CEN/TS 15634-2:2012

Foodstuffs - Detection of food allergens by molecular biological methods - Part 2: Celery (Apium graveolens) - Qualitative determination of a specific DNA sequence in cooked sausages by real-time PCR

This Technical Specification specifies a method for the qualitative detection of celery (Apium graveolens) in emulsion-type sausages (e.g. frankfurter, wiener).
Real-time PCR detection of celery is based on a 101 bp (base pair) 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 ground celery seeds or alternatively celery root powder, both at a 1000 mg/kg level. 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 [2].

Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 2: Sellerie (Apium graveolens) - Qualitative Bestimmung einer spezifischen DNA-Sequenz in Brühwürsten mittels Real-time-PCR

Diese Technische Spezifikation legt ein Verfahren für den qualitativen Nachweis von Sellerie (Apium graveolens) in Brühwürsten (z. B. Frankfurter, Wiener) fest. Der Nachweis von Sellerie mittels Real-time-PCR beruht auf der Vervielfältigung einer 101 bp (Basenpaar) langen Sequenz aus dem Gen der Mannitoldehydrogenase (GenBank Acc. No. AF067082) von Sellerie (Apium graveolens). Das Verfahren wurde an mit Sellerie dotierten Brühwürsten (Bayrischer Leberkäse) validiert. Zu diesem Zweck wurde ein Fleischbrät bestehend aus Massenanteilen von 50 % Schweinefleisch, 25 % Schweinefett, 23 % zerstoßenem Eis und 1,8 % einer Mischung aus Natriumchlorid, Nitrit, Nitrat, Phosphaten und Ascorbaten nach einem Standardverfahren für Brühwürste hergestellt. Das Fleischbrät wurde mit gemahlener Selleriesaat oder alternativ mit Sellerieknollenpulver jeweils auf die Dotierungsstufe 1 000 mg/kg aufgestockt. Niedrigere Dotierungsstufen wurden durch Verdünnung mit selleriefreiem Fleischbrät erhalten. Das Brät wurde in Gefäße abgefüllt und für 60 min auf 65 °C erwärmt [2].

Produits alimentaires - Détection des allergènes alimentaires par des méthodes d'analyse de biologie moléculaire - Partie 2: Céleri (Apium graveolens) - Détermination qualitative d'une séquence d'ADN spécifique dans des saucisses cuites par PCR en temps réel

La présente Spécification technique décrit une méthode de détection qualitative du céleri (Apium graveolens)
dans des saucisses émulsionnées (par exemple, saucisses de Francfort ou de Vienne).
La détection du céleri par PCR en temps réel repose sur une séquence de 101 pb (paires de base) provenant
du gène de la mannitol déshydrogénase (n° d'accès GenBank AF067082) du céleri (Apium graveolens).
La méthode a été validée pour les saucisses émulsionnées (saucissons bavarois de type « Leberkäse »)
supplémentées en céleri. Pour cela, préparer une pâte de viande contenant des fractions massiques de 50 %
de viande de porc, 25 % de graisse de porc, 23 % de glace pilée et 1,8 % d’un mélange de chlorure de
sodium, nitrite, nitrate, phosphates et ascorbates en respectant le mode opératoire normalisé relatif aux
saucisses émulsionnées. La pâte de viande a été supplémentée en graines de céleri broyées ou en poudre
de racine de céleri, à une teneur de 1 000 mg/kg. Des taux de supplémentation moins élevés ont été obtenus
par dilution dans de la pâte de viande exempte de céleri. La pâte a été embossée dans des boyaux et
chauffée à 65 °C pendant 60 min [2].

Živila - Odkrivanje prisotnosti alergenov v živilih z metodami molekularne biologije - 2. del: Zelena (Apium graveolens) - Kvalitativno določanje specifičnega niza DNK v obarjenih klobasah s PCR v realnem času

Ta tehnična specifikacija določa metodo za kvalitativno odkrivanje zelene (Apium graveolens) v klobasah na osnovi emulzije (npr. frankfurtska klobasa, hrenovka). Odkrivanje zelene s PCR v realnem času temelji na nizu 101 baznega para gena manitol dehidrogenaze (GenBank dost. št. AF067082) zelene (Apium graveolens). Metoda je bila potrjena na klobasah na osnovi emulzije (bavarskih »Leberk?se«), ki vsebujejo zeleno. Mesna masa, ki je vsebovala masne deleže 50 % prašičjega mesa, 25 % prašičje maščobe, 23 % zdrobljenega ledu in 1,8 % mešanice natrijevega klorida, nitrita, nitrata, fosfatov in askorbatov, je bila za ta namen pripravljena po standardnem postopku za klobase na osnovi emulzije. Mesni masi so bila dodana mleta semena zelene ali korenina zelene v prahu, in sicer 1000 mg/kg. Manjši delež zelene je bil dosežen z redčenjem z maso brez zelene. Masa je bila nadevana v ovoje in 60 min segrevana na 65 °C.

