Foodstuffs - Detection of food allergens by molecular biological methods - Part 1: General considerations

This document provides the overall framework for detection of sequences corresponding to species containing allergens using the polymerase chain reaction (PCR). It relates to the requirements for the specific amplification of target nucleic acid sequences (DNA) and for the confirmation of the identity of the amplified nucleic acid sequence.
Guidelines, minimum requirements and performance criteria laid down in this document are intended to ensure that comparable and reproducible results are obtained in different laboratories. This document has been established for food matrices.

Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 1: Allgemeine Betrachtungen

Dieses Dokument legt den Gesamtrahmen für den Nachweis von Sequenzen, die Arten entsprechen, die Allergene enthalten, durch Polymerase-Kettenreaktion (PCR) fest. Er bezieht sich auf die Anforderungen an die spezifische Amplifikation von Target-Nukleinsäuresequenzen (DNA) und an die Bestätigung der Identi-tät der amplifizierten Nukleinsäuresequenz.
Die in diesem Dokument angeführten Leitlinien, Mindestanforderungen und Leistungskriterien sind dafür vorgesehen, sicherzustellen, dass in verschiedenen Laboratorien vergleichbare und reproduzierbare Ergebnisse erhalten werden. Dieses Dokument wurde für Lebensmittelmatrices erarbeitet.

Produits alimentaires - Détection des allergènes alimentaires par des méthodes d’analyse de biologie moléculaire - Partie 1: Considérations générales

Le présent document fournit un cadre général pour la détection des séquences correspondant aux espèces qui contiennent des allergènes, à l’aide de la méthode d’amplification en chaîne par polymérase (méthode PCR). Il traite des exigences requises pour l’amplification spécifique de séquences cibles d’acide nucléique (ADN) et la confirmation de l’identité de la séquence d’acide nucléique amplifiée.
Les lignes directrices, les exigences minimales et les critères de performances établis dans le présent document sont destinés à assurer que des laboratoires différents obtiendront des résultats comparables et reproductibles. Le présent document a été élaboré pour les matrices alimentaires.

Živila - Odkrivanje prisotnosti alergenov v živilih z molekularno biološkimi metodami - 1. del: Splošne ugotovitve

Ta dokument podaja splošen okvir za odkrivanje zaporedij v vrstah, ki vsebujejo alergene, s polimerazno verižno reakcijo (PCR). Navezuje se na zahteve za specifično povečanje ciljnih zaporedij nukleinske kisline (DNA) in za potrditev identitete povečanega zaporedja nukleinske kisline.
Smernice, minimalne zahteve in merila učinkovitosti iz tega dokumenta so namenjeni zagotavljanju, da se v različnih laboratorijih pridobijo primerljivi in ponovljivi rezultati. Ta dokument je bil pripravljen za zmesi živil.

General Information

Status
Published
Public Enquiry End Date
09-Sep-2018
Publication Date
06-Nov-2019
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
16-Oct-2019
Due Date
21-Dec-2019
Completion Date
07-Nov-2019

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SLOVENSKI STANDARD
SIST EN 15634-1:2019
01-december-2019
Nadomešča:
SIST EN 15634-1:2009
Živila - Odkrivanje prisotnosti alergenov v živilih z molekularno biološkimi
metodami - 1. del: Splošne ugotovitve

Foodstuffs - Detection of food allergens by molecular biological methods - Part 1:

General considerations
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 1: Allgemeine Betrachtungen
Produits alimentaires - Détection des allergènes alimentaires par des méthodes
d’analyse de biologie moléculaire - Partie 1: Considérations générales
Ta slovenski standard je istoveten z: EN 15634-1:2019
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
SIST EN 15634-1:2019 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 15634-1:2019
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SIST EN 15634-1:2019
EN 15634-1
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN 15634-1:2009
English Version
Foodstuffs - Detection of food allergens by molecular
biological methods - Part 1: General considerations

Produits alimentaires - Détection des allergènes Lebensmittel - Nachweis von Lebensmittelallergenen

alimentaires par des méthodes d'analyse de biologie mit molekularbiologischen Verfahren - Teil 1:

moléculaire - Partie 1 : Considérations générales Allgemeine Betrachtungen
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 15634-1:2019 E

worldwide for CEN national Members.
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SIST EN 15634-1:2019
EN 15634-1:2019 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 General requirements for laboratories .................................................................................................. 7

