1、BSI Standards PublicationPD CEN/TS 15634-4:2016Foodstuffs Detection offood allergens by molecularbiological methodsPart 4: Peanut (Arachis hypogaea) Qualitative detection of a specific DNAsequence in chocolate by real-time PCRPD CEN/TS 15634-4:2016 PUBLISHED DOCUMENTNational forewordThis Published D
2、ocument is the UK implementation of CEN/TS15634-4:2016.The UK participation in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not pur
3、port to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2016. Published by BSI StandardsLimited 2016ISBN 978 0 580 90304 5ICS 07.100.30; 67.190Compliance with a British Standard cannot confer immunity fromlegal ob
4、ligations.This Published Document was published under the authority of theStandards Policy and Strategy Committee on 30 April 2016.Amendments issued since publicationDate Text affectedPD CEN/TS 15634-4:2016TECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 15634-4 March 2
5、016 ICS 07.100.30; 67.190 English Version Foodstuffs - Detection of food allergens by molecular biological methods - Part 4: Peanut (Arachis hypogaea) - Qualitative detection of a specific DNA sequence in chocolate by real-time PCR Produits alimentaires - Dtection dallergnes alimentaires par des mth
6、odes de biologie molculaire - Partie 4 : Arachide (Arachis hypogaea) - Dtection qualitative dune squence dADN spcifique dans du chocolat, par PCR en temps rel Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 4: Erdnuss (Arachis hypogaea) - Qualitativer Na
7、chweis 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 members of CEN will be r
8、equested 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 f
9、orm. 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,
10、 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 COMM
11、ITTEE 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 15634-4:2016 EPD CEN/
12、TS 15634-4:2016CEN/TS 15634-4:2016 (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 . 5 4.1 DNA extraction . 6 4.2 PCR
13、 6 5 Procedure. 6 5.1 General 6 5.2 Sample preparation 6 5.3 DNA extraction with CTAB . 6 5.4 DNA purification by means of solid phase extraction 7 5.5 Measuring the mass concentration and purity of the extracted DNA 7 5.6 Real-time PCR . 8 6 Validation status and performance criteria 9 6.1 General
14、9 6.2 Detection . 10 6.3 Reliability of the method . 10 6.3.1 Setup of the interlaboratory study 10 6.3.2 Results 10 6.3.3 Specificity 11 7 Test report 11 Bibliography . 13 PD CEN/TS 15634-4:2016CEN/TS 15634-4:2016 (E) 3 European foreword This document (CEN/TS 15634-4:2016) has been prepared by Tech
15、nical 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
16、such patent rights. 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
17、 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 Specifi
18、cation; Part 5: Mustard (Sinapis alba) 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
19、 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, Portuga
20、l, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. PD CEN/TS 15634-4:2016CEN/TS 15634-4:2016 (E) 4 1 Scope This Technical Specification describes a procedure for the qualitative detection of peanut (Arachis hypogaea) in chocolate using real-time PCR based on t
21、he gene for the peanut allergen Ara h 2 4, 5. 2 Principle The total DNA is extracted from the sample and the DNA content estimated. A sequence specific to peanut from the gene for Ara h 2 is multiplicated using real-time PCR. The amplicon with a length of 86 base pairs (bp) formed in this way is det
22、ected by annealing a sequence-specific probe and generating a fluorescence signal 4. 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 a
23、ppropriate reagents in water and 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 (Na2EDTA). 3.1.4 Cetyltrimethylammoniumbromide (CTAB). 3.1.5 Hydrochloric acid, mass f
24、raction 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 al
25、cohol (3.1.6). Commercially available 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), Na2EDTA (c = 0,02 mol/l). Adjust the pH value with hydrochl
26、oric acid to pH = 8,0. 3.1.14 Ethanol solution, = 70 %. 3.1.15 Proteinase K solution, = 20 mg/ml. PD CEN/TS 15634-4:2016CEN/TS 15634-4:2016 (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)
27、 and Na2-EDTA (c = 0,000 1 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 for DNA purification by means of solid phase extraction, including spin filter co
28、lumns or plates or also with vacuum operated systems. Commercially available kits can also be used. Observe the manufacturers data for this. 3.3 Real-time PCR reagents 3.3.1 PCR master mix1), containing reaction buffers, dNTPs, MgCl2and Hotstart Taq polymerase. 3.3.2 Oligonucleotides, 10 mol each. 3
29、.3.2.1 Peanut (AR-58 F), gCA gCA gTg ggA ACT CCA Agg AgA CA. 3.3.2.2 Peanut (AR-143 R), gCA TgA gAT gTT gCT CgC Ag. 3.3.2.3 Peanut probe (AR-103 T), FAM CgA gAg ggC gAA CCT gAg gCC TAMRA or BHQ1. 3.3.3 Negative PCR control, conducted with DNA-free water instead of the DNA extract from the sample. 3.
