CEN/TS 15634-4:2016

SLOVENSKI STANDARD
01-maj-2016
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Foodstuffs - Detection of food allergens by molecular biological methods - Part 4: Peanut
(Arachis hypogaea) - Qualitative detection of a specific DNA sequence in chocolate by
real-time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 4: Erdnuss (Arachis hypogaea) - Qualitativer Nachweis einer
spezifischen DNA-Sequenz in Schokolade mittels Real-time PCR
Produits alimentaires - Détection d’allergènes alimentaires par des méthodes de biologie
moléculaire - Partie 4 : Arachide (Arachis hypogaea) - Détection qualitative d’une
séquence d’ADN spécifique dans du chocolat, par PCR en temps réel
Ta slovenski standard je istoveten z: CEN/TS 15634-4:2016
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
67.190 ýRNRODGD Chocolate
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

CEN/TS 15634-4
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
March 2016
TECHNISCHE SPEZIFIKATION
ICS 07.100.30; 67.190
English Version
Foodstuffs - Detection of food allergens by molecular
biological methods - Part 4: Peanut (Arachis hypogaea) -
Qualitative detection of a specific DNA sequence in
chocolate by real-time PCR
Produits alimentaires - Détection d'allergènes Lebensmittel - Nachweis von Lebensmittelallergenen
alimentaires par des méthodes de biologie moléculaire mit molekularbiologischen Verfahren - Teil 4: Erdnuss
- Partie 4 : Arachide (Arachis hypogaea) - Détection (Arachis hypogaea) - Qualitativer Nachweis einer
qualitative d'une séquence d'ADN spécifique dans du spezifischen DNA-Sequenz in Schokolade mittels Real-
chocolat, par PCR en temps réel time PCR
This Technical Specification (CEN/TS) was approved by CEN on 11 February 2016 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, Former Yugoslav Republic of Macedonia, 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

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15634-4:2016 E
worldwide for CEN national Members.

Contents Page
European foreword . 3
1 Scope . 4
2 Principle . 4
3 Reagents . 4
3.1 DNA extraction with CTAB . 4
3.2 DNA purification by means of solid phase extraction . 5
3.3 Real-time PCR reagents . 5
4 Apparatus and equipment . 5
4.1 DNA extraction . 6
4.2 PCR . 6
5 Procedure. 6
5.1 General . 6
5.2 Sample preparation . 6
5.3 DNA extraction with CTAB . 6
5.4 DNA purification by means of solid phase extraction . 7
5.5 Measuring the mass concentration and purity of the extracted DNA . 7
5.6 Real-time PCR . 8
6 Validation status and performance criteria . 9
6.1 General . 9
6.2 Detection . 10
6.3 Reliability of the method . 10
6.3.1 Setup of the interlaboratory study . 10
6.3.2 Results . 10
6.3.3 Specificity . 11
7 Test report . 11
Bibliography . 13

