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

This document specifies a method for the detection of celery (Apium graveolens) in emulsion-type sausages (e.g. Frankfurter, Wiener).
Real-time PCR (polymerase chain reaction) 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 [1].
This document is intended to be used in addition to EN 15842 and FprEN 15634 1.

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

Dieses Dokument legt ein Verfahren für den Nachweis von Sellerie (Apium graveolens) in Brühwürsten (z. B. Frankfurter, Wiener) fest.
Der Nachweis von Sellerie mittels Real-time-PCR (Polymerase-Kettenreaktion) beruht auf einer 101 bp (Basenpaar) langen Sequenz aus dem Gen der Mannitoldehydrogenase (GenBank Acc. No. AF0670821) 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 [1].
Dieses Dokument ist für die Verwendung zusätzlich zu EN 15842 und EN 15634-1 vorgesehen.

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

Le présent document décrit une méthode de détection du céleri (Apium graveolens) dans des saucisses émulsionnées (par exemple, saucisses de Francfort ou de Vienne).
La détection du céleri par PCR (réaction de polymérisation en chaîne) 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, 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, a été préparée 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 [1].
Le présent document est destiné à être utilisé conjointement avec l’EN 15842 et l’EN 15634 1.

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

Ta dokument določa metodo za 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 »Leberkäse«), 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 [2].

General Information

Status
Published
Publication Date
01-Oct-2019
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
02-Oct-2019
Completion Date
02-Oct-2019

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SLOVENSKI STANDARD
SIST EN 15634-2:2019
01-december-2019
Nadomešča:
SIST-TS CEN/TS 15634-2:2012
Živila - Odkrivanje prisotnosti alergenov v živilih z molekularno biološkimi
metodami - 2. del: Zelena (Apium graveolens) - Odkrivanje specifičnega niza DNK
v obarjenih klobasah s PCR v realnem času

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
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen

Verfahren - Teil 2: Sellerie (Apium graveolens) - Nachweis 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) - Détection d'une

séquence d'ADN spécifique dans des saucisses cuites par PCR en temps réel
Ta slovenski standard je istoveten z: EN 15634-2:2019
ICS:
07.100.30 Mikrobiologija živil Food microbiology
67.120.10 Meso in mesni proizvodi Meat and meat products
SIST EN 15634-2:2019 en,fr,de

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

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SIST EN 15634-2:2019
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SIST EN 15634-2:2019
EN 15634-2
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2019
EUROPÄISCHE NORM
ICS 07.100.30; 67.120.10 Supersedes CEN/TS 15634-2:2012
English Version
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

Produits alimentaires - Détection des allergènes Lebensmittel - Nachweis von Lebensmittelallergenen

alimentaires par des méthodes d'analyse de biologie mit molekularbiologischen Verfahren - Teil 2: Sellerie

moléculaire - Partie 2 : Céleri (Apium graveolens) - (Apium graveolens) - Nachweis einer spezifischen

Détection d'une séquence d'ADN spécifique dans des DNA-Sequenz in Brühwürsten mittels Real-time-PCR

saucisses cuites par PCR en temps réel
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 15634-2:2019 E

worldwide for CEN national Members.
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SIST EN 15634-2:2019
EN 15634-2:2019 (E)
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

6 Apparatus and equipment ........................................................................................................................... 7

7 Procedure........................................................................................................................................................... 8

8 Expression of results ................................................................................................................................... 13

9 Validation ........................................................................................................................................................ 13

10 Test report ...................................................................................................................................................... 19

Bibliography ................................................................................................................................................................. 20

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SIST EN 15634-2:2019
EN 15634-2:2019 (E)
European foreword

This document (EN 15634-2:2019) 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 CEN/TS 15634-2:2012.

Significant technical changes between this standard and CEN/TS 15634-2:2012 are as follows:

a) the document was converted from a Technical Specification into a European Standard;

b) normative references added (2);
c) expression of results (8) updated;
d) requirements regarding the test report added (10)
e) 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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EN 15634-2:2019 (E)
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 method for the detection of celery (Apium graveolens) in emulsion-type

sausages (e.g. Frankfurter, Wiener).

