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 Technical Specification 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) [1]. 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 %.

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

Diese Technische Spezifikation 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 [1]. Ein Senfgehalt von 10 mg/kg oder höher und ein Sojagehalt von 10 mg/kg oder höher können mit einer Wahrscheinlichkeit > 95 % nachgewiesen werden.

Živila - Odkrivanje prisotnosti alergenov v živilih z metodami molekularne biologije - 5. del: Gorčica (Sinapis alba) in soja (Glycine max) - Kvalitativno odkrivanje specifičnega niza DNK v obarjenih klobasah s PCR v realnem času

Ta osnutek tehnične specifikacije določa postopek za kvalitativno odkrivanje specifične DNK iz bele gorčice (Sinapis alba) in soje (Glycine max) v obarjenih klobasah z enojno PCR v realnem času na osnovi genov MADS-D (gorčica) in lektina (soja). Odkriti je mogoče delež gorčice 10 mg/kg ali večji in delež soje 10 mg/kg ali večji z verjetnostjo > 95 %.

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Status
Published
Publication Date
21-Jun-2016
Current Stage
9093 - Decision to confirm - Review Enquiry
Completion Date
04-Sep-2019

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SLOVENSKI STANDARD
SIST-TS CEN/TS 15634-5:2016
01-september-2016

äLYLOD2GNULYDQMHSULVRWQRVWLDOHUJHQRYYåLYLOLK]PHWRGDPLPROHNXODUQHELRORJLMH

GHO*RUþLFD 6LQDSLVDOED LQVRMD *O\FLQHPD[ .YDOLWDWLYQRRGNULYDQMH
VSHFLILþQHJDQL]D'1.YREDUMHQLKNOREDVDKV3&5YUHDOQHPþDVX

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
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
Ta slovenski standard je istoveten z: CEN/TS 15634-5:2016
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
67.120.10 Meso in mesni proizvodi Meat and meat products
SIST-TS CEN/TS 15634-5:2016 en,fr,de

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

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SIST-TS CEN/TS 15634-5:2016
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SIST-TS CEN/TS 15634-5:2016
CEN/TS 15634-5
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
June 2016
TECHNISCHE SPEZIFIKATION
ICS 07.100.30; 67.120.10
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
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

This Technical Specification (CEN/TS) was approved by CEN on 18 April 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 15634-5:2016 E

worldwide for CEN national Members.
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Contents Page

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

1 Scope .................................................................................................................................................................... 4

2 Principle ............................................................................................................................................................. 4

3 Reagents ............................................................................................................................................................. 4

4 Apparatus and equipment ........................................................................................................................... 6

5 Procedure........................................................................................................................................................... 6

5.1 General ................................................................................................................................................................ 6

5.2 Sample preparation ........................................................................................................................................ 6

5.3 DNA extraction with CTAB ........................................................................................................................... 7

5.4 DNA purification by means of solid phase extraction ........................................................................ 7

5.5 Measuring the mass concentration of the extracted DNA and setting to target

concentration ................................................................................................................................................... 8

5.6 Real-time PCR ................................................................................................................................................... 8

5.7 Temperature/Time program ...................................................................................................................... 9

6 Validation status and performance criteria .......................................................................................... 9

6.1 General information on the interpretation of the real-time PCR .................................................. 9

6.2 Reliability of the method ........................................................................................................................... 10

6.2.1 Setup of the interlaboratory study ........................................................................................................ 10

6.2.2 Results of the interlaboratory study samples.................................................................................... 10

6.2.3 Qualitative interpretation......................................................................................................................... 10

7 Test report ...................................................................................................................................................... 12

Bibliography ................................................................................................................................................................. 13

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European foreword

This document (CEN/TS 15634-5:2016) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN.

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 detection of a specific DNA

sequence in cooked sausages by real-time PCR [Technical Specification].

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.

