1、April 2009DEUTSCHE NORM English price group 11No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 07.100.30; 67.050!$W$“15
2、20127www.din.deDDIN EN 15634-1Foodstuffs Detection of food allergens by molecular biological methods Part 1: General considerationsEnglish version of DIN EN 15634-1:2009-04Lebensmittel Nachweis von Lebensmittelallergenen mit molekularbiologischen Verfahren Teil 1: Allgemeine BetrachtungenEnglische F
3、assung DIN EN 15634-1:2009-04www.beuth.deDocument comprises 17 pagesDIN EN 15634-1:2009-04 2 National foreword This standard has been prepared by Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was
4、 the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Technical Committee NA 057-01-05 AA Lebensmittelallergene. Further parts of this standards series will be developed in the future. EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORME
5、N 15634-1January 2009ICS 07.100.30; 67.050English VersionFoodstuffs - Detection of food allergens by molecular biologicalmethods - Part 1: General considerationsProduits alimentaires - Dtection des allergnesalimentaires par des mthodes danalyse de biologiemolculaire - Partie 1: Considrations gnrales
6、Lebensmittel - Nachweis von Lebensmittelallergenen mitmolekularbiologischen Verfahren - Teil 1: AllgemeineBetrachtungenThis European Standard was approved by CEN on 1 December 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving thi
7、s EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtained on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (En
8、glish, French, German). A version in any other language made by translationunder the responsibility of a CEN member into its own language and notified to the CEN Management Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria,
9、 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 and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONC
10、OMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any form and by any means reservedworldwide for CEN national Members.Ref. No. EN 15634-1:2009: EEN 15634-1:2009 (E) 2 Contents Page Foreword 3Introdu
11、ction .41 Scope 52 Normative references 53 Terms and definitions .54 General laboratory requirements .75 Procedure .86 Isolation / Extraction of nucleic acid .87 Amplification of specific target sequences 98 Detection and confirmation of PCR products 129 Interpretation . 1210 Expression of the resul
12、ts . 1311 Test report . 14Bibliography . 15DIN EN 15634-1:2009-04 EN 15634-1:2009 (E) 3 Foreword This document (EN 15634-1:2009) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horitontal methods”, the secretariat of which is held by DIN. This European Standard shall be given th
13、e status of a national standard, either by publication of an identical text or by endorsement, at the latest by July 2009, and conflicting national standards shall be withdrawn at the latest by July 2009. Attention is drawn to the possibility that some of the elements of this document may be the sub
14、ject 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 implement this European Standard: Austria, Belgium, B
15、ulgaria, 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 and the United Kingdom. DIN EN 15634-1:2009-04 E
16、N 15634-1:2009 (E) 4 Introduction This European Standard describes the procedure to qualitatively detect and/or quantitate DNA as markers for potentially allergenic ingredients or constituents by analysing the nucleic acids extracted from the sample under study. The qualitative detection of DNA targ
17、ets is performed in order to get a yes or no answer to the question whether a certain DNA fragment is detected or not relative to appropriate controls and within the detection limits of the analytical method used and the test portion analysed. The quantitative detection of DNA targets is performed t
18、o express the quantity of DNA targets, relative to the quantity of a specific reference, appropriate calibrants and controls and within the dynamic range of the analytical method used and the test portion analysed. Appropriate procedures for extraction of nucleic acids are included in each method. T
19、he main focus of this European Standard will be on PCR based amplification methods. However, because of the rapid rate of technological change in this area, other amplification technologies and detection methods may be considered. DIN EN 15634-1:2009-04 EN 15634-1:2009 (E) 5 1 Scope This European St
20、andard provides the overall framework for detection of sequences corresponding to species containing allergens using the polymerase chain reaction (PCR). It relates to the requirements for the specific amplification of target nucleic acid sequences (DNA) and for the confirmation of the identity of t
21、he amplified nucleic acid sequence. Guidelines, minimum requirements and performance criteria laid down in the European Standard are intended to ensure that comparable and reproducible results are obtained in different laboratories. This European Standard has been established for food matrices. 2 No
22、rmative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. prEN 15842:2008, Foodstuffs De
23、tection of food allergens General considerations and validation of methods 3 Terms and definitions For the purposes of this document, the terms and definitions given in prEN 15842:2008 and the following apply. 3.1 Terms relative to extraction and purification of DNA 3.1.1 DNA extraction separation o
24、f DNA from the other components in a test sample EN ISO 24276:2006 1 NOTE The factors of major importance for the isolated DNA are: a) purity, b) amount or concentration and c) quality (integrity). 3.1.2 DNA purification method resulting in a higher purity of the extracted DNA NOTE In this context,
25、purity refers to the reduction of observable measurable effects of PCR inhibitors. 3.1.3 PCR quality DNA template of sufficient quality to be amplified by PCR 3.2 Terms relative to amplification of DNA 3.2.1 species (class/order/family/genus) specific target sequence sequence known to be specific fo
26、r the species DIN EN 15634-1:2009-04 EN 15634-1:2009 (E) 6 3.2.2 identification of nucleic acid sequences identification by comparison with a reference nucleic acid fragment/sequence NOTE Identification is possible by e.g. positive hybridisation with probe, matching restriction digest profiles or ma
27、tching nucleic acid sequences. 3.3 Definitions referring to controls 3.3.1 positive DNA target control reference DNA or DNA extracted from a certified reference material or known positive samples representative of the sequence or target under study NOTE The control is intended to demonstrate what th
