1、BRITISH STANDARDBS EN ISO 20837:2006Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens Requirements for sample preparation for qualitative detectionThe European Standard EN ISO 20837:2006 has the status of a British StandardICS 07
2、.100.30g49g50g3g38g50g51g60g44g49g42g3g58g44g55g43g50g56g55g3g37g54g44g3g51g40g53g48g44g54g54g44g50g49g3g40g59g38g40g51g55g3g36g54g3g51g40g53g48g44g55g55g40g39g3g37g60g3g38g50g51g60g53g44g42g43g55g3g47g36g58BS EN ISO 20837:2006This British Standard was published under the authority of the Standards
3、Policy and Strategy Committee on 28 April 2006 BSI 2006ISBN 0 580 48238 3National forewordThis British Standard is the official English language version of EN ISO 20837:2006. It is identical with ISO 20837:2006.The UK participation in its preparation was entrusted to Technical Committee AW/9, Microb
4、iology, which has the responsibility to: A list of organizations represented on this committee can be obtained on request to its secretary.Cross-referencesThe British Standards which implement international or European publications referred to in this document may be found in the BSI Catalogue under
5、 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 applic
6、ation. 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
7、 related international and European developments and promulgate them in the UK.Summary of pagesThis 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 dis
8、played in this document indicates when the document was last issued.Amendments issued since publicationAmd. No. Date CommentsEUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORMEN ISO 20837April 2006ICS 07.100.30English VersionMicrobiology of food and animal feeding stuffs - Polymerasechain reaction (PCR
9、) for the detection of food-borne pathogens -Requirements for sample preparation for qualitative detection(ISO 20837:2006)Microbiologie des aliments - Raction de polymrisation enchane (PCR) pour la dtection des micro-organismespathognes dans les aliments - Exigences relatives laprparation des chanti
10、llons pour la dtection qualitative(ISO 20837:2006)Mikrobiologie von Lebensmitteln und Futtermitteln -Polymerase-Kettenreaktion (PCR) zum Nachweis vonpathogenen Mikroorganismen in Lebensmitteln -Anforderungen an die Probenvorbereitung bei qualitativemNachweis (ISO 20837:2006)This European Standard wa
11、s 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 EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such natio
12、nalstandards 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 translationunder the responsibility of a CEN member into its own language and notifi
13、ed to the Central Secretariat has the same status as the officialversions.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,
14、 Norway, Poland, Portugal, Romania,Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: rue de Stassart, 36 B-1050 Brussels 2006 CEN All rights of exploitation in any for
15、m and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 20837:2006: EForeword This document (EN ISO 20837:2006) 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 Com
16、mittee 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. Ac
17、cording 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, Lith
18、uania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EN ISO 20837:2006Reference numberISO 20837:2006(E)INTERNATIONAL STANDARD ISO20837First edition2006-04-15Microbiology of food and animal feeding stuffs Polymera
19、se chain reaction (PCR) for the detection of food-borne pathogens Requirements for sample preparation for qualitative detection Microbiologie des aliments Raction de polymrisation en chane (PCR) pour la dtection des micro-organismes pathognes dans les aliments Exigences relatives la prparation des c
20、hantillons pour la dtection qualitative EN ISO 20837:2006ii iiiContents Page Foreword iv Introduction v 1 Scope . 1 2 Normative references . 1 3 Principle. 1 4 General laboratory requirements 2 5 Reagents, apparatus and equipment 2 6 Procedure 2 Annex A (informative) Standards concerning the enrichm
21、ent of microorganisms (bacteria) . 4 Annex B (informative) Method for DNA extraction from Gram-negative bacteria . 5 Bibliography . 7 EN ISO 20837:2006iv Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The
22、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-gover
23、nmental, 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. Th
24、e 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
25、. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 20837 was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/
26、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 20837:2006vIntroduction The detection of food-borne
27、pathogens by PCR is usually performed by means of the following successive (or simultaneous) steps: homogenization of the sample; (cultural) enrichment of the pathogen under study and sample treatment; nucleic acid extraction (optional); amplification of nucleic acids from the pathogen under study;
