SIST-TS CEN/TS 15634-2:2012

SLOVENSKI STANDARD
SIST-TS CEN/TS 15634-2:2012
01-april-2012
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Foodstuffs - Detection of food allergens by molecular biological methods - Part 2: Celery
(Apium graveolens) - Qualitative determination of a specific DNA sequence in cooked
sausages by real-time PCR
Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen
Verfahren - Teil 2: Sellerie (Apium graveolens) - Qualitative Bestimmung 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) - Détermination
qualitative d'une séquence d'ADN spécifique dans des saucisses cuites par PCR en
temps réel
Ta slovenski standard je istoveten z: CEN/TS 15634-2:2012
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-TS CEN/TS 15634-2:2012 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 15634-2:2012

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SIST-TS CEN/TS 15634-2:2012


TECHNICAL SPECIFICATION
CEN/TS 15634-2

SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION
February 2012
ICS 07.100.30; 67.050
English Version
Foodstuffs - Detection of food allergens by molecular biological
methods - Part 2: Celery (Apium graveolens) - Qualitative
determination of a specific DNA sequence in cooked sausages
by real-time PCR
Produits alimentaires - Détection des allergènes
Lebensmittel - Nachweis von Lebensmittelallergenen mit
alimentaires par des méthodes d'analyse de biologie molekularbiologischen Verfahren - Teil 2: Sellerie (Apium
moléculaire - Partie 2: Céleri (Apium graveolens) - graveolens) - Qualitative Bestimmung einer spezifischen
Détermination qualitative d'une séquence d'ADN spécifique DNA-Sequenz in Brühwürsten mittels Real-time-PCR
dans des saucisses cuites par PCR en temps réel
This Technical Specification (CEN/TS) was approved by CEN on 15 November 2011 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, 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.





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COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 15634-2: E
worldwide for CEN national Members.

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SIST-TS CEN/TS 15634-2:2012
CEN/TS 15634-2:2012 (E)
Contents Page
Foreword .3
1 Scope .4
2 Principle .4
3 Reagents .4
4 Apparatus and equipment .6
5 Analysis steps .6
6 Validation status and performance criteria . 11
7 Sample type and amounts . 14
8 Limit of detection . 14
9 Interferences . 14
10 Test report . 14
11 Method performance studies . 15
Bibliography . 17

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SIST-TS CEN/TS 15634-2:2012
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Foreword
This document (CEN/TS 15634-2:2012) 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.
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, 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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SIST-TS CEN/TS 15634-2:2012
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1 Scope
This Technical Specification specifies a method for the qualitative detection of celery (Apium graveolens) in
emulsion-type sausages (e.g. Frankfurter, Wiener).
Real-time PCR 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 [2].
2 Principle
Total DNA from emulsion-type sausages are isolated from the sample matrix. DNA is released from the
sample matrix using the cetyltrimethylammonium bromide (CTAB) approach. Potential PCR inhibitors are
removed from the isolated DNA by purification with solid phase columns. Real-time PCR is used to detect,
amplify and quantify a celery specific sequence. The real time PCR method involves a fluorescence approach
with a sequence specific hydrolysis probe [1], [2].
3 Reagents
3.1 General
The following general conditions for analysis shall be followed, unless specified differently. Use only analytical
grade reagents suitable for molecular biology. Reagents shall be stored in small aliquots to minimise 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.
3.2 Extraction reagents
3.2.1 Chloroform, CAS 66-67-3.
3.2.2 Ethanol, volume fraction ϕ = 70 %, CAS 64-17-5.
3.2.3 Ethylenediaminetetraacetic acid disodium salt (Na EDTA), CAS 6381-92-6.
2
3.2.4 Cetyltrimethylammoniumbromide (CTAB), CAS 57-09-0.
3.2.5 Hydrochloric acid, ϕ = 37 %, CAS 7647-01-0.
3.2.6 Isoamyl alcohol, CAS 123-51-3.
3.2.7 Isopropanol, CAS 67-63-0.
3.2.8 Proteinase K, EC 3.4.21.64.
3.2.9 Sodium chloride, CAS 7647-14-5.
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3.2.10 Sodium hydroxide, CAS 1310-73-2.
3.2.11 Tris(hydroxymethyl)aminomethane (TRIS), CAS 7-86-1.
3.2.12 Chloroform isoamyl alcohol mixture, 24 parts by volume of chloroform (3.2.1) are mixed with one
part by volume of isoamyl alcohol (3.2.6).
NOTE Similar mixtures available commercially may be used.
3.2.13 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). The pH shall be
2
adjusted to read 8,0 by adding hydrochloric acid.
3.2.14 Proteinase K solution (ρ = 20 mg/ml)
NOTE Store in the form of aliquots at -20 °C after dissolving. Do not autoclave.
3.2.15 TE buffer solution containing TRIS (c = 0,001 mol/l) and Na EDTA (c = 0,000 1 mol/l). The pH shall
2
be adjusted to read 8,0 by adding hydrochloric acid or sodium hydroxide solution.
3.3 DNA purification by means of solid phase extraction
For the DNA purification different methods may be used.
NOTE Several formats are commercially available, among them spin filter columns or plates. Commercially available
kits may be used as appropriate.
3.4 Real-time PCR reagents
1)
3.4.1 Concentrated PCR buffer solution (containing reaction buffers, dNTPs, MgCl and Hotstart Taq
2
polymerase).
3.4.2 Oligonucleotides, c = 20 µmol/l each.
3.4.2.1 General
For information on the DNA target sequence and validation of selectivity, see 6.3.
NOTE In the interlaboratory study, the participants received their primers and the probe from the same production lot.
3.4.2.2 Forward primer (iF), Cel-MDH iF 5’-CgA TgA gCg TgT ACT gAg TC – 3’.
3.4.2.3 Reverse primer (iR), Cel-MDH iR 5’-AAT Agg AAC TAA CAT TAA TCA TAC CAA AC – 3’.
2)
3.4.2.4 Cel-MDH probe 5’-FAM-AAC AgA TAA CgC TgA CTC ATC ACA CCg-TAMRA – 3’ .