5 Procedure........................................................................................................................................................... 8

6 Isolation/Extraction of nucleic acid ......................................................................................................... 9

7 Amplification of specific target sequences ......................................................................................... 10

8 Detection and confirmation of PCR products ..................................................................................... 12

9 Interpretation ................................................................................................................................................ 13

10 Expression of the results ........................................................................................................................... 14

11 Test report ...................................................................................................................................................... 14

Bibliography ................................................................................................................................................................. 15

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SIST EN 15634-1:2019
EN 15634-1:2019 (E)
European foreword

This document (EN 15634-1:2019) 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 EN 15634-1:2009.

Significant technical changes between this standard and EN 15634-1:2009 are as follows:

a) updated terms and definitions (3);
b) requirements regarding the preparation of samples changed (6.1);
c) clause 6.3 on DNA quantitation changed;
d) clause 7.2.1 on primer design changed;
e) requirements regarding quantitation of PCR products (8.2) changed;
f) clause on "Quality assurance requirements" deleted;
g) the test report should comply with EN ISO/IEC 17025;
h) 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.
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SIST EN 15634-1:2019
EN 15634-1:2019 (E)
Introduction

This document describes the procedure to qualitatively detect and/or quantitate DNA fragments as

markers for potentially allergenic ingredients or constituents by analysing the nucleic acids extracted

from the sample under study.

The qualitative detection of DNA targets is performed in order to get a yes or no answer to the question

whether a certain DNA sequence is detected or not relative to appropriate controls and within the

detection limits of the analytical method used and the test portion analysed.

The quantitative detection of DNA targets is performed to express the quantity of DNA targets, relative

to the quantity of a specific reference, appropriate calibrants and controls and within the dynamic range

of the analytical method used and the test portion analysed. Appropriate procedures for extraction of

nucleic acids are included in each method.

The main focus of this document will be on PCR based amplification methods. However, because of the

rapid rate of technological change in this area, other amplification technologies and detection methods

may be considered.
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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SIST EN 15634-1:2019
EN 15634-1:2019 (E)
1 Scope

This document provides the overall framework for detection of sequences corresponding to species

containing allergens using the polymerase chain reaction (PCR). It relates to the requirements for the

specific amplification of target nucleic acid sequences (DNA) and for the confirmation of the identity of

the amplified nucleic acid sequence.

Guidelines, minimum requirements and performance criteria laid down in European Standards are

intended to ensure that comparable and reproducible results are obtained in different laboratories. This

document has been established for food matrices.
This document is intended to be used in addition to EN 15842.
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 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 the following 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
3.1 Terms relative to extraction and purification of DNA
3.1.1
DNA extraction
separation of DNA from the other components in a test sample
Note 1 to entry: The factors of major importance for the isolated DNA are:
a) purity,
b) amount or concentration and
c) quality (integrity).
[SOURCE: EN ISO 24276:2006, 3.2.1, modified — note was added]
3.1.2
DNA purification

method resulting in a DNA intended to reduce observable measurable effects of PCR inhibitors

Note 1 to entry: In this context, purity refers to the reduction of observable measurable effects of PCR

inhibitors.
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SIST EN 15634-1:2019
EN 15634-1:2019 (E)
3.1.3
PCR quality DNA

DNA template of sufficient length, chemical purity, and structural integrity to be amplified by PCR

3.2 Terms relative to amplification of DNA
3.2.1
species specific target sequence
sequence known to be specific for the species or higher taxa
3.2.2
identification of amplified nucleic acid sequences

proof of identity of amplified nucleic acid sequences (amplicons) by comparison with a reference

nucleic acid fragment pattern or sequence
3.3 Definitions referring to controls
3.3.1
positive DNA target control

reference DNA, or DNA extracted from a certified reference material, or known positive samples

representative of the sequence or target under study

Note 1 to entry: The control is intended to demonstrate the result of analyses of test sample containing the

sequence under study.

