1、July 2017 English price group 22No part of this translation may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 07.100.30!%gu“2688261www.din.deDIN
2、EN ISO 15216-1Microbiology of the food chain Horizontal method for determination of hepatitis A virus and norovirus using realtime RTPCR Part 1: Method for quantification (ISO 152161:2017);English version EN ISO 152161:2017,English translation of DIN EN ISO 15216-1:2017-07Mikrobiologie der Lebensmit
3、telkette Horizontales Verfahren zur Bestimmung von HepatitisAVirus und Norovirus mittels RealtimeRTPCR Teil 1: Verfahren zur Quantifizierung (ISO 152161:2017);Englische Fassung EN ISO 152161:2017,Englische bersetzung von DIN EN ISO 15216-1:2017-07Microbiologie dans la chane alimentaire Mthode horizo
4、ntale pour la recherche des virus de lhpatite A et norovirus par la technique RTPCR en temps rel Partie 1: Mthode de quantification (ISO 152161:2017);Version anglaise EN ISO 152161:2017,Traduction anglaise de DIN EN ISO 15216-1:2017-07SupersedesDIN CEN ISO/TS 152161(DIN SPEC 100511):201308www.beuth.
5、deDocument comprises 58 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.06.17 DIN EN ISO 15216-1:2017-07 2 A comma is used as the decimal marker. National foreword This document (EN ISO 15216-1:2017) has been prepared by Techni
6、cal Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany), in collaboration with Technical Committee ISO/TC 34 “Food products”. The responsible German body involved in its preparation was DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards C
7、ommittee Food and Agricultural Products), Working Committee NA 057-01-06 AA “Microbiology of the food chain”. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 5725-2 DIN ISO 5725-2 ISO 7218 DIN EN ISO 7218 ISO 17468 DIN EN ISO 17468 ISO
8、20838 DIN EN ISO 20838 ISO 22119 DIN EN ISO 22119 ISO 22174 DIN EN ISO 22174 Amendments: This standard differs from DIN CEN ISO/TS 15216-1 (DIN SPEC 10051-1):2013-08 as follows: a) the use of linear dsDNA molecules for quantification has been prescribed; b) the use of a suitable buffer for dilution
9、of control materials has been prescribed; c) the method for generating process control virus RNA for the standard curve has been changed; d) breakpoints with defined temperature and time parameters in the extraction methods have been added; e) the terminology has been changed from amplification effi
10、ciency to RT-PCR inhibition; f) extra real-time RT-PCR reactions for negative controls have been added; g) precision data and results of an interlaboratory study have been added. Previous editions DIN CEN ISO/TS 15216-1 (DIN SPEC 10051-1): 2013-08 DIN EN ISO 15216-1:2017-07 3 National Annex NA (info
11、rmative) Bibliography DIN EN ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations DIN EN ISO 17468, Microbiology of the food chain Technical requirements and guidance on establishment or revision of a standardized reference metho
12、d DIN EN ISO 20838, 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 DIN EN ISO 22119, Microbiology of food and animal feeding stuffs Real-time polymerase chai
13、n reaction(PCR) for thedetection of food-borne pathogens General requirements and definitions 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 DIN ISO 5725-2, Accuracy (true
14、ness and precision) of measurement methods and results Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method DIN EN ISO 15216-1:2017-07 4 This page is intentionally blank EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 15216-1 March
15、 2017 ICS 07.100.30 Supersedes CEN ISO/TS 15216-1:2013English Version Microbiology of the food chain - Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR - Part 1: Method for quantification(ISO 15216-1:2017) Microbiologie dans la chaine alimentaire - Mthode
16、 horizontale pour la recherche des virus de lhpatite A et norovirus par la technique RT-PCR en temps rel - Partie 1: Mthode de quantification(ISO 15216-1:2017) Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zur Bestimmung von Hepatitis-A-Virus und Norovirus mittels Real-time-RT-PCR - T
17、eil 1: Verfahren zur Quantifizierung (ISO 15216-1:2017) This European Standard was approved by CEN on 23 February 2017. 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
18、 any alteration. Up-to-date lists and bibliographical references concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official versions (English, French, German). A version in any other lan
19、guage made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denma
20、rk, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN CO
21、MMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2017 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 15216-1:2017 EEurop
22、ean foreword 4Introduction 61 Scope . 72 Normative references 73 Terms and definitions . 74 Principle 94.1 Virus extraction 94.2 RNA extraction . 104.3 Real-time RT-PCR . 104.4 Control materials . 104.4.1 Process control virus . 104.4.2 Double-stranded DNA (dsDNA) control . 104.4.3 EC RNA control .
