1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationFoodstuffs Detection of food allergens by molecular biological methodsPart 2: Celery (Apium graveolens) Qualitative determination of a specific DNA sequence in cooked sausages by
2、 real-time PCRPD CEN/TS 15634-2:2012National forewordThis Published Document is the UK implementation of CEN/TS 15634-2:2012. The UK participation in its preparation was entrusted to Technical CommitteeAW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee c
3、an be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2012Published by BSI Standards Limited 2012 ISBN 978 0 580 75517 0 ICS 07.100.30; 6
4、7.050Compliance with a British Standard cannot confer immunity from legal obligations.This Published Document was published under the authority of the Standards Policy and Strategy Committee on 29 February 2012.Amendments issued since publicationAmd. No. Date Text affectedPUBLISHED DOCUMENTPD CEN/TS
5、 15634-2:2012TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 15634-2 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 specif
6、ic DNA sequence in cooked sausages by real-time PCR Produits alimentaires - Dtection des allergnes alimentaires par des mthodes danalyse de biologie molculaire - Partie 2: Cleri (Apium graveolens) - Dtermination qualitative dune squence dADN spcifique dans des saucisses cuites par PCR en temps rel L
7、ebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 2: Sellerie (Apium graveolens) - Qualitative Bestimmung einer spezifischen DNA-Sequenz in Brhwrsten mittels Real-time-PCR This Technical Specification (CEN/TS) was approved by CEN on 15 November 2011 for pro
8、visional 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 annou
9、nce 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 th
10、e 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, Portu
11、gal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17, B-1000 Brussels 2012 CEN All rights of exploitation in any form and by an
12、y means reserved worldwide for CEN national Members. Ref. No. CEN/TS 15634-2: EPD CEN/TS 15634-2:2012CEN/TS 15634-2:2012 (E) 2 Contents Page Foreword 31 Scope 42 Principle 43 Reagents .44 Apparatus and equipment 65 Analysis steps 66 Validation status and performance criteria 117 Sample type and amou
13、nts . 148 Limit of detection 149 Interferences . 1410 Test report . 1411 Method performance studies . 15Bibliography . 17PD CEN/TS 15634-2:2012CEN/TS 15634-2:2012 (E) 3 Foreword This document (CEN/TS 15634-2:2012) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal method
14、s”, 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 Reg
15、ulations, 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, Luxembou
16、rg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 15634-2:2012CEN/TS 15634-2:2012 (E) 4 1 Scope This Technical Specification specifies a method for the qualitative detection of celery (Apium graveolens)
17、 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
18、“Leberkse”) 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. T
19、he 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 isola
20、ted 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 se
21、quence. 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. R
22、eagents 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 diffe
23、rently. 3.2 Extraction reagents 3.2.1 Chloroform, CAS 66-67-3. 3.2.2 Ethanol, volume fraction = 70 %, CAS 64-17-5. 3.2.3 Ethylenediaminetetraacetic acid disodium salt (Na2EDTA), CAS 6381-92-6. 3.2.4 Cetyltrimethylammoniumbromide (CTAB), CAS 57-09-0. 3.2.5 Hydrochloric acid, = 37 %, CAS 7647-01-0. 3.
24、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. PD CEN/TS 15634-2:2012CEN/TS 15634-2:2012 (E) 5 3.2.10 Sodium hydroxide, CAS 1310-73-2. 3.2.11 Tris(hydroxymethyl)aminomethane (TRIS), CAS 7-86-1. 3.2.12 Chlorof
25、orm 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 = 20 g/l), sodium chloride (substan
26、ce concentration c = 1,4 mol/l), TRIS (c = 0,1 mol/l), Na2EDTA (c = 0,02 mol/l). The pH shall be adjusted to read 8,0 by adding hydrochloric acid. 3.2.14 Proteinase K solution ( = 20 mg/ml) NOTE Store in the form of aliquots at -20 C after dissolving. Do not autoclave. 3.2.15 TE buffer solution cont
27、aining TRIS (c = 0,001 mol/l) and Na2EDTA (c = 0,000 1 mol/l). The pH shall 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 comme
28、rcially available, among them spin filter columns or plates. Commercially available kits may be used as appropriate. 3.4 Real-time PCR reagents 3.4.1 Concentrated PCR buffer solution 1)(containing reaction buffers, dNTPs, MgCl2and Hotstart Taq polymerase). 3.4.2 Oligonucleotides, c = 20 mol/l each.
29、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 Re
30、verse primer (iR), Cel-MDH iR 5-AAT Agg AAC TAA CAT TAA TCA TAC CAA AC 3. 3.4.2.4 Cel-MDH probe 5-FAM-AAC AgA TAA CgC TgA CTC ATC ACA CCg-TAMRA 3 2). 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 tha
31、n 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. PD CEN/TS 15634-2:2012CEN/TS 15634-2:2012 (E) 6 4 Apparatus and equipment 4.
32、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 DN
33、A 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. 4.2.3 Centrifuge suitable for centrifuging 50 ml centrifuge tubes at 8
34、000 g 3). 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 Suitab
35、le 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. b
36、y 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-degra
37、ding agents, hypochlorite solution, hot water and detergents. 3) g = 9,81 m s-2PD CEN/TS 15634-2:2012CEN/TS 15634-2:2012 (E) 7 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 a
38、dequately. 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 l of Proteinase K solution (3.2.14) and mix by inversion, pipetting or vortexing Incubate a
39、nd 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 l of chloroform isoamyl alcohol mixture (3.2.12) in a 2 ml reaction vial (tube B). Add 700 l of supernatant from tube A to tube B and mix thoroughly for 30 s. Centrifuge for 15 min
40、 at approximately 14 500 g at room temperature. Place 500 l of isopropanol (3.2.7) in a 1,5 ml reaction vial (tube C). Add 500 l 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
41、 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 l ethanol (3.2.2) and swirl several times. Centrifuge for 5 min at approximately 14 500 g at room temperature. Carefully remov
42、e 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 l of TE buffer solution (3.2.15). NOTE It is acce
43、ptable 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 manufacture
44、r 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. PD CEN/TS 15634-2:2012CEN/TS 15634-2:2012 (E) 8 5.3.2 Quantification and normalization of DNA concentration
45、 The concentration of a DNA aliquot can be determined by means of UV spectrophotometers at a wavelength of 260 nm (concentration in ng/l = 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 val
46、ues 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/l, e. g. by diluting wi
47、th 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 l (20 l PCR mix and 5 l DNA extract) with the reagents indicated in Table 1. The final concentrations of the reagents given
48、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
49、.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