[SOURCE: EN ISO 24276:2006, 3.4.1, modified — replaced organism by target, note was changed]

3.3.2
negative DNA target control

reference DNA, or DNA extracted from a certified negative (blank matrix) reference material, or known

negative sample not containing the sequence under study

Note 1 to entry The control is intended to demonstrate the result of analyses of test samples not containing the

sequence under study.
[SOURCE: EN ISO 24276:2006, 3.4.2, modified — note was changed]
3.3.3
PCR inhibition control

control containing known amounts of defined template DNA added in the same or less amount as

analyte DNA to the reaction

Note 1 to entry: This control allows the detection of the presence of soluble PCR inhibitors, particularly

necessary in case of non-specific or none amplification and of quantitative PCR.
3.3.4
amplification reagent control
no template control
control containing all the reagents, except extracted test sample template DNA

Note 1 to entry: Instead of the template DNA, a corresponding volume of nucleic acid free water or buffer is

added to the reaction.
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SIST EN 15634-1:2019
EN 15634-1:2019 (E)
3.3.5
extraction blank control

control performing all steps of the extraction procedure, except addition of the test portion, e.g. by

substitution of water for the test portion

Note 1 to entry: It is used to detect possible contaminating nucleic acid in a buffer or chemicals used during

extraction.
3.3.6
positive extraction control

control sample to demonstrate that the nucleic acid extraction procedure has been performed in a way

that will allow extraction and subsequent amplification of the target nucleic acid, i.e. by using a sample

material known to contain the target nucleic acid

Note 1 to entry: The DNA amount is usually e.g. tenfold over the LOD of the method to ensure amplification.

4 General requirements for laboratories
4.1 General

In addition to EN ISO/IEC 17025, also EN 15842 dealing with general considerations and validation

criteria of methods exists.
4.2 Laboratory organization

Compliance with applicable requirements with respect to safety regulations should be followed and

manufacturer’s safety recommendation should be taken into account.

Accidental contamination of DNA can originate from dust or spreading aerosols. As a consequence, the

organization of the work area in the laboratory is logically based on:

— the systemic containment of the methodological steps involved in the analysis, and

— a forward flow principle for sample handling.

A minimum of three separately designated work areas with their own apparatus is required:

a) a work area for extraction of the nucleic acid from the test portion (sample);

b) a work area dedicated to the setup of PCR/amplification reactions; and

c) a work area dedicated for subsequent processing including analysis and characterization of the

amplified DNA segments.

If dust particle producing grinding techniques are used, this shall be carried out in a separate work area.

Physical separation through the use of different rooms is the most effective and preferable way of

ensuring separated work areas, but other physical or biochemical methods may be used as a protection

against contamination provided their effectiveness is comparable.

Staff should preferably wear different sets of lab coats at each dedicated work area. They shall also wear

disposable gloves. Gloves and lab coats should be changed at appropriated frequencies.

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SIST EN 15634-1:2019
EN 15634-1:2019 (E)
4.3 Apparatus and equipment

The laboratory should use properly maintained equipment suitable for the method employed, e.g.

according to the requirements outlined by EN ISO/IEC 17025. In addition to standard laboratory

equipment, additional apparatus are described in the specific methods.

Apparatus and equipment should be maintained according to manufacturer’s instructions. Calibration

systems should be available and calibration routinely performed for equipment which could impact the

data produced, according to laboratory quality assurance programs.
4.4 Material and reagents

For the analysis, unless otherwise stated, use only analytically pure reagents suitable for molecular

biology, free from DNA and DNAses. Reagents and solutions should be stored at room temperature,

unless otherwise specified. PCR reagents should be stored in small aliquots to minimize the risk of

contamination. The water used shall be double distilled or equivalent, free from DNA and nucleases

(nucleic acid based grade). Solutions are prepared by dissolving the appropriate reagents in water and

autoclaved unless specified differently. Filtration can be performed for obtaining sterile solutions, when

autoclaving is not possible.

In order to avoid contamination, all the appliances and the parts of equipment in contact with the

samples, as well as work surfaces should be easy to clean and decontaminate. Work surfaces cleaning

and decontamination is recommended before use
...

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