23、104.5 Test results. 115 Reagents 115.1 General 115.2 Reagents used as supplied . 115.3 Prepared reagents . 126 Equipment and consumables . 137 Sampling . 158 Procedure 158.1 General laboratory requirements 158.2 Virus extraction . 158.2.1 Process control virus material 158.2.2 Negative process contr
24、ol 158.2.3 Food surfaces . 158.2.4 Soft fruit, leaf, stem and bulb vegetables 158.2.5 Bottled water .168.2.6 Bivalve molluscan shellfish 178.3 RNA extraction . 178.4 Real-time RT-PCR . 188.4.1 General requirements .188.4.2 Real-time RT-PCR analysis 189 Interpretation of results 209.1 General 209.2 C
25、onstruction of standard curves 209.3 Calculation of RT-PCR inhibition 219.4 Calculation of extraction efficiency 219.5 Sample quantification 2210 Expression of results .2311 Precision 2311.1 Interlaboratory study . 2311.2 Repeatability 2311.3 Reproducibility limit . 2412 Test report 24Contents PageD
26、IN EN ISO 15216-1:2017-07 EN ISO 15216-1:2017 (E) 2Foreword 5Annex D (informative) Real-time RT-PCR primers and hydrolysis probes for the detection of HAV, norovirus GI and GII and mengo virus (process control) 30Annex E (informative) Growth of mengo virus strain MC0for use as a process control 33An
27、nex F (informative) RNA extraction using the NucliSENSsystem .34Annex G (informative) Generation of dsDNA control stocks .36Annex H (informative) Generation of EC RNA stocks 39Annex I (informative) Typical optical plate layout41Annex J (informative) Method validation studies and performance characte
28、ristics 43Bibliography .54DIN EN ISO 15216-1:2017-07 EN ISO 15216-1:2017 (E)3Annex A (normative) Diagram of procedure .25Annex B (normative) Composition and preparation of reagents and buffers 26Annex C (informative) Real-time RT-PCR mastermixes and cycling parameters 29European foreword This docume
29、nt (EN ISO 15216-1:2017) 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 “Food products“. This European Standard shall be given the status of a national standard, ei
30、ther by publication of an identical text or by endorsement, at the latest by September 2017 and conflicting national standards shall be withdrawn at the latest by September 2017. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN
31、 and/or CENELEC shall not be held responsible for identifying any or all such patent rights. This document supersedes CEN ISO/TS 15216-1:2013. This document has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN-CENELEC
32、 Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Icela
33、nd, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 15216-1:2017 has been approved by CEN as EN ISO 15216-1:2017 without any
34、 modification. DIN EN ISO 15216-1:2017-07 EN ISO 15216-1:2017 (E) 4 ForewordISO (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
35、 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
36、 the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed f
37、or the different types of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).Attention is drawn to the possibility that some of the elements of this document may be the subject of patent ri
38、ghts. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO list of patent declarations received (see www .iso .org/ patents).Any trade name used in
39、 this document is information given for the convenience of users and does not constitute an endorsement. For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the Wor
40、ld Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www .iso .org/ iso/ foreword .html.This document was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 275, in collaboration with ISO Technical Committee ISO/T
41、C 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the agreement on technical cooperation between ISO and CEN (Vienna Agreement).This first edition cancels and replaces ISO/TS 15216-1:2013, which has been technically revised with the following changes: use of linear dsDNA molec
42、ules for quantification prescribed; use of a suitable buffer for dilution of control materials prescribed; change to the method for generating process control virus RNA for the standard curve; addition of breakpoints with defined temperature and time parameters in the extraction methods; change in t
43、erminology from amplification efficiency to RT-PCR inhibition; addition of extra real-time RT-PCR reactions for negative controls; addition of precision data and results of interlaboratory study.A list of all parts in the ISO 15216 series can be found on the ISO website.DIN EN ISO 15216-1:2017-07 EN
44、 ISO 15216-1:2017 (E)5 IntroductionHepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness. No routine methods exist for culture of norovirus, and HAV culture methods are not appropriate for routine application to food matrices. Detection is therefore reliant on
45、molecular methods using the reverse-transcriptase polymerase chain reaction (RT-PCR). As many food matrices contain substances that are inhibitory to RT-PCR, it is necessary to use an extraction method that produces highly clean RNA preparations that are fit for purpose. For food surfaces, viruses a
46、re removed by swabbing. For soft fruit, leaf, stem and bulb vegetables, virus extraction is by elution with agitation followed by precipitation with PEG/NaCl. For bottled water, adsorption and elution using positively charged membranes followed by concentration by ultrafiltration is used and for biv
47、alve molluscan shellfish (BMS), viruses are extracted from the tissues of the digestive glands using treatment with a proteinase K solution. For all matrices that are not covered by this document, it is necessary to validate this method. All matrices share a common RNA extraction method based on vir
48、us capsid disruption with chaotropic reagents followed by adsorption of RNA to silica particles. Real-time RT-PCR monitors amplification throughout the real-time RT-PCR cycle by measuring the excitation of fluorescently labelled molecules. In real-time RT-PCR with hydrolysis probes, the fluorescent label is attached to a sequence-specific nucleotide probe that also enables simultaneous confirmation of target template. These modifications increase the sensitivity and specificity of the real-time RT-PCR method, and obviate the need for additional amplification product confirma