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.
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1 Scope

This Technical Specification 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) [1]. 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 Principle

The DNA of the sample is extracted and is set to a definite concentration after photometric

measurement. 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 is multiplicated from

the sample DNA by real-time PCR. The amplicons formed are detected and verified by annealing a

sequence-specific probe and generating a fluorescence signal [2].
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 (Na EDTA).
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 8,0.
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3.1.14 Ethanol solution, φ = 70 %.
3.1.15 Proteinase K solution, ρ = 20 mg/ml.
Store aliquots of the solution at −20 °C.

3.1.16 TE buffer solution, containing TRIS (c = 0,01 mol/l) and Na -EDTA (c = 0,001 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, among them spin filter columns or plates or also using

vacuum operated systems. Suitable, commercially available kits can be used. Observe the

manufacturer's data for this.
3.3 Real-time PCR reagents:

3.3.1 Universal master mix (2 ×) for the real-time PCR, containing thermostable DNA polymerase

(for hot-start PCR) and PCR buffer solution (containing reaction buffers, dNTPs, MgCl and Hotstart

Taq polymerase), double concentrated.
3.3.2 Oligonucleotides [2]:
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
5´– FAM – AAC Cgg TAg CgT TgC CAg CTT Cg – TAMRA-3´
Mustard (Sinapis alba) MADS D protein [2]
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
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.
3.4 Controls:

3.4.1 Negative PCR control, conducted with DNA-free water instead of the DNA extract from the

sample and without PCR inhibitors.

1) Ready-to-use reagents or single components may be used as a PCR master mix. Quantitect Multiplex Mastermix

(2×), available from QIA- GEN, Hilden; was used within the interlaboratory study.

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3.4.2 Extraction blank 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.

3.4.3 Positive PCR control, sample containing the target sequence, which shall be treated in the

same way as the samples to be examined.
4 Apparatus and equipment

General aspects are described in EN ISO 24276 [3]. In addition to the usual laboratory facilities, the

following equipment is required.

Due to the high sensitivity of the PCR analytics and the risk of DNA contamination resulting from it, the

use of aerosol protected filter tips is obligatory.
4.1 DNA extraction:
4.1.1 Suitable reaction vials, 1,5 ml and 2 ml, Nucleic acid-free.
4.1.2 50 ml centrifuge tubes, Nucleic acid-free.
4.1.3 Thermostat or water bath, preferably with shaker function.
4.1.4 Centrifuge, suitable for centrifuging 50 ml centrifuge tubes at 8 000 g .

4.1.5 Centrifuge, suitable for centrifuging 1,5 ml and 2 ml reaction vials at 16 000 g.

4.1.6 Apparatus and/or material for grinding the sample, e.g. a blender.
4.1.7 UV spectrometer, suitable for estimating the amount of DNA.
4.2 PCR:
4.2.1 Suitable PCR tubes.
4.2.2 Microcentrifuge for PCR tubes.

4.2.3 Real-time PCR equipment, suitable for excitation and for emission measurement of

fluorescence-marked oligonucleotides.
5 Procedure
5.1 General
General aspects are described in EN ISO 24276 [3].
5.2 Sample preparation

Ensure that a representative sample is made available to the laboratory for investigation.

The sample shall be transported and stored so that damage and/or changes are prevented.

Ensure that the test sample is representative of the laboratory sample, e.g. by milling or homogenizing.

2) g = 9,81 m · s .
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5.3 DNA extraction with CTAB

Measures and work steps to be considered for the DNA extraction are described in EN ISO 21571 [4].

An extraction blank control (3.4.2) shall be performed in parallel.
— Weigh 2 g of the homogenized test sample into 50 ml centrifuge tubes;
— Add 10 ml of CTAB buffer (3.1.13);
— Add 30 µl of Proteinase K (3.1.15) and mix;
— Incubate and shake for 90 min at 65 °C;
— Centrifuge for 5 min at 6 000 g to 8 0
...

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