1) 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.
2) 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.
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4 Apparatus and equipment
4.1 General
General aspects are described in EN 15634-1 [3]. In addition to the usual laboratory facilities, the following
equipment is required.
NOTE 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.
4.2 DNA extraction
4.2.1 Suitable reaction vials with a capacity of 1,5 ml and 2 ml, sterile; 50 ml centrifuge tube, sterile.
4.2.2 Thermostat or water bath, preferably with shaker function.
3)
4.2.3 Centrifuge suitable for centrifuging 50 ml centrifuge tubes at 8 000 g .
4.2.4 Centrifuge suitable for centrifuging 1,5 ml and 2 ml reaction vials at 14 500 g.
4.2.5 Equipment and/or material for grinding the sample, e.g., a kitchen blender.
4.2.6 UV spectrophotometer or other detection instruments suitable for estimating the amount of DNA.
4.3 PCR
4.3.1 Suitable PCR tubes
4.3.2 Microcentrifuge for PCR tubes
4.3.3 Real-time PCR equipment suitable for excitation and for emission measurement of fluorescence-
marked oligonucleotides.
5 Analysis steps
5.1 General
General aspects are described in EN 15634-1 [3].
5.2 Sample preparation
It should be ensured, e.g. by milling or homogenizing, that the test sample is representative of the laboratory
sample.
In order to minimise 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.

-2
3) g = 9,81 m × s
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5.3 Preparation of extracts
5.3.1 DNA extraction with CTAB and DNA purification
In parallel to the test samples, the controls listed in 5.4.6 and 5.4.7 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 (3.2.13).
 Add 30 µl of Proteinase K solution (3.2.14) and mix by inversion, pipetting or vortexing.
 Incubate and shake for 90 min at a temperature of 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 (3.2.12) 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 (3.2.7) in a 1,5 ml reaction vial (tube C).
 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 (3.2.2) and swirl 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 (3.2.15).
NOTE 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 available.
The purified DNA extract may be stored for a short period of time (approx. 1 week) at 4 °C. For long-term
storage of several months a temperature of -18 °C should be maintained.
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5.3.2 Quantification and normalization of DNA concentration
The concentration of a DNA aliquot can be determined by means of 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.
NOTE 1 The DNA concentration may also be estimated using other suitable procedures.
NOTE 2 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.
5.4 Procedure: Real-time PCR set-up
5.4.1 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 5.4.3 to 5.4.7 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.
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 [4].
Table 1 — Reaction mix for real-time PCR
Reagent Final substance
concentration

a) 2x PCR buffer solution (3.4.1) 1x
b) Primer Cel-MDH iF 0,3 µmol/l
c) Primer Cel-MDH iR 0,3 µmol/l
d) Probe Cel-MDH probe 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
per PCR reaction into the reaction vials.
 For each of the sample reactions, pipette 5 µl of sample DNA extract into the PCR mix.
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 For the positive DNA target control (5.4.2), pipette 5 µl of a celery reference DNA into the PCR mix.
 For the negative DNA target control (5.4.3), pipette 5 µl of a celery free sample DNA extract into the
PCR mix.
 For the PCR inhibition controls (5.4.4), pipette one aliquot of celery control DNA (e.g. 100 copies of the
279 bp amplicon described in reference [1]) to an aliquot of (each of) the extracted sample DNA.
 For the amplification reagent control (5.4.5), pipette 5 µl of water into the PCR mix.
 For the extraction blank control (5.4.6), pipette 5 µl of the corresponding control extract into the PCR
mix.
 For the positive extraction control (5.4.7), 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
5.4.8.
5.4.2 Positive control for DNA targets
In general, there are four types of positive controls for DNA targets:
1. Reference DNA
2. DNA extracted from a certified reference material
3. Known positive sample representative of the sequence
4. Target under study.
NOTE The control is intended to demonstrate what the positive test samples are expected to look like.
5.4.3 Negative control for DNA targets
In general, there are three types of negative controls for DNA targets
1. Reference DNA
2. DNA extracted from a certified negative reference material (blank matrix)
3. Known negative sample not containing the sequence under study.
NOTE The control is intended to demonstrate the result of negative test samples (i. e. samples not containing the
sequence under study).
5.4.4 PCR inhibition control
Control DNA which is added to an aliquot of the extracted nucleic acid sample in a specified amount or
number of copies. This aliquot is then used in a separate reaction batch in order to check the influence of co-
extracted
...