28、e result of analyses of test sample containing the sequence under study will be. 3.3.2 negative DNA target control reference DNA or DNA extracted from a certified negative (blank matrix) reference material or known negative sample not containing the sequence under study NOTE The control is intended
29、to demonstrate what the result of analyses of test samples not containing the sequence under study will be. 3.3.3 PCR inhibition control control containing known amounts of positive template DNA added in the same amount as analyte DNA to the reaction NOTE This control allows the determination of the
30、 presence of soluble PCR inhibitors, particularly necessary in case of negative amplification and of quantitative PCR. 3.3.4 amplification reagent control control containing all the reagents, except extracted test sample template DNA NOTE 1 Instead of the template DNA, a corresponding volume of nucl
31、eic acid free water is added to the reaction. NOTE 2 The water used should be double distilled or equivalent, free from DNA and nucleases (molecular biology grade). 3.3.5 extraction blank control control performing all steps of the extraction procedure, except addition of the test portion, e.g. by s
32、ubstitution of water for the test portion NOTE 1 It is used to demonstrate the absence of contaminating nucleic acid during extraction. NOTE 2 The water used should be double distilled or equivalent, free from DNA and nucleases (molecular biology grade). 3.3.6 positive extraction control control sam
33、ple meant to demonstrate that the nucleic acid extraction procedure has been performed in a way that will allow for extraction and subsequent amplification of the target nucleic acid, i.e. by using a sample material known to contain the target nucleic acid NOTE Information about controls can be foun
34、d in EN ISO 24276. DIN EN 15634-1:2009-04 EN 15634-1:2009 (E) 7 4 General laboratory requirements 4.1 General A draft European Standard dealing with General considerations and validation criteria of methods was adopted as prEN 15842:2008. 4.2 Laboratory organisation Compliance with applicable requir
35、ements with respect to safety regulations and manufacturers safety recommendation shall be followed. Accidental contamination of DNA can originate from dust or spreading aerosols. As a consequence, the organisation of the work area in the laboratory is logically based on: the systemic containment of
36、 the methodological steps involved in the production of the results, and a forward flow principle for sample handling. A minimum of three separately designated work areas with their own apparatus is required: a) a work area for extraction of the nucleic acid from the test portion (sample); b) a work
37、 area dedicated to the set up of PCR/amplification reactions; and c) a work area dedicated for subsequent processing including analysis and characterisation of the amplified DNA segments. If dust particle producing grinding techniques are used, this has to be carried out in a separate work area. Phy
38、sical separation through the use of different rooms is the most effective and preferable way of ensuring separated work areas, but other physical or biochemical methods may be used as a protection against contamination provided their effectiveness is comparable. Staff shall wear different sets of la
39、b coats at each dedicated work area. They shall also wear disposable gloves. Gloves and lab coats should be changed at appropriated frequencies. 4.3 Apparatus and equipment The laboratory should use properly maintained equipment suitable for the method employed, e.g. according to the requirements ou
40、tlined by EN ISO/IEC 17025 2. In addition to standard laboratory equipment, additional apparatus are described in the specific methods. Apparatus and equipment shall be maintained according to manufacturers instructions. Calibration systems shall be available and calibration routinely performed for
41、equipment which may impact the data produced, according to laboratory quality assurance programs. 4.4 Material and reagents For the analysis, unless otherwise stated, use only analytically pure reagents suitable for molecular biology, free from DNA and DNAses. Reagents and solutions should be stored
42、 at room temperature, unless otherwise specified. PCR reagents should be stored in small aliquots to minimize the risk of contamination. The water used shall be double distilled or equivalent, free from DNA and nucleases (molecular biology grade). Solutions are prepared by dissolving the appropriate
43、 reagents in water and autoclaved unless specified differently. Ultra-filtration devices can be used, when autoclaving is not possible. DIN EN 15634-1:2009-04 EN 15634-1:2009 (E) 8 In order to avoid contamination, all the appliances and the parts of equipment in contact with the samples, as well as
44、work surfaces should be easy to clean and decontaminate. Work surfaces cleansing and decontamination is recommended before use and additionally when/where there is a suspicion for contamination. Sterile techniques could be adopted in the PCR set up area powder-free gloves, sterilised plastic-ware, s
45、terilised glassware, autoclaved reagents, disposable plastic-wares, aerosol-protected pipette tips could be used. 5 Procedure 5.1 Principle Qualitative analysis consists of specific extraction and detection of target nucleic acid sequences in the test portion. A qualitative result shall clearly demo
46、nstrate the presence or absence of the target (class, order, family, genus or species) under study, relative to appropriate controls and within the detection limits of the analytical method used and test portion analysed. Quantitative analysis consists, in addition, of the quantitation of target DNA
47、 sequences in the test portion. 5.2 General The analysis of food allergens by molecular biology methodology usually involves the following stages: isolation (/extraction) of nucleic acid; amplification and detection of specific target sequences; confirmation of the specificity of the amplified fragm
48、ent; and quantitation of the amplified fragments relative to calibrants. NOTE In the case of real-time PCR analysis, amplification, detection and confirmation occur simultaneously. 6 Isolation / Extraction of nucleic acid 6.1 Preparation of sample Further information can be found in prEN 15842:2008
49、and in the respective method standard protocols. It is recommended that from each laboratory sample two test portions should be prepared. 6.2 DNA extraction The basic principle of DNA extraction consists of releasing the DNA present in the matrix and further, concurrently or subsequently, purifying the DNA from polymerase chain reaction inhibitors. The DNA extraction /purification is described in each method, taking into account examples of matrices given in each method. 6.3 DNA quantitation I