General Information

• Status: Withdrawn
• Publication Date: 31-Jan-2012
• Withdrawal Date: 01-Oct-2019
• ICS: 07.100.30 - Food microbiology
• ICS: 67.050 - General methods of tests and analysis for food products
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 12 - Food allergens
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Completion Date: 02-Oct-2019
• Ref Project: SIST-TS CEN/TS 15634-2:2012 - Foodstuffs - Detection of food allergens by molecular biological methods - Part 2: Celery (Apium graveolens) - Qualitative determination of a specific DNA sequence in cooked sausages by real-time PCR

Relations

• Replaced By: EN 15634-2:2019 - 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
• Effective Date: 17-May-2017

Standards Content (Sample)

SLOVENSKI STANDARD
01-april-2012
äLYLOD2GNULYDQMHSULVRWQRVWLDOHUJHQRYYåLYLOLK]PHWRGDPLPROHNXODUQHELRORJLMH
GHO=HOHQD$SLXPJUDYHROHQV.YDOLWDWLYQRGRORþDQMHVSHFLILþQHJDQL]D'1.
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Foodstuffs - Detection of food allergens by molecular biological methods - Part 2: Celery
(Apium graveolens) - Qualitative determination of a specific DNA sequence in cooked
sausages by real-time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 2: Sellerie (Apium graveolens) - Qualitative Bestimmung 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) - Détermination
qualitative d'une séquence d'ADN spécifique dans des saucisses cuites par PCR en
temps réel
Ta slovenski standard je istoveten z: CEN/TS 15634-2:2012
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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL SPECIFICATION
CEN/TS 15634-2
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
February 2012
ICS 07.100.30; 67.050
English Version
Foodstuffs - Detection of food allergens by molecular biological
methods - Part 2: Celery (Apium graveolens) - Qualitative
determination of a specific DNA sequence in cooked sausages
by real-time PCR
Produits alimentaires - Détection des allergènes
Lebensmittel - Nachweis von Lebensmittelallergenen mit
alimentaires par des méthodes d'analyse de biologie molekularbiologischen Verfahren - Teil 2: Sellerie (Apium
moléculaire - Partie 2: Céleri (Apium graveolens) - graveolens) - Qualitative Bestimmung einer spezifischen
Détermination qualitative d'une séquence d'ADN spécifique DNA-Sequenz in Brühwürsten mittels Real-time-PCR
dans des saucisses cuites par PCR en temps réel
This Technical Specification (CEN/TS) was approved by CEN on 15 November 2011 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom.

EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15634-2: E
worldwide for CEN national Members.

Contents Page
Foreword .3
1 Scope .4
2 Principle .4
3 Reagents .4
4 Apparatus and equipment .6
5 Analysis steps .6
6 Validation status and performance criteria . 11
7 Sample type and amounts . 14
8 Limit of detection . 14
9 Interferences . 14
10 Test report . 14
11 Method performance studies . 15
Bibliography . 17

