1、 DEUTSCHE NORM August 2006DIN EN ISO 20838 ICS 07.100.30 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) English version of DIN EN ISO 20838:
2、2006-08 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)Englische Fassung DIN EN ISO 20838:2006-08 Document comprises
3、12 pages 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 !,pIs“01.07 977
4、3880DIN EN ISO 20838:2006-08 2 National foreword This standard was prepared by Working Group WG 6 “Microbial contamination” (Secretariat: France) of Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: Germany), in collaboration with ISO/TC 34/SC 9 “Microbiology” (Secretar
5、iat: France), 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), T
6、echnical Committees Mikrobiologische Lebensmitteluntersuchung einschlielich Schnellverfahren and Polymerase-Kettenreaktion zum Nachweis von Mikroorganismen. The DIN Standards corresponding to the International Standards referred to in clause 2 of the EN are as fol-lows: ISO 16140 DIN EN ISO 16140 IS
7、O 22174 DIN EN ISO 22174 National Annex NA (informative) Bibliography DIN EN ISO 16140, Microbiology of food and animal feeding stuffs Protocol for the validation of alternative methods DIN EN ISO 22174, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection
8、 of food-borne pathogens General requirements and definitions EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE 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 -Requiremen
9、ts for amplification and detection for qualitative methods (ISO 20838:2006) Microbiologie des aliments - Raction de polymrisation en chane (PCR) pour la dtection des micro-organismes pathognes dans les aliments - Exigences relatives lamplification et la dtection pour les mthodes qualitatives (ISO 20
10、838:2006) 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
11、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 obtaine
12、d 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 Secreta
13、riat 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, Portu
14、gal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG Management Centre: rue de Stassart, 36 B-1050 Brussels 2006 CEN All rights of exploitation in any form and by any mean
15、s reserved worldwide for CEN national Members. Ref. No. EN ISO 20838:2006: EEN ISO 20838:2006 (E) 2 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 Techni
16、cal 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 2
17、006. 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, Latvi
18、a, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom. Contents Page Foreword2 Introduction .3 1 Scope 4 2 Normative references 4 3 Terms and definitions .4 4 Principle4 5 Reagents.5 6 Apparatus and equipmen
19、t 6 7 Procedure .7 8 Interpretation9 9 Performance .9 10 Test report 9 Bibliography 10 EN ISO 20838:2006 (E) 3 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 d
20、etermination 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 foods
21、tuffs, 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-base
22、d 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 Microbiolo
23、gy 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) Requirement
24、s 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 posi
25、tion 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 licen
26、ces 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 Fos
27、ter City, CA 94404 USA and Roche Molecular Systems, Inc. Licensing Department 1145 Atlantic Avenue Alameda, CA 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 respo
28、nsible for identifying any or all such patent rights. EN ISO 20838:2006 (E) 4 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
29、 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 cha
30、in 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 Internat
31、ional 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 environm
32、ental 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 re
33、ferenced 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
34、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
35、 sequences in the test samples. Specificity can be at genus, species or a lower taxonomic level. EN ISO 20838:2006 (E) 5 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
36、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.
37、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
38、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 manufacturers instructions. Each
39、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 manufacturers instructi
40、ons. 5.3 Reaction buffer The appropriate buffer should be used according to the enzyme manufacturers 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 accord
41、ing to the manufacturers 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.
42、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 20838:2006
43、(E) 6 5.7 Magnesium chloride (MgCl2) 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 PCR method should be of appropriate quality. 5.9 Optional additional re
44、agents 5.9.1 Mineral oil This is dispensed onto the reaction mix to minimize evaporation during thermal cycling. 5.9.2 Facilitators These are substances, such as polyethylene glycol or bovine serum albumin, which can be added to the PCR reaction to reduce inhibition by matrix-derived substances1. 5.
45、9.3 RNase inhibitors These are added to an RT-PCR to prevent degradation of target RNA by RNase enzymes, which may have contaminated the reagents or plastic ware used in the extraction procedure. RNase inhibitors are commercially available. 5.9.4 Reagents to prevent carry over of PCR products As a f
46、urther guard against contamination, a decontamination system (based on psoralene, dUTP and UNG, for example) may be included in the PCR system to minimize the risk of carryover contamination from PCR products produced during previous PCR reactions. 6 Apparatus and equipment This shall be in accordan
47、ce with ISO 22174. In addition to standard laboratory equipment, the following apparatus and equipment shall be used. 6.1 Thermal cycler apparatus, capable of reproducibly and accurately performing the temperature and time cycles described in a PCR method. 6.2 Pipettes, with filter tips. At least th
48、ree sets of pipettes are required, one of each dedicated to sample preparation, master mix preparation, post-amplification steps. NOTE The use of filter tips is not necessary in the post-amplification steps. 6.3 Reaction vessels, suitable for use in thermal cyclers, and which can be repeatedly heate
49、d to 100 C and cooled to 4 C without damage. EN ISO 20838:2006 (E) 7 6.4 System for detection of PCR products, comprising a) apparatus for agarose or polyacrylamide gel electrophoresis and, if necessary, a UV radiation source for recording visualization of amplified DNA, or b) apparatus for nucleic acid column chromatography and the appropriate detection system, or c) solid phases loaded with a specific probe and apparatus for detecting PCR products, or d) other equally suitable systems. 7 Procedure 7.1 PCR amplificat
