BS EN ISO 20838-2006

BRITISH STANDARD BS EN ISO 20838:2006 Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection of food-borne pathogens — Requirements for amplification and detection for qualitative methods The European Standard EN ISO 20838:2006 has the status of a British Standard ICS 07.100.30 BS EN ISO 20838:2006 This British Standard was published under the authority of the Standards Policy and Strategy Committee on 28 April 2006 © BSI 2006 ISBN 0 580 48239 1 National foreword This British Standard is the official English language version of EN ISO 20838:2006. It is identical with ISO 20838:2006. The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under the section entitled “International Standards Correspondence Index”, or by using the “Search” facility of the BSI Electronic Catalogue or of British Standards Online. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. — aid enquirers to understand the text; — present to the responsible international/European committee any enquiries on the interpretation, or proposals for change, and keep UK interests informed; — monitor related international and European developments and promulgate them in the UK. Summary of pages This document comprises a front cover, an inside front cover, the EN ISO title page, the EN ISO foreword page, the ISO title page, pages ii to vi, pages 1 to 7 and a back cover. The BSI copyright notice displayed in this document indicates when the document was last issued. Amendments issued since publication Amd. No. Date CommentsEUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM EN ISO 20838 April 2006 ICS 07.100.30 English Version Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - Requirements for amplification and detection for qualitative methods (ISO 20838:2006) Microbiologie des aliments - Réaction de polymérisation en chaîne (PCR) pour la détection des micro-organismes pathogènes dans les aliments - Exigences relatives à l amplification et à la détection pour les méthodes qualitatives (ISO 20838:2006) Mikrobiologie von Lebensmitteln und Futtermitteln - Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Anforderungen an Amplifikation und Nachweis bei qualitativen Verfahren (ISO 20838:2006) This European Standard was approved by CEN on 13 April 2006. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the Central Secretariat or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other language made by translation under the responsibility of a CEN member into its own language and notified to the Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, 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 STANDARDIZATION COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels © 2006 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 20838:2006: E Foreword This document (EN ISO 20838:2006) has been prepared by Technical Committee CEN/TC 275 “Food analysis - Horizontal methods“, the secretariat of which is held by DIN, in collaboration with Technical Committee ISO/TC 34 “Agricultural food products“. 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 October 2006, and conflicting national standards shall be withdrawn at the latest by October 2006. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, 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. EN ISO 20838:2006 Reference number ISO 20838:2006(E)INTERNATIONAL STANDARD ISO 20838 First edition 2006-04-15 Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection of food-borne pathogens — Requirements for amplification and detection for qualitative methods Microbiologie des aliments — Réaction de polymérisation en chaîne (PCR) pour la détection des micro-organismes pathogènes dans les aliments — Exigences relatives à l amplification et à la détection pour les méthodes qualitatives EN ISO 20838:2006 ii iii Contents Page Foreword iv Introduction v 1 Scope. 1 2 Normative references. 1 3 Terms and definitions. 1 4 Principle. 1 5 Reagents 2 6 Apparatus and equipment . 3 7 Procedure 4 8 Interpretation. 6 9 Performance 6 10 Test report. 6 Bibliography . 7 EN ISO 20838:2006iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. ISO 20838 was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 275, Food analysis — Horizontal methods, in collaboration with Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). EN ISO 20838:2006v Introduction The amplification and detection of target nucleic acid sequences is performed to determine whether certain nucleic acid sequences are present or not in the test portion. This determination is relative to appropriate controls and within the detection limits of the analytical method used and test portion analysed. This International Standard describes the procedure used to detect food-borne microorganisms, including pathogens, by analysing nucleic acids extracted from foodstuffs, feed and environmental samples, or from cultures or cell suspensions prepared from the foodstuff. Appropriate procedures for sample preparation, culturing of microorganisms and extraction of nucleic acids are described in ISO 20837. The main focus of this International Standard is 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. This International Standard is related to a series of standards and a Technical Specification under the general title Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitions (ISO 22174) Requirements for sample preparation for qualitative detection (ISO 20837) Performance testing for thermal cyclers (ISO/TS 20836) Requirements for