Real-time PCR (polymerase chain reaction) 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

[1].
This document is intended to be used in addition to EN 15842 and EN 15634-1.
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 considerations

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 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 emulsion-type sausages is isolated from the sample using a cetyltrimethylammonium

bromide (CTAB) method. Potential PCR inhibitors are removed from the DNA extracted by purification

with solid phase columns. Real-time PCR is used to detect a celery specific sequence. The real time PCR

method involves a fluorescence detection with a sequence specific hydrolysis probe [1], [2].

1) NCBI-GeneBank® is an example of a suitable search tool for free use. This information is given for the convenience of

users of this document and does not constitute an endorsement by CEN.
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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. Reagents should be stored in small aliquots to

minimize 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.
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, φ = 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.
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), c = 0,1 mol/l,
— Na EDTA (5.2.3), 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
Store in the form of aliquots at −20 °C after dissolving. Do not autoclave.
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5.2.15 TE buffer solution, containing:
— TRIS (5.2.11), c = 0,001 mol/l, and
— Na EDTA (5.2.3), c = 0,000 1 mol/l.

The pH is adjusted to 8,0 by adding hydrochloric acid (5.2.5) or sodium hydroxide solution (5.2.10).

5.3 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.
5.4 Real-time PCR reagents

5.4.1 Concentrated PCR buffer solution (containing reaction buffers, dNTPs, MgCl and Hotstart

Taq polymerase).
5.4.2 Oligonucleotides, c = 20 µmol/l each.
5.4.2.1 Forward primer (iF), Cel-MDH iF 5’-CgA TgA gCg TgT ACT gAg TC – 3’.

5.4.2.2 Reverse primer (iR), Cel-MDH iR 5’-AAT Agg AAC TAA CAT TAA TCA TAC CAA AC – 3’.

5.4.2.3 Cel-MDH probe 5’-FAM-AAC AgA TAA CgC TgA CTC ATC ACA CCg-TAMRA – 3’ .
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.

6.2 DNA extraction

6.2.1 Suitable reaction vials with a capacity of 1,5 ml and 2 ml, sterile; 50 ml centrifuge tube, sterile.

6.2.2 Thermostat or water bath, preferably with shaker function.
6.2.3 Centrifuge suitable for centrifuging 50 ml centrifuge tubes at 8 000 g .

6.2.4 Centrifuge suitable for centrifuging 1,5 ml and 2 ml reaction vials at 14 500 g.

6.2.5 Equipment and/or material for grinding the sample, e.g. a kitchen blender.

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.

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) 2
g = 9,81 m × s
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6.2.6 UV spectrophotometer or other detection instruments suitable for estimating the amount of

DNA.
6.3 PCR
6.3.1 Suitable PCR tubes
6.3.2 Microcentrifuge for PCR tubes

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

fluorescence-marked oligonucleotides.
7 Procedure
7.1 General
General aspects are described in EN 15634-1.

In general, factors compromising DNA detection methods, include PCR inhibitors, acidic pH and/or

extensive heat treatment of the food commodity and reduction or elimination of the DNA during the

production process. The detection of celery root powder is reduced compared to equivalent amounts of

celery seed as demonstrated by the inter-laboratory study.
7.2 Sample preparation

It should be ensured, that the test sample taken after milling or homogenizing is representative of the

laboratory sample.

In order to minimize 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.
7.3 Preparation of extracts
7.3.1 DNA extraction with CTAB and DNA purification

In parallel to the test samples, the controls listed in 7.4.6 and 7.4.7 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 (5.2.13).

— Add 30 µl of Proteinase K solution (5.2.14) and mix by inversion, pipetting or vortexing.

— Incubate and shake for 90 min at 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 (5.2.12) 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 (5.2.7) in a 1,5 ml reaction vial (tube C).
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— 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.

— Fill the reaction vial with 500 µl ethanol (5.2.2) and mix several times.
— Centrifuge for 5 min at approximately 14 500 g at room temperature.

— Carefully remove and discard the supernatant using a pipette or by gently pouring out.

