FprEN 15634-5
(Main)Foodstuffs - Detection of food allergens by molecular biological methods - Part 5: Mustard (Sinapis alba) and soya (Glycine max) - Qualitative detection of a specific DNA sequence in cooked sausages by real-time PCR
Foodstuffs - Detection of food allergens by molecular biological methods - Part 5: Mustard (Sinapis alba) and soya (Glycine max) - Qualitative detection of a specific DNA sequence in cooked sausages by real-time PCR
This document specifies a procedure for the qualitative detection of species specific DNA from white mustard (Sinapis alba) and soya (Glycine max) in cooked sausages using singleplex real-time PCR based on the genes MADS-D (mustard) and lectin (soya).
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 5: Senf (Sinapis alba) sowie Soja (Glycine max) - Qualitativer Nachweis einer spezifischen DNA-Sequenz in Brühwürsten mittels Real-time PCR
Dieses Dokument legt ein Verfahren zum qualitativen Nachweis einer speziesspezifischen DNA von weißem Senf (Sinapis alba) und Soja (Glycine max) in Brühwurst mittels Singleplex-Real time-PCR auf Basis der Gene MADS D (Senf) und Lectin (Soja) fest.
Produits alimentaires - Détection des allergènes alimentaires par des méthodes d’analyse de biologie moléculaire - Partie 5 : Moutarde (Sinapis alba) et soja (Glycine max) - Détection qualitative d’une séquence d’ADN spécifique dans des saucisses cuites, par PCR en temps réel
Le présent document décrit une méthode de détection qualitative d´ADN spécifique de la moutarde blanche (Sinapis alba) et du soja (Glycine max) dans des saucisses cuites, par PCR (réaction de polymérisation en chaîne) singleplex en temps réel reposant sur les gènes de MADS-D (moutarde) et de lectine (soja).
Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 5. del: Gorčica (Sinapis alba) in soja (Glycine max) - Kvalitativno določanje specifičnega zaporedja DNK v obarjenih klobasah s PCR v realnem času
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
oSIST prEN 15634-5:2022
01-januar-2022
Živila - Določevanje alergenov v živilih z molekularno biološkimi metodami - 5. del:
Gorčica (Sinapis alba) in soja (Glycine max) - Kvalitativno določanje specifičnega
zaporedja DNK v obarjenih klobasah s PCR v realnem časuFoodstuffs - Detection of food allergens by molecular biological methods - Part 5:
Mustard (Sinapis alba) and soya (Glycine max) - Qualitative detection of a specific DNA
sequence in cooked sausages by real-time PCRLebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 5: Senf (Sinapis alba) sowie Soja (Glycine max) - Qualitativer Nachweis
einer spezifischen DNA-Sequenz in Brühwürsten mittels Real-time PCRProduits alimentaires - Détection des allergènes alimentaires par des méthodes
d'analyse de biologie moléculaire - Partie 5 : Moutarde (Sinapis alba) et soja (Glycine
max) - Détection qualitative d´une séquence d´ADN spécifique dans des saucissescuites, par PCR en temps réel
Ta slovenski standard je istoveten z: prEN 15634-5
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
67.120.10 Meso in mesni proizvodi Meat and meat products
oSIST prEN 15634-5:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN 15634-5:2022
DRAFT
EUROPEAN STANDARD
prEN 15634-5
NORME EUROPÉENNE
EUROPÄISCHE NORM
November 2021
ICS 07.100.30; 67.120.10 Will supersede CEN/TS 15634-5:2016
English Version
Foodstuffs - Detection of food allergens by molecular
biological methods - Part 5: Mustard (Sinapis alba) and
soya (Glycine max) - 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 5: Senf
moléculaire - Partie 5 : Moutarde (Sinapis alba) et soja (Sinapis alba) sowie Soja (Glycine max) - Qualitativer
(Glycine max) - 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 réelThis 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, Turkey 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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 15634-5:2021 E
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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 DNA purification by means of solid phase extraction ........................................................................ 7
5.4 Real-time PCR reagents ................................................................................................................................. 7
6 Apparatus and equipment ........................................................................................................................... 8
6.1 General ................................................................................................................................................................ 8
6.2 DNA extraction ................................................................................................................................................. 8
6.3 PCR ........................................................................................................................................................................ 8
7 Procedure........................................................................................................................................................... 8
7.1 General ................................................................................................................................................................ 8
7.2 Sample preparation ........................................................................................................................................ 8
7.3 Preparation of extracts ................................................................................................................................. 9
7.3.1 DNA extraction with CTAB and DNA purification ................................................................................ 9
7.3.2 Quantification and normalization 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 Amplification reagent control ................................................................................................................. 11
7.4.3 Extraction blank control ............................................................................................................................ 11
7.4.4 Positive extraction control ....................................................................................................................... 11
7.4.5 Temperature/time programme (real-time PCR) ............................................................................. 11
7.4.6 Accept/Reject criteria ................................................................................................................................ 12
7.4.7 Identification ................................................................................................................................................. 12
8 Validation ........................................................................................................................................................ 12
8.1 General ............................................................................................................................................................. 12
8.2 Inter-laboratory validation ...................................................................................................................... 13
8.2.1 Setup of the inter-laboratory study ....................................................................................................... 13
8.2.2 Inter-laboratory validation results ....................................................................................................... 13
8.2.3 Qualitative interpretation......................................................................................................................... 13
9 Test report ...................................................................................................................................................... 15
Bibliography ................................................................................................................................................................. 16
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European foreword
This document (prEN 15634-5:2021) has been prepared by Technical Committee CEN/TC 275 “Food
analysis – Horizontal methods”, the secretariat of which is held by DIN.This document is currently submitted to the CEN Enquiry.
