1、Foodstuffs Detection of food allergens by molecular biological methodsPart 5: Mustard (Sinapis alba) and soya (Glycine max) Qualitative detection of a specific DNA sequence in cooked sausages by real-time PCRPD CEN/TS 15634-5:2016BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05
2、/2013 15:06National forewordThis Published Document is the UK implementation of CEN/TS15634-5:2016.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 be obtained onrequest to
3、 its secretary.This publication does not purport to include all the necessary provisions ofa contract. Users are responsible for its correct application. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 90303 8ICS 07.100.30; 67.120.10Compliance with a Brit
4、ish Standard cannot confer immunity fromlegal obligations.This Published Document was published under the authority of theStandards Policy and Strategy Committee on 31 July 2016. Amendments/corrigenda issued since publicationDate Text affectedPUBLISHED DOCUMENTPD CEN/TS 15634-5:2016TECHNICAL SPECIFI
5、CATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN/TS 15634-5 June 2016 ICS 07.100.30; 67.120.10 English Version Foodstuffs - Detection of food allergens by molecular biological methods - Part 5: Mustard (Sinapis alba) and soya (Glycine max) - Qualitative detection of a specific DNA sequence
6、 in cooked sausages by real-time PCR Lebensmittel - Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren - Teil 5: Senf (Sinapis alba) sowie Soja (Glycine max) - Qualitativer Nachweis einer spezifischen DNA-Sequenz in Brhwrsten mittels Real-time PCR This Technical Specification (C
7、EN/TS) was approved by CEN on 18 April 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 requested to submit their comments, particularly on the question whether the CEN/TS can be converted into a E
8、uropean 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
9、 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland
10、, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey andUnited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Managemen
11、t 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 15634-5:2016 EPD CEN/TS 15634-5:2016CEN/TS 15634-5:2016 (E) 2 Contents Page European foreword . 3 1 Scope 4 2 Principle . 4 3 Reag
12、ents . 4 4 Apparatus and equipment . 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 PCR
13、. 8 5.7 Temperature/Time program 9 6 Validation status and performance criteria 9 6.1 General information on the interpretation of the real-time PCR 9 6.2 Reliability of the method . 10 6.2.1 Setup of the interlaboratory study 10 6.2.2 Results of the interlaboratory study samples 10 6.2.3 Qualitativ
14、e interpretation. 10 7 Test report 12 Bibliography . 13 PD CEN/TS 15634-5:2016CEN/TS 15634-5:2016 (E) 3 European foreword This document (CEN/TS 15634-5:2016) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”, the secretariat of which is held by DIN. EN 15634, F
15、oodstuffs 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; P
16、art 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 (Sinapi
17、s alba) and soya (Glycine max) Qualitative detection of a specific DNA sequence in cooked sausages by real-time PCR Technical Specification. 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 ide
18、ntifying 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, Forme
19、r 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 the United Kingdom. PD CEN/TS 15634-5:2016CEN/TS 15634-5:2016
20、(E) 4 1 Scope This Technical Specification specifies a procedure for the qualitative detection of species specific DNA from white mustard (Sinapis alba) and soya (Glycine max) in cooked sausages using singleplex real-time PCR based on the genes MADS-D (mustard) and lectin (soya) 1. A mustard content
21、 of 10 mg/kg or greater and a soya content of 10 mg/kg or greater can be detected with a probability of 95 %. 2 Principle The DNA of the sample is extracted and is set to a definite concentration after photometric measurement. A 74 base pair (bp) long sequence of the DNA for the MADS-D protein of Si