— Dry the extracted DNA in order to remove the remaining traces of ethanol, e.g. by inverting tube C

and allowing to blot dry on paper towels.
— Dissolve the dried DNA extract in 100 µl of TE buffer solution (5.2.15).

It is acceptable to use a commercially available kit instead of the DNA extraction procedure described

above, if it is ensured that comparable or better results are obtained.

— Purify the DNA extract using e.g. solid phase extraction. For commercial kits the instructions given

by the respective kit manufacturer are considered.

The purified DNA extract may be stored for a short period of time (approximately 1 week) at 4 °C. For

long-term storage of several months the storage temperature should be −18 °C.
7.3.2 Quantification and normalization of DNA concentration

The concentration of a DNA aliquot can be determined by UV spectrophotometers at a wavelength of

260 nm (concentration in ng/µl = 50 × optical density × dilution factor of the measured aliquot).

In order to check its purity, the sample can in addition be measured at 280 nm. The ratio of the values

for optical density at 260 nm and 280 nm should be approximately 1,8.
The DNA concentration may also be estimated using other suitable procedures.

NOTE In the interlaboratory trial the DNA extracts have been adjusted to a mass concentration of

approximately 20 ng/µl, e.g. by diluting with water.
7.4 Real-time PCR set-up
7.4.1 Reaction mix for real-time PCR

As an example, the procedure is described in the following for a total reaction volume of 25 µl (20 µl

PCR mix and 5 µl DNA extract) with the reagents indicated in Table 1. The final concentrations of the

reagents given in Table 1 have been proved to be suitable. The PCR may also be carried out with larger

volumes, if the solutions are adapted correspondingly.

In parallel to the test samples, the controls listed in 7.4.3 to 7.4.7 should be carried out adequately.

Prior to use, the reagents should be gently thawed e.g. on an ice/cooling block and centrifuged briefly. If

needed, during preparation of the PCR mix the reagents should be stored in an ice bath or cooling block.

Care shall be taken to carefully mix any reagents immediately before pipetting.
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A PCR mix should be prepared or set up containing all the components except for the DNA extract. The

required amount of PCR mix is determined by the number of reactions to be carried out plus a pipetting

reserve of approximately 10 %.

Two replicates of each DNA extract should be analysed by PCR. For each reaction, 5 µl of DNA extract

should be used.

NOTE In order to exclude false-negative results occurring due to PCR inhibition or highly degraded DNA,

suitability of the isolate DNA can be checked by, e.g. amplification of universal sequences from plants [3].

Table 1 — Reaction mix for real-time PCR
Final substance
Reagent
concentration
a) 2x PCR buffer solution (5.4.1) 1x
b) Primer Cel-MDH iF 0,3 µmol/l
c) Primer Cel-MDH iR 0,3 µmol/l
d) Probe Cel-MDH 0,2 µmol/l
e) Water
f) DNA extract (approx. 20 ng/µl, max.
50 ng/µl)

— Mix the PCR reagents (see Table 1, a) to e)), centrifuge shortly (not longer than 10 s) and pipette

20 µl of this mixture in each reaction vial.

— For each of the sample reactions, pipette 5 µl of sample DNA extract (see Table 1, f) into the PCR

mix.

— For the positive DNA target control (7.4.2), pipette 5 µl of a celery reference DNA into the PCR

mix.

— For the negative DNA target control (7.4.3), pipette 5 µl of a celery free sample DNA extract into

the PCR mix.

— For the PCR inhibition controls (7.4.4), pipette one aliquot of celery control DNA (e.g. 100 copies

of the 279 bp amplicon described in reference [2]) to an aliquot of the extracted sample DNA.

— For the amplification reagent control (7.4.5), pipette 5 µl of water into the PCR mix.

— For the extraction blank control (7.4.6), pipette 5 µl of the corresponding control extract into the

PCR mix.

— For the positive extraction control (7.4.7), pipette 5 µl of DNA extract of a reference sample

containing celery into the PCR mix.

— Place the reaction vials in the PCR device and start the temperature/time programme as described

in 7.4.8.
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7.4.2 Positive control for DNA targ
...

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