Specification into a European Standard;
b) normative references added (2);
c) expression of results (8) updated;
d) requirements regarding the test report added (10)
e) updated bibliography.
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, Turkey 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; and
— ‘can’ indicates a possibility and/or a capability.
1 Scope
This document specifies a method for the detection of celery (Apium graveolens) in emulsion-type
sausages (e.g. Frankfurter, Wiener).
Real-time PCR (polymerase chain reaction) detection of celery is based on an 101 bp (base pair)
1)
sequence from the gene of the mannitol dehydrogenase (GenBank Acc. No. AF067082 of celery (Apium
graveolens).
The method has been validated on emulsion-type sausages (Bavarian “Leberkäse”) spiked with celery.
For this purpose meat batter containing mass fractions of 50 % pork meat, 25 % pork fat, 23 % crushed
ice and 1,8 % of a mixture of sodium chloride, nitrite, nitrate, phosphates and ascorbates was prepared
according to a standard procedure for emulsion-type sausage. The meat batter was spiked with either
ground celery seeds or celery root powder to 1000 mg/kg. Lower spiking levels were obtained by
diluting with celery-free meat batter. The batter was stuffed into casings and heated at 65 °C for 60 min
[1].
This document is intended to be used in addition to EN 15842 and EN 15634-1.
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 emulsion-type sausages is isolated from the sample using a cetyltrimethylammonium
bromide (CTAB) method. Potential PCR inhibitors are removed from the DNA extracted by purification
with solid phase columns. Real-time PCR is used to detect a celery specific sequence. The real time PCR
method involves a fluorescence detection with a sequence specific hydrolysis probe [1], [2].

1) NCBI-GeneBank® 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 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. Reagents should be stored in small aliquots to
minimize the risk of contamination. 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, φ = 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.
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), 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).
5.2.14 Proteinase K solution, ρ = 20 mg/ml
Store in the form of aliquots at −20 °C after dissolving. Do not autoclave.
5.2.15 TE buffer solution, containing:
— TRIS (5.2.11), c = 0,001 mol/l, and
— 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.
5.4 Real-time PCR reagents
2)
5.4.1 Concentrated PCR buffer solution (containing reaction buffers, dNTPs, MgCl and Hotstart
Taq polymerase).
5.4.2 Oligonucleotides, c = 20 µmol/l each.
5.4.2.1 Forward primer (iF), Cel-MDH iF 5’-CgA TgA gCg TgT ACT gAg TC – 3’.
5.4.2.2 Reverse primer (iR), Cel-MDH iR 5’-AAT Agg AAC TAA CAT TAA TCA TAC CAA AC – 3’.
3)
5.4.2.3 Cel-MDH probe 5’-FAM-AAC AgA TAA CgC TgA CTC ATC ACA CCg-TAMRA – 3’ .
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.
6.2 DNA extraction
6.2.1 Suitable reaction vials with a capacity of 1,5 ml and 2 ml, sterile; 50 ml centrifuge tube, sterile.
6.2.2 Thermostat or water bath, preferably with shaker function.
4)
6.2.3 Centrifuge suitable for centrifuging 50 ml centrifuge tubes at 8 000 g .
6.2.4 Centrifuge suitable for centrifuging 1,5 ml and 2 ml reaction vials at 14 500 g.
6.2.5 Equipment and/or material for grinding the sample, e.g. a kitchen blender.

2)
Ready-to-use reagent mixtures or single components may be used for the PCR buffer solution as long as they
give results comparable to or better than the ones stated for the collaborative trial.
3)
FAM: 6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine; equivalent reporter and/or quencher
dyes may be used if they are shown to give comparable or better results.
4) 2
g = 9,81 m × s
6.2.6 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.
7 Procedure
7.1 Ge
...