1、BSI Standards Publication Foodstuffs Detection of food allergens by molecular biological methods Part 3: Hazelnut (Corylus avellana) Qualitative detection of a specific DNA sequence in chocolate by real-time PCR PD CEN/TS 15634-3:2016National foreword This Published Document is the UK implementation
2、 of CEN/TS 15634-3:2016. The UK participation in its preparation was entrusted to Technical Committee AW/275, Food analysis - Horizontal methods. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the ne
3、cessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2016. Published by BSI Standards Limited 2016 ISBN 978 0 580 90302 1 ICS 07.100.30; 67.190 Compliance with a British Standard cannot confer immunity from legal obligations. This Pub
4、lished Document was published under the authority of the Standards Policy and Strategy Committee on 30 April 2016. Amendments/corrigenda issued since publication Date Text affected PUBLISHED DOCUMENT PD CEN/TS 15634-3:2016 TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/T
5、S 15634-3 March 2016 ICS 07.100.30; 67.190 English Version Foodstuffs - Detection of food allergens by molecular biological methods - Part 3: Hazelnut (Corylus avellana) - Qualitative detection of a specific DNA sequence in chocolate by real-time PCR Produits alimentaires - Dtection dallergnes alime
6、ntaires par des mthodes de biologie molculaire - Partie 3: Noisette (Corylus avellana) - Dtection qualitative dune squence dADN spcifique dans du chocolat, par PCR en temps rel Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 3: Haselnuss (Corylus avellan
7、a) - Qualitativer Nachweis einer spezifischen DNA-Sequenz in Schokolade mittels Real-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 memb
8、ers 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
9、 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, Cyp
10、rus, 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 Ki
11、ngdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR 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 worldwide for CEN national Members. Ref. No. CEN/TS 1
12、5634-3:2016 E PD CEN/TS 15634-3:2016CEN/TS 15634-3:2016 (E) 2 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 . 6 4.1 DNA e
13、xtraction . 6 4.2 PCR 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 8 6 Validation stat
14、us and performance criteria . 10 6.1 General . 10 6.2 Detection . 10 6.3 Reliability of the method . 11 7 Test report 12 Bibliography . 13 PD CEN/TS 15634-3:2016CEN/TS 15634-3:2016 (E) 3 European foreword This document (CEN/TS 15634-3:2016) has been prepared by Technical Committee CEN/TC 275 “Food a
15、nalysis - 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, Food
16、stuffs 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
17、 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 a
18、lba) 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 Specifica
19、tion: 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, Sp
20、ain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 15634-3:2016CEN/TS 15634-3:2016 (E) 4 1 Scope This Technical Specification describes a procedure for the qualitative detection of hazelnut (Corylus avellana) in chocolate. DNA is extracted from the chocolate and a specific DNA sequen
21、ce for hazelnut detected from the gene for corA 1 4, 5. 2 Principle The total DNA is extracted from the sample and the DNA content estimated. A 152 bp long sequence from the gene for corA 1 is multiplicated using real-time PCR. The amplicon formed in this way is detected by annealing a sequence-spec
22、ific probe and generating a fluorescence signal 4, 5. 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 an
23、d autoclaving, unless indicated differently. 3.1 DNA extraction with CTAB 3.1.1 Chloroform. 3.1.2 Ethanol, volume fraction = 96 %. 3.1.3 Ethylenediaminetetraacetic acid disodium salt (Na 2EDTA). 3.1.4 Cetyltrimethylammoniumbromide (CTAB). 3.1.5 Hydrochloric acid, mass fraction w = 37 %. 3.1.6 Isoamy
24、l 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 av
25、ailable and comparable mixtures can be used. 3.1.13 CTAB extraction buffer solution, containing CTAB (mass concentration = 20 g/l), sodium chloride (substance concentration c = 1,4 mol/l), TRIS (c = 0,1 mol/l), Na 2EDTA (c = 0,02 mol/l). Adjust the pH value with hydrochloric acid to pH = 8,0. 3.1.14
26、 Ethanol solution, = 70 %. 3.1.15 Proteinase K solution, = 20 mg/ml. PD CEN/TS 15634-3:2016CEN/TS 15634-3:2016 (E) 5 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 2-EDTA (c = 0,000 1 m
27、ol/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 For the DNA purification, different methods may be used. Various systems are commercially available for DNA purification by means of solid phase extract
28、ion, including spin filter columns or plates or also with vacuum operated systems. Commercially available kits can also be used. Observe the manufacturers data for this (see also 6.3.1). 3.3 Real-time PCR reagents 3.3.1 PCR master mix 1 ) , containing reaction buffer, dNTPs, MgCl 2 and Hotstart Taq
29、polymerase. 3.3.2 Oligonucleotides, 5 mol each. 3.3.2.1 Hazelnut iF, 5 TAC AgC ATC ATC gAg ggA ggT C 3. 3.3.2.2 Hazelnut iR, 5 CTC CTC ATT gAT TgA AgC gTT g 3. 3.3.2.3 Hazelnut probe, 5 FAM AgA Tgg Cgg CAg CCC CTC AT TAMRA 3 2 )3.3.3 Negative PCR control, conducted with DNA-free water instead of the