22、napis alba (NCBI accession no. Y08626) or a 81 bp long sequence from the soya lectin gene is multiplicated from the sample DNA by real-time PCR. The amplicons formed are detected and verified by annealing a sequence-specific probe and generating a fluorescence signal 2. 3 Reagents As a rule, analyti
23、cal 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 and autoclaving, unless indicated differently. 3.1 DNA extraction with CTAB: 3.1.1 Ch
24、loroform. 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 fraction w = 37 %. 3.1.6 Isoamyl alcohol. 3.1.7 Isopropanol. 3.1.8 Proteinase K. 3.1.9 Sodium chloride. 3.1.10 Sod
25、ium 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 available and comparable mixtures can be used. 3.1.13 CTAB extraction buffer solution
26、, containing CTAB (mass concentration = 20 g/l), sodium chloride (substance concentration c = 1,4 mol/l), TRIS (c = 0,1 mol/l), Na2-EDTA (c = 0,02 mol/l). Adjust the pH value with hydrochloric acid to 8,0. PD CEN/TS 15634-5:2016CEN/TS 15634-5:2016 (E) 5 3.1.14 Ethanol solution, = 70 %. 3.1.15 Protei
27、nase K solution, = 20 mg/ml. Store aliquots of the solution at 20 C. 3.1.16 TE buffer solution, containing TRIS (c = 0,01 mol/l) and Na2-EDTA (c = 0,001 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 extractio
28、n: Various systems are commercially available, among them spin filter columns or plates or also using vacuum operated systems. Suitable, commercially available kits can be used. Observe the manufacturers data for this. 3.3 Real-time PCR reagents: 3.3.1 Universal master mix (2 ) for the real-time PCR
29、, containing thermostable DNA polymerase (for hot-start PCR) and PCR buffer solution1)(containing reaction buffers, dNTPs, MgCl2and Hotstart Taq polymerase), double concentrated. 3.3.2 Oligonucleotides 2: Primers and probes for the real-time PCR are shown in Table 1. Table 1 Primers and probes for t
30、he real-time PCR Name DNA sequence of the oligonucleotide Soya lectin gene Lectin-F 5 TCC ACC CCC ATC CAC ATT T 3 Lectin-R 5 ggC ATA gAA ggT gAA gTT gAA ggA 3 Lectin probe 5 FAM AAC Cgg TAg CgT TgC CAg CTT Cg TAMRA-3aMustard (Sinapis alba) MADS D protein 2 MADS D-F 5 TGA AAA CTC TCT TCC CCT CTT AGG
31、3 MADS D-R 5 ACA AAT GCA CAC AAG ACA GAG ATA TAG A 3; MADS D probe 5 FAM TAC ATG ATG CTT ACC TCG C TAMRA 3aaFAM: 6-carboxyfluorescein, TAMRA: 6-carboxytetramethylrhodamine; equivalent reporter and/or quencher dyes may be used if they are shown to give comparable or better results. 3.4 Controls: 3.4.
32、1 Negative PCR control, conducted with DNA-free water instead of the DNA extract from the sample and without PCR inhibitors. 1) Ready-to-use reagents or single components may be used as a PCR master mix. Quantitect Multiplex Mastermix (2), available from QIA- GEN, Hilden; was used within the interla
33、boratory study. PD CEN/TS 15634-5:2016CEN/TS 15634-5:2016 (E) 6 3.4.2 Extraction blank 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. 3.4.3 Positive PCR control, sample
34、containing the target sequence, which shall be treated in the same way as the samples to be examined. 4 Apparatus and equipment General aspects are described in EN ISO 24276 3. In addition to the usual laboratory facilities, the following equipment is required. Due to the high sensitivity of the PCR
35、 analytics and the risk of DNA contamination resulting from it, the use of aerosol protected filter tips is obligatory. 4.1 DNA extraction: 4.1.1 Suitable reaction vials, 1,5 ml and 2 ml, Nucleic acid-free. 4.1.2 50 ml centrifuge tubes, Nucleic acid-free. 4.1.3 Thermostat or water bath, preferably w
36、ith shaker function. 4.1.4 Centrifuge, suitable for centrifuging 50 ml centrifuge tubes at 8 000 g2). 4.1.5 Centrifuge, suitable for centrifuging 1,5 ml and 2 ml reaction vials at 16 000 g. 4.1.6 Apparatus and/or material for grinding the sample, e.g. a blender. 4.1.7 UV spectrometer, suitable for e