European foreword
This document (CEN/TS 15634-4:2016) 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.
EN 15634, Foodstuffs — Detection of food allergens by molecular biological methods, is currently
composed with the following parts:
— Part 1: General considerations;
— Part 2: Celery (Apium graveolens) — Qualitative determination of a specific DNA sequence in cooked
sausages by real-time PCR [Technical Specification];
— Part 3: Hazelnut (Corylus avellana) — Qualitative detection of a specific DNA sequence in chocolate
by real-time PCR [Technical Specification];
— Part 4: Peanut (Arachis hypogaea) — Qualitative detection of a specific DNA sequence in chocolate
by real-time PCR [Technical Specification];
— Part 5: Mustard (Sinapis alba) and soya (Glycine max) — Qualitative dectection of a specific DNA
sequence in cooked sausages by real-time PCR [Technical Specification].
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, Former Yugoslav Republic of Macedonia,
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 describes a procedure for the qualitative detection of peanut (Arachis
hypogaea) in chocolate using real-time PCR based on the gene for the peanut allergen Ara h 2 [4], [5].
2 Principle
The total DNA is extracted from the sample and the DNA content estimated. A sequence specific to
peanut from the gene for Ara h 2 is multiplicated using real-time PCR. The amplicon with a length of 86
base pairs (bp) formed in this way is detected by annealing a sequence-specific probe and generating a
fluorescence signal [4].
3 Reagents
As a rule, analytical grade chemical reagents suitable for molecular biology shall be used. The water
used shall be double distilled or equivalent quality. Solutions should be prepared by dissolving the
appropriate reagents in water and autoclaving, unless indicated differently.
3.1 DNA extraction with CTAB
3.1.1 Chloroform.
3.1.2 Ethanol, volume fraction φ = 96 %.
3.1.3 Ethylenediaminetetraacetic acid disodium salt (Na2EDTA).
3.1.4 Cetyltrimethylammoniumbromide (CTAB).
3.1.5 Hydrochloric acid, mass fraction w = 37 %.
3.1.6 Isoamyl alcohol.
3.1.7 Isopropanol.
3.1.8 Proteinase K.
3.1.9 Sodium chloride.
3.1.10 Sodium hydroxide.
3.1.11 Tris(hydroxymethyl)aminomethane (TRIS).
3.1.12 Chloroform isoamyl alcohol mixture.
Mix 24 parts by volume of chloroform (3.1.1) with one part by volume of isoamyl alcohol (3.1.6).
Commercially available and comparable mixtures can be used.
3.1.13 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). Adjust
the pH value with hydrochloric acid to pH = 8,0.
3.1.14 Ethanol solution, φ = 70 %.
3.1.15 Proteinase K solution, ρ = 20 mg/ml.
The freshly produced Proteinase K solution should be stored in the form of aliquots at -20 °C.
3.1.16 TE buffer solution, containing TRIS (c = 0,001 mol/l) and Na -EDTA (c = 0,000 1 mol/l). Adjust
the pH value with hydrochloric acid or sodium hydroxide solution to pH = 8,0.
3.2 DNA purification by means of solid phase extraction
Various systems are commercially available for DNA purification by means of solid phase extraction,
including spin filter columns or plates or also with vacuum operated systems. Commercially available
kits can also be used. Observe the manufacturer's data for this.
3.3 Real-time PCR reagents
)
3.3.1 PCR master mix , containing reaction buffers, dNTPs, MgCl and Hotstart Taq polymerase.
3.3.2 Oligonucleotides, 10 µmol each.
3.3.2.1 Peanut (AR-58 F), gCA gCA gTg ggA ACT CCA Agg AgA CA.
3.3.2.2 Peanut (AR-143 R), gCA TgA gAT gTT gCT CgC Ag.
3.3.2.3 Peanut probe (AR-103 T), FAM – CgA gAg ggC gAA CCT gAg gCC – TAMRA or – BHQ1.
3.3.3 Negative PCR control, conducted with DNA-free water instead of the DNA extract from the
sample.
3.3.4 Negative extraction control, performing all steps of the DNA extraction procedure, except
addition of the test portion, e.g. by substitution of a corresponding amount of water for the test portion.
3.3.5 Negative process control, sample of the food matrix without target sequence, which passes
through all steps of the analytical process (blank sample).
)
3.3.6 Positive PCR control reaction containing the target DNA in a specified quantity or number of
copies.
3.3.7 Positive process control, sample of the food matrix with known quantity of peanut, which
passes through all steps of the analytical process.
3.3.8 External amplification control (inhibition control), control DNA that is added to an aliquot
of the extracted nucleic acid in a specified quantity or number of copies and used in a separate reaction
to check the influence of co-extracted substances from the sample matrix on the amplification.
4 Apparatus and equipment
General aspects are described in EN ISO 24276 [3].

1)
Ready-to-use reagents or single components may be used as a PCR master mix, insofar as they provide
comparable or better results.
2)
DNA for the positive PCR control is extracted from phenotypically identified pure peanuts as described in 5.3
and 5.4. DNA mass concentration is determined as described in 5.5.
Plastic and glass materials shall be sterilised and free of DNA before use. In addition, the use of aerosol
protected filter tips is obligatory due to the high sensitivity of the PCR analytics and the resultant risk of
DNA contamination.
In addition to the usual laboratory facilities, the following equipment is required.
4.1 DNA extraction
4.1.1 Suitable reaction vials, 1,5 ml and 2 ml, DNA-free.
4.1.2 50 ml centrifug
...