1、BS EN15634-1:2009ICS 07.100.30; 67.050NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBRITISH STANDARDFoodstuffs Detectionof food allergens bymolecular biologicalmethodsPart 1: General considerationsThis British Standardwas published under theauthority of the StandardsPolicy an
2、d StrategyCommittee on 31 January2009 BSI 2009ISBN 978 0 580 58241 7Amendments/corrigenda issued since publicationDate CommentsBS EN 15634-1:2009National forewordThis British Standard is the UK implementation of EN 15634-1:2009.The UK participation in its preparation was entrusted to TechnicalCommit
3、tee AW/-/3, Food analysis - Horizontal methods.A list of organizations represented on this committee can be obtained onrequest to its secretary.This publication does not purport to include all the necessary provisionsof a contract. Users are responsible for its correct application.Compliance with a
4、British Standard cannot confer immunityfrom legal obligations.BS EN 15634-1:2009EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN 15634-1January 2009ICS 07.100.30; 67.050English VersionFoodstuffs - Detection of food allergens by molecular biologicalmethods - Part 1: General considerationsProduits al
5、imentaires - Dtection des allergnesalimentaires par des mthodes danalyse de biologiemolculaire - Partie 1: Considrations gnralesLebensmittel - Nachweis von Lebensmittelallergenen mitmolekularbiologischen Verfahren - Teil 1: AllgemeineBetrachtungenThis European Standard was approved by CEN on 1 Decem
6、ber 2008.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards may be obtain
7、ed on application to the CEN Management Centre or to any CEN member.This European Standard exists in three official versions (English, 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
8、 Centre has the same status as theofficial versions.CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland,France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Pol
9、and, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT 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 m
10、eans reservedworldwide for CEN national Members.Ref. No. EN 15634-1:2009: EBS EN 15634-1:2009EN 15634-1:2009 (E) 2 Contents Page Foreword 3Introduction .41 Scope 52 Normative references 53 Terms and definitions .54 General laboratory requirements .75 Procedure .86 Isolation / Extraction of nucleic a
11、cid .87 Amplification of specific target sequences 98 Detection and confirmation of PCR products 129 Interpretation . 1210 Expression of the results . 1311 Test report . 14Bibliography . 15BS EN 15634-1:2009EN 15634-1:2009 (E) 3 Foreword This document (EN 15634-1:2009) has been prepared by Technical
12、 Committee CEN/TC 275 “Food analysis - Horitontal methods”, the secretariat of which is held by DIN. This European Standard shall be given the 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 s
13、hall 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 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 Re
14、gulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Ne
15、therlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN 15634-1:2009EN 15634-1:2009 (E) 4 Introduction This European Standard describes the procedure to qualitatively detect and/or quantitate DNA as markers for potentially allergenic
16、 ingredients or constituents by analysing the nucleic acids extracted from the sample under study. The qualitative detection of DNA targets 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 th
17、e detection limits of the analytical method used and the test portion analysed. The quantitative detection of DNA targets is performed to 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 ana
18、lytical method used and the test portion analysed. Appropriate procedures for extraction of nucleic acids are included in each method. The 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 a
19、mplification technologies and detection methods may be considered. BS EN 15634-1:2009EN 15634-1:2009 (E) 5 1 Scope This European Standard provides the overall framework for detection of sequences corresponding to species containing allergens using the polymerase chain reaction (PCR). It relates to t
20、he requirements for the specific amplification of target nucleic acid sequences (DNA) and for the confirmation of the identity of the amplified nucleic acid sequence. Guidelines, minimum requirements and performance criteria laid down in the European Standard are intended to ensure that comparable a
21、nd reproducible results are obtained in different laboratories. This European Standard has been established for food matrices. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For u
22、ndated references, the latest edition of the referenced document (including any amendments) applies. prEN 15842:2008, Foodstuffs Detection 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
23、prEN 15842:2008 and the following apply. 3.1 Terms relative to extraction and purification of DNA 3.1.1 DNA extraction separation of 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
24、 and c) quality (integrity). 3.1.2 DNA purification method resulting in a higher purity of the extracted DNA NOTE In this context, 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
