1、 DEUTSCHE NORM August 2006DIN EN ISO 20837 ICS 07.100.30 Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens Requirements for sample preparation for qualitative detection (ISO 20837:2006) English version of DIN EN ISO 20837:2006-08
2、 Mikrobiologie von Lebensmitteln und Futtermitteln Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln Anforderungen an die Probenvorbereitung fr den qualitativen Nachweis (ISO 20837:2006) Englische Fassung DIN EN ISO 20837:2006-08 Document comprises 12 pages
3、 No part of this standard may be reproduced without prior permission of DIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany, has the exclusive right of sale for German Standards (DIN-Normen). English price group 10 www.din.de www.beuth.de !,pIr“01.07 9773879DIN
4、EN ISO 20837:2006-08 2 National foreword This standard was prepared by Working Group WG 6 “Microbial contamination” (Secretariat: France) of Tech-nical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: Germany), in collaboration with ISO/TC 34/SC 9 “Microbiology” (Secretariat: Fr
5、ance), in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und land-wirtschaftliche Produkte (Foodstuffs and Agricultural Products Standards Committee), Technica
6、l Committees Mikrobiologische Lebensmitteluntersuchung einschlielich Schnellverfahren and Polymerase-Kettenreaktion zum Nachweis von Mikroorganismen. The DIN Standard corresponding to the International Standard referred to in clause 2 of the EN is as follows: ISO 22174 DIN EN ISO 22174 National Anne
7、x NA (informative) Bibliography DIN EN ISO 22174, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitions EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 20837 April 2006 ICS 07.100.30 Engl
8、ish Version Microbiology of food and animal feeding stuffs - Polymerase chain reaction (PCR) for the detection of food-borne pathogens -Requirements for sample preparation for qualitative detection (ISO 20837:2006) Microbiologie des aliments - Raction de polymrisation en chane (PCR) pour la dtection
9、 des micro-organismes pathognes dans les aliments - Exigences relatives la prparation des chantillons pour la dtection qualitative (ISO 20837:2006) Mikrobiologie von Lebensmitteln und Futtermitteln - Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Anfor
10、derungen an die Probenvorbereitung fr den qualitativen Nachweis (ISO 20837:2006) 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 nat
11、ional 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 a
12、ny 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, Finl
13、and, 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 COMIT EUROPEN DE NORMALISATION EUROPISCHES K
14、OMITEE FR 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 20837:2006: EEN ISO 20837:2006 (E) 2 Foreword This document (EN ISO 20837:2006) has been prepared by
15、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
16、 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 Standar
17、d: 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. Contents Page Fo
18、reword2 Introduction .3 1 Scope 4 2 Normative references 4 3 Principle4 4 General laboratory requirements.5 5 Reagents, apparatus and equipment.5 6 Procedure .5 Annex A (informative) Standards concerning the enrichment of microorganisms (bacteria) 7 Annex B (informative) Method for DNA extraction fr
19、om Gram-negative bacteria 8 Bibliography 10 EN ISO 20837:2006 (E) 3 Introduction The detection of food-borne 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
20、treatment; nucleic acid extraction (optional); amplification of nucleic acids from the pathogen under study; 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
21、specific method for sample preparation is given in Annex B. 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:
22、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 Stand
23、ardization (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 Bi
24、osystems, 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
25、 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
26、 94501 USA 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 20837:2006 (E) 4 WARNING The use of this standard
27、 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 regu
28、latory 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 quantity for PCR. It provides a description of the general principles involved. References to
29、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 could also be applied to feed and agricultural/environmental matrices with some adaptation
30、s, 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 latest edition of the referenced document (including any amendments) applies. ISO 22174
31、: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 sample preparation methods described is to obtain samples or nucleic acids of suitable qua
32、lity 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. Enrichment and sample treatment should allow the detection of low numbers of target micro
33、organisms 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 compatible with PCR amplification. EN ISO 20837:2006 (E) 5 3.2 Enrichment and sample treatmen
34、t 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 acid extraction consist of releasing DNA present in the bacteria and concurrent or sub
35、sequent 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 laboratory. 4 General laboratory requirements Sample treatment should be carried out in
36、 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 treatment for bacteria Food samples should be enriched according to the corresponding In
37、ternational 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 described in International Standards. Some enrichment media recommended in Interna
38、tional 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 suppress the growth of competing background micro-organisms (e.g. by addition of se
39、lective 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 of lack of PCR response, more rigorous methods such as boiling, the use of chelati
40、ng 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 to reduce the high calcium content of dairy products which can be inhibitory. 6.2 N
41、ucleic 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 2
42、0837:2006 (E) 6 b) precipitate the resulting peptides with organic solvents (e.g. a mixture of phenol and chloroform) to leave the DNA in the aqueous phase; c) purify the DNA solution and concentrate further by ethanol precipitation in the presence of monovalent cations; d) collect the precipitated
43、DNA by centrifugation; e) wash the DNA with ethanol and resuspend in buffer e.g. tris(hydroxymethyl)aminomethane/EDTA buffer (Tris-EDTA buffer) or Tris buffer. A DNA co-precipitant such as glycogen, polyethylene glycol (PEG) or transfer RNA (t-RNA) may be used to improve the DNA recovery during the
44、precipitation steps. Only co-precipitants without any nuclease activity and without PCR inhibitors/competitors, and without any sequence homology with potential PCR targets under study may be used. NOTE Using vacuum freeze dryers to dry the DNA pellets obtained after a precipitation step can cause c
45、ross contamination. DNA may be released by thermal cell disruption (e.g. by boiling for 10 min). After boiling, centrifuge the chilled sample and use the supernatant for PCR. Before boiling, to facilitate cell disruption, enzymatic treatment may be applied (e.g. lysozyme, mutanolysin, for use with G
46、ram-positive bacteria) followed by a protease/proteinase incubation. Other methods such as vigorous agitation with beads may be required when the organism has a particularly tough cell wall (e.g. Mycobacterium spp.). Any other method including commercial kits may be used for nucleic acid extraction
47、provided the results are at least comparable. 6.2.1.2 DNA quality and quantity The quality and yield of DNA extracted using a given method on a given matrix should be both repeatable and reproducible in terms of amplification by PCR, provided enough DNA is present in the matrix. In particular, the m
48、ethod used shall allow the recovery of DNA fragments with average size equal to or larger than the PCR products under study. The concentration and purity of the DNA isolated may be estimated by fluorometric methods or by gel electrophoresis. Purified DNA may be quantified by spectrophotometric metho
49、ds. A rapid way to estimate the quality and amount of DNA is agarose gel electrophoresis, followed by ethidium bromide staining and fluorescence under ultraviolet (UV) light (see Reference 1). For some sample preparation methods (e.g. boiling), it is necessary to use the nucleic acid solution directly after the preparation, when the nucleic acids released are not stable. In general, repeated freezing and thawing of nucleic acid solutions should be avoided. Use appropriate plastic ware to store low copy numbers of nucleic ac