Foreword
This document (CEN/TS 15634-2:2012) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: 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, Romania, Slovakia, Slovenia,
Spain, Sweden, Switzerland, Turkey and the United Kingdom.
1 Scope
This Technical Specification specifies a method for the qualitative detection of celery (Apium graveolens) in
emulsion-type sausages (e.g. Frankfurter, Wiener).
Real-time PCR detection of celery is based on an 101 bp (base pair) 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 [2].
2 Principle
Total DNA from emulsion-type sausages are isolated from the sample matrix. DNA is released from the
sample matrix using the cetyltrimethylammonium bromide (CTAB) approach. Potential PCR inhibitors are
removed from the isolated DNA by purification with solid phase columns. Real-time PCR is used to detect,
amplify and quantify a celery specific sequence. The real time PCR method involves a fluorescence approach
with a sequence specific hydrolysis probe [1], [2].
3 Reagents
3.1 General
The following general conditions for analysis shall be followed, unless specified differently. Use only analytical
grade reagents suitable for molecular biology. Reagents shall be stored in small aliquots to minimise 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.
3.2 Extraction reagents
3.2.1 Chloroform, CAS 66-67-3.
3.2.2 Ethanol, volume fraction ϕ = 70 %, CAS 64-17-5.
3.2.3 Ethylenediaminetetraacetic acid disodium salt (Na EDTA), CAS 6381-92-6.
3.2.4 Cetyltrimethylammoniumbromide (CTAB), CAS 57-09-0.
3.2.5 Hydrochloric acid, ϕ = 37 %, CAS 7647-01-0.
3.2.6 Isoamyl alcohol, CAS 123-51-3.
3.2.7 Isopropanol, CAS 67-63-0.
3.2.8 Proteinase K, EC 3.4.21.64.
3.2.9 Sodium chloride, CAS 7647-14-5.
3.2.10 Sodium hydroxide, CAS 1310-73-2.
3.2.11 Tris(hydroxymethyl)aminomethane (TRIS), CAS 7-86-1.
3.2.12 Chloroform isoamyl alcohol mixture, 24 parts by volume of chloroform (3.2.1) are mixed with one
part by volume of isoamyl alcohol (3.2.6).
NOTE Similar mixtures available commercially may be used.
3.2.13 CTAB extraction buffer solution containing CTAB (mass concentration ρ = 20 g/l), sodium chloride
(substance concentration c = 1,4 mol/l), TRIS (c = 0,1 mol/l), Na EDTA (c = 0,02 mol/l). The pH shall be
adjusted to read 8,0 by adding hydrochloric acid.
3.2.14 Proteinase K solution (ρ = 20 mg/ml)
NOTE Store in the form of aliquots at -20 °C after dissolving. Do not autoclave.
3.2.15 TE buffer solution containing TRIS (c = 0,001 mol/l) and Na EDTA (c = 0,000 1 mol/l). The pH shall
be adjusted to read 8,0 by adding hydrochloric acid or sodium hydroxide solution.
3.3 DNA purification by means of solid phase extraction
For the DNA purification different methods may be used.
NOTE Several formats are commercially available, among them spin filter columns or plates. Commercially available
kits may be used as appropriate.
3.4 Real-time PCR reagents
1)
3.4.1 Concentrated PCR buffer solution (containing reaction buffers, dNTPs, MgCl and Hotstart Taq
polymerase).
3.4.2 Oligonucleotides, c = 20 µmol/l each.
3.4.2.1 General
For information on the DNA target sequence and validation of selectivity, see 6.3.
NOTE In the interlaboratory study, the participants received their primers and the probe from the same production lot.
3.4.2.2 Forward primer (iF), Cel-MDH iF 5’-CgA TgA gCg TgT ACT gAg TC – 3’.
3.4.2.3 Reverse primer (iR), Cel-MDH iR 5’-AAT Agg AAC TAA CAT TAA TCA TAC CAA AC – 3’.
2)
3.4.2.4 Cel-MDH probe 5’-FAM-AAC AgA TAA CgC TgA CTC ATC ACA CCg-TAMRA – 3’ .

1) 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.
2) 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 Apparatus and equipment
4.1 General
General aspects are described in EN 15634-1 [3]. In addition to the usual laboratory facilities, the following
equipment is required.
NOTE 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.
4.2 DNA extraction
4.2.1 Suitable reaction vials with a capacity of 1,5 ml and 2 ml, sterile; 50 ml centrifuge tube, sterile.
4.2.2 Thermostat or water bath, preferably with shaker function.
3)
4.2.3 Centrifuge suitable for centrifuging 50 ml centrifuge tubes at 8 000 g .
4.2.4 Centrifuge suitable for centrifuging 1,5 ml and 2 ml reaction vials at 14 500 g.
4.2.5 Equipment and/or material for grinding the sample, e.g., a kitchen blender.
4.2.6 UV spectrophotometer or other detection instruments suitable for estimating the amount of DNA.
4.3 PCR
4.3.1 Suitable PCR tubes
4.3.2 Microcentrifuge for PCR tubes
4.3.3 Real-time PCR equipment suitable for excitation and for emission measurement of fluorescence-
marked oligonucleotides.
5 Analysis steps
5.1 General
General aspects are described in EN 15634-1 [3].
5.2 Sample preparation
It should be ensured, e.g. by milling or homogenizing, that the test sample is representative of the laboratory
sample.
In order to minimise the risk of carry-over contaminations, all equipment should be cleaned extensively prior to
proceeding with the next sample. Examples of cleaning products or techniques include: DNA-degrading
agents, hypochlorite solution, hot water and detergents.

-2
3) g = 9,81 m × s
5.3 Preparation of extracts
5.3.1 DNA extraction with CTAB and DNA purification
In parallel to the test samples, the controls listed in 5.4.6 and 5.4.7 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 50 ml centrifuge tubes (tube A).
 Add 10 ml of CTAB buffer (3.2.13).
 Add 30 µl of Proteinase K solution (3.2.14) and mix by inversion, pipetting or vortexing.
 Incubate and shake for 90 min at a temperature of 65 °C.
 Centrifuge for 5 min at 6 000 g to 8 000 g at room temperature.
 Place 500 µl of chloroform isoamyl alcohol mixture (3.2.12) 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 approximately 14 500 g at room temperature.
 Place 500 µl of isopropanol (3.2.7) 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 by inversion,
pipetting or vortexing.
 Incubate tube C for 30 min at room temperature.
 Centrifuge for 15 min at approximately 14 500 g at room temperature.
 Carefully remove and discard the supernatant using a pipette or by gently pouring out.
...