30、 DNA extract from the sample and without PCR inhibitors. 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
31、of the food matrix without target sequence, which passes through all steps of the analytical process. 3.3.6 Positive PCR control 3 ) , 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 haze
32、lnut, 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-ex
33、tracted substances from the sample matrix on the amplification. 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)FAM: 6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine; equivalent reporter dyes and/or quenc
34、her dyes may be used if they are shown to give comparable or better results. 3) DNA for the positive PCR control is extracted from phenotypically identified pure hazelnuts as described in 5.3 and 5.4. DNA mass concentration is determined as described in 5.5.PD CEN/TS 15634-3:2016CEN/TS 15634-3:2016
35、(E) 6 4 Apparatus and equipment General aspects are described in EN ISO 24276 3. Plastic and glass materials shall be sterilized 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
36、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 centrifuge tubes, sterile. 4.1.3 Thermostat or water bath, preferably with shaker function. 4.1.4 Centrifug
37、e, suitable for centrifuging 50 ml centrifuge tubes at 8 000 g 4 ) . 4.1.5 Centrifuge, suitable for centrifuging 1,5 ml and 2 ml reaction vials at 14 500 g. 4.1.6 Apparatus and/or material for grinding the sample, e.g. blender or mill. 4.1.7 UV spectrometer or other detection instruments, suitable f
38、or 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. NOTE Laboratories participating in the interlaboratory study used the f
39、ollowing real-time PCR equipment: Rotor Gene 2000 or 3000, Stratagene Mx 3005P, ABI PRISM 7000 or 7500, ABI PRISM 7700 or 7900HT and Roche LightCycler . 5)5 Procedure 5.1 General General aspects are described in EN ISO 24276 3. 5.2 Sample preparation Ensure, e.g. by milling or homogenizing, that the
40、 test sample is representative of the laboratory sample. 4)g = 9,81 m s2 5 ) Rotor Gene 2000 and 3000, Stratagene Mx 3005P, ABI PRISM 7000 and 7500, ABI PRISM 7700 and 7900HT and Roche LightCycler are examples of suitable products available commercially. This information is given for the convenience
41、 of users of this Technical Specification and does not constitute an endorsement by CEN of these products. Equivalent products may be used if they can be shown to lead to the same results.PD CEN/TS 15634-3:2016CEN/TS 15634-3:2016 (E) 7 5.3 DNA extraction with CTAB Measures and work steps to be consi
42、dered for the DNA extraction are described in EN ISO 21571 2. It is acceptable to use a commercially available kit instead of the DNA extraction procedure described below, if it is ensured that comparable or better results are obtained. In parallel to the test samples, carry out the controls listed
43、in 3.3.4, 3.3.5 and 3.3.7 adequately. Prepare every sample twice in accordance with the following scheme: Weigh 2 g of the sample into 50 ml centrifuge tubes; Add 10 ml of CTAB extraction buffer solution (3.1.13); Add 30 l of Proteinase K solution (3.1.15) and mix; Incubate and shake for 90 min at 6
44、5 C; Centrifuge for 5 min at 6 000 g to 8 000 g; Place 500 l of chloroform isoamyl alcohol mixture (3.1.12) in a 2 ml reaction vial; Add 700 l of supernatant and mix thoroughly for 30 s; Centrifuge for 15 min at about 14 500 g; Place 500 l of cold isopropanol (3.1.7) in a 1,5 ml reaction vial; Add 5
45、00 l of supernatant (aqueous phase) and mix carefully; Incubate for 30 min at room temperature; Centrifuge for 15 min at about 14 500 g; Carefully remove and discard the supernatant; Fill the reaction vial with 500 l of ethanol (3.1.2) and swirl the reaction vial several times; Centrifuge for 5 min
46、at about 14 500 g; Carefully remove and discard the supernatant; Dry the extracted DNA; Dissolve the dried DNA extract in 100 l of TE buffer solution (3.1.16). 5.4 DNA purification by means of solid phase extraction Purify DNA extract according to the instructions given by the respective kit manufac
47、turer. The DNA extract can be stored at 4 C for a short period. If storage times exceed more than one week, the DNA extracts should be stored at temperatures of 18 C. PD CEN/TS 15634-3:2016CEN/TS 15634-3:2016 (E) 8 5.5 Measuring the mass concentration of the extracted DNA and setting to target conce
48、ntration The mass concentration of a DNA aliquot can be determined by means of a UV spectrometer at 260 nm. Calculate the DNA mass concentration as follows: (DNA) 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
49、 at 280 nm. The ratio of the values for optical density at wavelengths of 260 nm and 280 nm should be approximately 1,8. The DNA mass concentration may also be estimated using other suitable procedures. Set the DNA extract to a mass concentration of approximately 20 ng/l by diluting with sterile water. 5.6 Real-time PCR NOTE 1 In order to exclude false negative results occurring due to PCR inhibition or highly degraded DNA