25、relative to amplification of DNA 3.2.1 species (class/order/family/genus) specific target sequence sequence known to be specific for the species BS EN 15634-1:2009EN 15634-1:2009 (E) 6 3.2.2 identification of nucleic acid sequences identification by comparison with a reference nucleic acid fragment/
26、sequence NOTE Identification is possible by e.g. positive hybridisation with probe, matching restriction digest profiles or matching 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 k
27、nown positive samples representative of the sequence or target under study NOTE The control is intended to demonstrate what the 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 (b
28、lank matrix) reference material or known negative sample not containing the sequence under study NOTE The control is intended 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 p
29、ositive template DNA added in the same amount as analyte DNA to the reaction NOTE This control allows the determination of the presence of soluble PCR inhibitors, particularly necessary in case of negative amplification and of quantitative PCR. 3.3.4 amplification reagent control control containing
30、all the reagents, except extracted test sample template DNA NOTE 1 Instead of the template DNA, a corresponding volume of nucleic 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 ext
31、raction blank control control performing all steps of the extraction procedure, except addition of the test portion, e.g. by substitution 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
32、 distilled or equivalent, free from DNA and nucleases (molecular biology grade). 3.3.6 positive extraction control control sample 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 nucl
33、eic acid, i.e. by using a sample material known to contain the target nucleic acid NOTE Information about controls can be found in EN ISO 24276. BS EN 15634-1:2009EN 15634-1:2009 (E) 7 4 General laboratory requirements 4.1 General A draft European Standard dealing with General considerations and val
34、idation criteria of methods was adopted as prEN 15842:2008. 4.2 Laboratory organisation Compliance with applicable requirements with respect to safety regulations and manufacturers safety recommendation shall be followed. Accidental contamination of DNA can originate from dust or spreading aerosols.
35、 As a consequence, the organisation of the work area in the laboratory is logically based on: the systemic containment of 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 the
36、ir own apparatus is required: a) a work area for extraction of the nucleic acid from the test portion (sample); b) a work 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 s
37、egments. If dust particle producing grinding techniques are used, this has to be carried out in a separate work area. Physical 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 use
38、d as a protection against contamination provided their effectiveness is comparable. Staff shall wear different sets of lab 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 la
39、boratory should use properly maintained equipment suitable for the method employed, e.g. according to the requirements outlined 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
40、 according to manufacturers instructions. Calibration systems shall be available and calibration routinely performed for 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 ana
41、lytically pure reagents suitable for molecular biology, free from DNA and DNAses. Reagents and solutions should be stored 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 o
42、r equivalent, free from DNA and nucleases (molecular biology grade). Solutions are prepared by dissolving the appropriate reagents in water and autoclaved unless specified differently. Ultra-filtration devices can be used, when autoclaving is not possible. BS EN 15634-1:2009EN 15634-1:2009 (E) 8 In
43、order to avoid contamination, all the appliances and the parts of equipment in contact with the samples, as well as 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 cont
44、amination. Sterile techniques could be adopted in the PCR set up area powder-free gloves, sterilised plastic-ware, sterilised glassware, autoclaved reagents, disposable plastic-wares, aerosol-protected pipette tips could be used. 5 Procedure 5.1 Principle Qualitative analysis consists of specific ex
45、traction and detection of target nucleic acid sequences in the test portion. A qualitative result shall clearly demonstrate 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 m
46、ethod used and test portion analysed. Quantitative analysis consists, in addition, of the quantitation of target DNA 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 ac
47、id; amplification and detection of specific target sequences; confirmation of the specificity of the amplified fragment; 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 I
48、solation / Extraction of nucleic acid 6.1 Preparation of sample Further information can be found in prEN 15842:2008 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
49、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 It may be performed by either physical (e.g. measure of absorbance at a specific wavelength), chemical-physical (e.g. use of intercalating or binding agents able to emit fluorescence), enzyma