28、detection of the amplified DNA from the pathogen under study. References to International Standards concerning enrichment of bacteria from food matrices are given in Annex A. An example of a specific method for sample preparation is given in Annex B. This International Standard is related to a serie
29、s 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 detectio
30、n (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 involv
31、e 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 te
32、chnology. 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 ob
33、tained 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 20837:2006vi Attention is drawn to the possibility that some of the elements of this docume
34、nt 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 20837:20061Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens Requir
35、ements for sample preparation for qualitative detection 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 e
36、stablish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. 1 Scope This International Standard provides criteria and examples for sample preparation in order to obtain PCR-compatible samples or nucleic acids of suitable quality and quanti
37、ty for PCR. It provides a description of the general principles involved. References to standards concerning the enrichment of microorganisms are given in Annex A, and a detailed method for DNA extraction is given in Annex B. This International Standard has been established for food matrices, but co
38、uld also be applied to feed and agricultural/environmental matrices with some adaptations, if necessary. 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
39、 latest edition of the referenced document (including any amendments) applies. 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 Principle 3.1 General The objective of the sa
40、mple preparation methods described is to obtain samples or nucleic acids of suitable quality and quantity for PCR. NOTE The quality of nucleic acids depends for example on the chemical purity, the average length of the molecules and the structural integrity of the extracted nucleic acid molecules. E
41、nrichment and sample treatment should allow the detection of low numbers of target microorganisms and the reduction of PCR inhibitory substances. Physical, chemical or biochemical procedures, least destructive to the nucleic acid integrity, should render the sample or the nucleic acid solution compa
42、tible with PCR amplification. EN ISO 20837:20062 3.2 Enrichment and sample treatment Sample treatment may start directly from the sample or after enrichment as described in the standards listed in Annex A or other appropriate standards. 3.3 Nucleic acid extraction The basic principles of nucleic aci
43、d extraction consist of releasing DNA present in the bacteria and concurrent or subsequent removal of PCR inhibitors. An example of a DNA extraction/purification method is provided in Annex B. This method is only an example and should be further modified by the users to meet the purpose of each labo
44、ratory. 4 General laboratory requirements Sample treatment should be carried out in separate working areas/rooms as specified in ISO 22174:2005, 6.3. 5 Reagents, apparatus and equipment See Annexes A and B of this International Standard and also ISO 22174. 6 Procedure 6.1 Enrichment and sample treat
45、ment for bacteria Food samples should be enriched according to the corresponding International Standards or other appropriate standards. Other enrichment media found to be more PCR compatible could be used, if they have been shown, through validation, to have performance at least comparable to those
46、 described in International Standards. Some enrichment media recommended in International Standards contain less PCR-inhibitory substances than others, which should be carefully considered in connection with the choice of sample preparation method. For some products, special care should be taken to
47、suppress the growth of competing background micro-organisms (e.g. by addition of selective chemicals or antibiotics). Least destructive methods, such as a simple dilution, centrifugation, protein digestion, filtration, density centrifugation, immunomagnetic separation, etc. may be tried. In the case
48、 of lack of PCR response, more rigorous methods such as boiling, the use of chelating agents or harsh chemicals, such as chloroform and ethanol, or kits with similar actions may be tried. Simple physical methods may be used to reduce the fat content of high-fat samples. Chelating agents may be used
49、to reduce the high calcium content of dairy products which can be inhibitory. 6.2 Nucleic acid extraction 6.2.1 DNA extraction 6.2.1.1 DNA release and purification Several DNA extraction principles may be combined. For example, the following steps may be carried out: a) degrade the proteins in the cell extract with proteases (e.g. Proteinase K) and RNA with ribonucleases; EN ISO 20837:20063b) precipitate the resulting peptides with organic solvents (e.g. a mixture of phenol and chloroform) to leave the DNA in the aqueo