amplification and detection for qualitative methods (ISO 20838) The International Organization for Standardization (ISO) draws attention to the fact that it is claimed that compliance with this document may involve the use of one or more patents concerning the PCR technology. ISO takes no position concerning the evidence, validity and scope of these patent rights. ISO has been informed that Applied Biosystems, Roche Molecular Systems, Inc. and F. Hoffman-La Roche Ltd. hold patent rights concerning the PCR technology. The companies have assured ISO that they are willing to negotiate licences under reasonable and non-discriminatory terms and conditions with applicants throughout the world. In this respect, the statements of the holders of these patent rights are registered with ISO. Information may be obtained from: Licensing Department Applied Biosystems 850 Lincoln Centre Drive Foster City, CA 94404 USA and Roche Molecular Systems, Inc. Licensing Department 1145 Atlantic Avenue Alameda, CA 94501 USA EN ISO 20838:2006vi Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights. EN ISO 20838:20061 Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection of food-borne pathogens — Requirements for amplification and detection for qualitative methods WARNING — The use of this standard may involve hazardous materials, operations and equipment. This standard does not purport to address all of the safety problems associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 1 Scope This International Standard provides the overall framework for qualitative methods for the detection of food- borne pathogens using the polymerase chain reaction (PCR). It covers the general requirements for the specific amplification of target nucleic acid sequences and the detection and confirmation of the identity of the amplified nucleic acid sequence. Guidelines, minimum requirements and performance characteristics described in this International Standard are intended to ensure that comparable and reproducible results are obtained in different laboratories. This International Standard has been established for food-borne pathogens in or isolated from food and feed matrices, but can also be applied to other matrices, for example environmental samples, or to the detection of other microorganisms under investigation. 2 Normative 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. ISO 16140, Microbiology of food and animal feeding stuffs — Protocol for the validation of alternative methods ISO 22174:2005, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection of food-borne pathogens — General requirements and definitions 3 Terms and definitions For the purposes of this document, the terms and definitions given in ISO 22174 apply. 4 Principle For the purposes of this International Standard, qualitative analysis consists of screening and/or specific detection of target nucleic acid sequences in the test samples. Specificity can be at genus, species or a lower taxonomic level. EN ISO 20838:20062 A qualitative result shall clearly demonstrate the presence or absence of the target sequence under study, relative to appropriate controls and within the detection limits of the analytical method used and test portion analysed. The analysis generally consists of a) amplification by PCR of specific target sequences, b) detection of the PCR product, c) confirmation of the identity of the PCR product, and/or d) confirmation by a standardized microbiological cultural method (e.g. an International Standard). 5 Reagents In all cases, analytically pure reagents suitable for molecular biological applications shall be used. It is generally advisable to take aliquots of the reaction solutions required for a PCR method and to store them under appropriate conditions, for example at –20 °C. 5.1 DNA polymerase A thermostable polymerase (possibly including reverse transcriptase activity) is used for PCR. This may be a purified, native enzyme, or a purified, genetically engineered recombinant form of the enzyme. It should be used according to the manufacturer’s instructions. Each DNA polymerase may need different experimental conditions, e.g. buffer, temperature. 5.2 Reverse transcriptase This enzyme is used for transcription of RNA in complementary single-stranded DNA (cDNA) able to be amplified by a subsequent PCR. It should be used according to the manufacturer’s instructions. 5.3 Reaction buffer The appropriate buffer should be used according to the enzyme manufacturer’s instructions. Ready to use reagents are commercially available. Materials used for preparation of a PCR buffer shall be stable with respect to storage and cycling conditions. It should be used according to the manufacturer s instructions. 5.4 Deoxyribonucleoside triphosphates (dNTP), for PCR Solutions containing molecular biology grade dATP, dCTP, dGTP, dTTP and/or dUTP, as appropriate, shall be used. They shall be stable during storage and under PCR conditions. They are commercially available. 5.5 Primers The primers should be selected based on a sequence specifically to detect the DNA of the target microorganism. 5.6 Water For the amplification reaction water that is DNase- and RNase-free should be used at all times. Suitable ultra pure water is available commercially. EN ISO 20838:20063 5.7 Magnesium chloride (MgCl 2 ) This is supplied either as a component of the reaction buffer or as a separate solution. 5.8 Chemicals for detection of PCR products The chemicals used for the detection system described in a