30、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 of the food matrix without target sequence, which passes th
31、rough all steps of the analytical process (blank sample). 3.3.6 Positive PCR control2)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 peanut, which passes through all steps of the analyti
32、cal 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-extracted substances from the sample matrix on the am
33、plification. 4 Apparatus and equipment General aspects are described in EN ISO 24276 3. 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) DNA for the positive PCR control is extracted from phenotypically identified
34、pure peanuts as described in 5.3 and 5.4. DNA mass concentration is determined as described in 5.5. PD CEN/TS 15634-4:2016CEN/TS 15634-4:2016 (E) 6 Plastic and glass materials shall be sterilised and free of DNA before use. In addition, the use of aerosol protected filter tips is obligatory due to t
35、he high sensitivity of the PCR analytics and the resultant risk of 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 Thermos
36、tat or water bath, preferably with shaker function. 4.1.4 Centrifuge, suitable for centrifuging 50 ml centrifuge tubes at 8 000 g3). 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.
37、4.1.7 UV spectrometer or other detection instruments, 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. NOTE La
38、boratories participating in the interlaboratory trial used the following real-time PCR equipment: Rotor Gene 6000, Stratagene Mx 3005P, ABI PRISM7500, ABI PRISM7900HT and Roche LightCycler1,5.4)5 Procedure 5.1 General General aspects are described in EN ISO 24276 3. 5.2 Sample preparation Ensure e.g
39、. by milling or homogenizing, that the test sample is representative of the laboratory sample. 5.3 DNA extraction with CTAB Measures and work steps to be considered for the DNA extraction are described in EN ISO 21571 2. 3)g = 9,81 m s24) Rotor Gene 6000, Stratagene Mx 3005P, ABI PRISM 7500, ABI PRI
40、SM 7900HT and Roche LightCycler 1.5 are examples of suitable products available commercially. This information is given for the convenience 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 t
41、o lead to the same results. PD CEN/TS 15634-4:2016CEN/TS 15634-4:2016 (E) 7 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 co
42、ntrols listed 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 f
43、or 90 min at 65 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 reacti
44、on vial; Add 500 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; Centrif
45、uge for 5 min 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 the DNA extract according to the instructions given by the resp
46、ective kit manufacturer. The DNA extract can be stored cooled (approximately 4 C) for a short period. If storage times exceed more than one week, the DNA extracts should be stored at temperatures of 95 %. PD CEN/TS 15634-4:2016CEN/TS 15634-4:2016 (E) 10 6.2 Detection The target sequence is considere
47、d to be detected, if an increase in the measured fluorescence can be observed that is caused by the amplification, by means of peanut-specific primers and the specific probe. The qualitative results of two test samples shall not be contradictory; otherwise, the analysis shall be repeated. no increas
48、e in fluorescence caused by amplification can be observed in the PCR controls without added peanut DNA (negative DNA control, negative process control); and if the expected Ctvalues are obtained in the positive controls and, if appropriate, in the inhibition control. The manufacturers instructions f
49、or the relevant analysis software shall be observed for the analysis. 6.3 Reliability of the method 6.3.1 Setup of the interlaboratory study The reliability of the method was validated in an interlaboratory study with a total of 13 participants. Every participant received 10 coded samples of dark chocolate that have been fortified with mass fractions w = (0, 2, 5, 10 and 20) mg/kg peanut (every concentration double). The homogeneity of the samples was