This document will supersede CEN/TS 15634-5:2016.
In comparison with the previous edition, the following technical modifications have been made:
a) the document was converted from a Technical Specification into a European Standard;
b) normative references and terms and definitions clause added;c) PCR controls moved from Clause 3 “Reagents” to Clause 7 “Procedure”;
d) added new subclause 7.4.6 “Accept/Reject criteria”;
e) restructured clauses in alignment with EN 15634-2:2019.
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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.
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1 Scope
This document specifies a procedure for the qualitative detection of species specific DNA from white
mustard (Sinapis alba) and soya (Glycine max) in cooked sausages using singleplex real-time PCR based
on the genes MADS-D (mustard) and lectin (soya).A mustard content of 10 mg/kg or greater and a soya content of 10 mg/kg or greater can be detected with
a probability of > 95 %.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
considerationsEN 15842, Foodstuffs - Detection of food allergens - General considerations and validation of methods
3 Terms and definitionsFor 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 cooked sausages is extracted from the sample using a cetyltrimethylammoniumbromide
(CTAB) method. Potential PCR inhibitors are removed from the DNA extracted by purification with solid
phase columns and the DNA content is estimated. Real-time PCR is used to detect a 74 base pair (bp) long
sequence of the DNA for the MADS-D protein of Sinapis alba (NCBI accession no. Y08626 ) or a 81 bp
long sequence from the soya lectin gene. The real-time PCR method involves a fluorescence detection
with sequence specific hydrolysis probes [1].1) NCBI-GeneBank® is an example of a suitable search tool for free use. This information is given for the convenience of users
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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
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, 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).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), 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).
5.2.14 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 TE buffer solution, containing— TRIS (5.2.11), c = 0,01 mol/l
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— Na EDTA (5.2.3), c = 0,00 1 mol/l.
The pH is adjusted to 8,0 by adding hydrochloric acid (5.2.5) or sodium hydroxide solution (5.2.10).
5.2.16 0,2 × TE buffer solution, one part by volume of TE buffer solution (5.2.15) are mixed with four
parts water5.3 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. Follow the manufacturer's data for this (see also 8.3.1).
5.4 Real-time PCR reagents5.4.1 PCR master mix (2 × ) for real-time PCR, containing reaction buffer, dNTPs, MgCl2 and Hotstart
Taq polymerase, double concentrated.Ready to use reagents or single components may be used as a PCR master mix, insofar as they provide
comparable or better results.NOTE QuantiTect® Multiplex Mastermix (2 × ) was used within the interlaboratory study.
5.4.2 Oligonucleotides [1]:Primers and probes for the real-time PCR are shown in Table 1.
Table 1 — Primers and probes for the real-time PCR
Name DNA sequence of the oligonucleotide
Soya lectin gene
Lectin-F 5´– TCC ACC CCC ATC CAC ATT T – 3´
Lectin-R 5´– ggC ATA gAA ggT gAA gTT gAA ggA – 3´
Lectin probe a
5´– FAM – AAC Cgg TAg CgT TgC CAg CTT Cg – TAMRA-3´
Mustard (Sinapis alba) MADS D protein
MADS D-F 5´– TGA AAA CTC TCT TCC CCT CTT AGG – 3´
MADS D-R 5´– ACA AAT GCA CAC AAG ACA GAG ATA TAG A – 3´;
MADS D probe a
5´– FAM – TAC ATG ATG CTT ACC TCG C – TAMRA – 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.QuantiTect® Multiplex Mastermix available from QIAGEN GmbH, Hilden is an example of a suitable product
available commercially. This information is given for the convenience of users of this document and does not
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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. Plastic and glass
materials shall be sterilized and free of DNA before use.6.2 DNA extraction
6.2.1 Suitable reaction vials, 1,5 ml an
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