37、stimating 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. 5 Procedure 5.1 General General aspects are described in EN ISO 24276 3. 5
38、.2 Sample preparation Ensure that a representative sample is made available to the laboratory for investigation. The sample shall be transported and stored so that damage and/or changes are prevented. Ensure that the test sample is representative of the laboratory sample, e.g. by milling or homogeni
39、zing. 2) g = 9,81 m s-2. PD CEN/TS 15634-5:2016CEN/TS 15634-5:2016 (E) 7 5.3 DNA extraction with CTAB Measures and work steps to be considered for the DNA extraction are described in EN ISO 21571 4. An extraction blank control (3.4.2) shall be performed in parallel. Weigh 2 g of the homogenized test
40、 sample into 50 ml centrifuge tubes; Add 10 ml of CTAB buffer (3.1.13); Add 30 l of Proteinase K (3.1.15) and mix; Incubate and shake for 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 2 ml reaction vials; Add 700 l of supern
41、atant and mix thoroughly for 30 s; Centrifuge for 15 min at approximately 16 000 g; Place 500 l of cold isopropanol (3.1.7) in 1,5 ml reaction vials; Add 500 l of supernatant (aqueous phase) and mix carefully; Incubate for 30 min at room temperature; Centrifuge for 15 min at approximately 16 000 g;
42、Carefully remove and discard the supernatant; Fill the reaction vial with 500 l ethanol solution, = 70 % (3.1.14) and swirl the reaction vial several times; Centrifuge for 5 min at about 16 000 g; Remove supernatant carefully and discard; Dry the extracted DNA at 50 C (alternatively in vacuum); Diss
43、olve the dried DNA extract in 100 l of TE buffer solution (3.1.16). 5.4 DNA purification by means of solid phase extraction Purification shall be performed by means of solid phase extraction. In the interlaboratory study, the QIAQuickPCR Purification Kit3)(Qiagen, Hilden) was used corresponding to t
44、he manufacturers information. Other suitable methods for the DNA purification by means of solid phase extraction can be applied. 3) QIAQuick PCR Purification Kit is an example of a suitable product available commercially. This information is given for the convenience of users of this European Standa
45、rd and does not constitute an endorsement by CEN of this product. PD CEN/TS 15634-5:2016CEN/TS 15634-5:2016 (E) 8 5.5 Measuring the mass concentration of the extracted DNA and setting to target concentration The mass concentration of a DNA aliquot can be determined by means of a UV spectrometer at a
46、 wavelength of 260 nm. Calculate the DNA mass concentration as follows: (DNA) in g/ml = 50 optical density dilution factor of the measured aliquot NOTE Other suitable methods for the DNA mass concentration determination can be applied. In order to check its purity, the sample can in addition be meas
47、ured at 280 nm. The ratio of the values for optical density at 260 nm and 280 nm should be approximately 1,8. The DNA extracts are brought to a mass concentration of approximately 250 ng/l by dilution, e g. with sterile water. If one or more of the DNA samples exhibit a DNA mass concentration below
48、250 ng/l, repeat the extraction for these sample(s). If the majority of the DNA solutions exhibit a mass concentration below 250 ng/l and/or the mass concentration 250 ng/l still cannot be attained even after repeated extraction, use the DNA solutions for the real-time PCR analysis as they are. 5.6
49、Real-time PCR Prior to use, the gently thawed reagents should be carefully mixed and centrifuged briefly. Store the reagents cooled in the ice bath or cooling block while preparing the PCR batch. To prevent false-negative results due to co-extracted PCR inhibitors or highly degraded DNA, the amplifiability of the extracted DNA should be assessed prior to the analysis via real-time PCR. NOTE This can for example be
