1、BS EN ISO 15216-1:2017Microbiology of the food chain Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCRPart 1: Method for quantification (ISO 15216-1:2017)BSI Standards PublicationWB11885_BSI_StandardCovs_2013_AW.indd 1 15/05/2013 15:06BS EN ISO 15216-1:201
2、7 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 15216-1:2017. It supersedes PD CEN ISO/TS 15216-1:2013 which is withdrawn.The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology.A list of organizations represented on
3、 this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. The British Standards Institution 2017.Published by BSI Standards Limited 2017ISBN 978 0 580 87672 1
4、 ICS 07.100.30 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 April 2017.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN S
5、TANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 15216-1 March 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 15
6、216-1:2017) Microbiologie dans la chaine alimentaire - Mthode 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 vo
7、n Hepatitis A-Virus und Norovirus mittels Real-time-RT-PCR - Teil 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 th
8、is 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 CEN-CENELEC Management Centre or to any CEN member. This European Standard exists in three official
9、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 CEN-CENELEC Management Centre has the same status as the official versions. CEN members are the national standards bodies of Aust
10、ria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, 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, Spai
11、n, Sweden, Switzerland, Turkey and United Kingdom. EUROPEAN COMMITTEE 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
12、 for CEN national Members. Ref. No. EN ISO 15216-1:2017 EBS EN ISO 15216-1:2017EN ISO 15216-1:2017 (E) 3 European foreword This document (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 collabo
13、ration with Technical Committee ISO/TC 34 “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 September 2017 and conflicting national standards shall be withdrawn at the latest by Sep
14、tember 2017. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN 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
15、prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croa
16、tia, Cyprus, Czech Republic, Denmark, 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, Turk
17、ey 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 modification. BS EN ISO 15216-1:2017ISO 15216-1:2017(E)Foreword vIntroduction vi1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 34.1 Virus
18、extraction 34.2 RNA extraction 44.3 Real-time RT-PCR 44.4 Control materials 44.4.1 Process control virus 44.4.2 Double-stranded DNA (dsDNA) control 44.4.3 EC RNA control 44.5 Test results 55 Reagents 55.1 General . 55.2 Reagents used as supplied 55.3 Prepared reagents 66 Equipment and consumables 77
19、 Sampling 98 Procedure. 98.1 General laboratory requirements . 98.2 Virus extraction 98.2.1 Process control virus material . 98.2.2 Negative process control . 98.2.3 Food surfaces 98.2.4 Soft fruit, leaf, stem and bulb vegetables 98.2.5 Bottled water .108.2.6 Bivalve molluscan shellfish 118.3 RNA ex
20、traction . 118.4 Real-time RT-PCR . 128.4.1 General requirements .128.4.2 Real-time RT-PCR analysis 129 Interpretation of results 149.1 General 149.2 Construction of standard curves 149.3 Calculation of RT-PCR inhibition 159.4 Calculation of extraction efficiency 159.5 Sample quantification 1610 Exp
21、ression of results .1711 Precision 1711.1 Interlaboratory study . 1711.2 Repeatability 1711.3 Reproducibility limit . 1812 Test report 18Annex A (normative) Diagram of procedure .19Annex B (normative) Composition and preparation of reagents and buffers 20Annex C (informative) Real-time RT-PCR master
22、mixes and cycling parameters 23 ISO 2017 All rights reserved iiiContents PageBS EN ISO 15216-1:2017ISO 15216-1:2017(E)Annex D (informative) Real-time RT-PCR primers and hydrolysis probes for the detection of HAV, norovirus GI and GII and mengo virus (process control) 24Annex E (informative) Growth o
23、f mengo virus strain MC0for use as a process control 27Annex F (informative) RNA extraction using the NucliSENSsystem .28Annex G (informative) Generation of dsDNA control stocks .30Annex H (informative) Generation of EC RNA stocks 33Annex I (informative) Typical optical plate layout35Annex J (inform
24、ative) Method validation studies and performance characteristics 37Bibliography .48iv ISO 2017 All rights reservedBS EN ISO 15216-1:2017ISO 15216-1:2017(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The
25、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
26、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. The procedures used to develop this document and those intended for its further maintenance are described in
27、 the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for 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
28、 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. Details of any patent rights identified during the development of the document will be in the Introduction and/or on the ISO l
29、ist of patent declarations received (see www .iso .org/ patents).Any trade name used in 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 r
30、elated to conformity assessment, as well as information about ISOs adherence to the World 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 (C
31、EN) Technical Committee CEN/TC 275, in collaboration with ISO 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).This first edition cancels and replaces ISO/TS 15216-1:2013, w
32、hich has been technically revised with the following changes: use of linear dsDNA molecules 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 breakpoi
33、nts with defined temperature and time parameters in the extraction methods; change in terminology 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 part
34、s in the ISO 15216 series can be found on the ISO website. ISO 2017 All rights reserved vBS EN ISO 15216-1:2017ISO 15216-1:2017(E)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 cultu
35、re methods are not appropriate for routine application to food matrices. Detection is therefore reliant on 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 extractio
36、n method that produces highly clean RNA preparations that are fit for purpose. For food surfaces, viruses are 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
37、elution using positively charged membranes followed by concentration by ultrafiltration is used and for bivalve 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 docume
38、nt, it is necessary to validate this method. All matrices share a common RNA extraction method based on virus 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 e
39、xcitation 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 rea
40、l-time RT-PCR method, and obviate the need for additional amplification product confirmation steps post real-time RT-PCR. Due to the complexity of the method, it is necessary to include a comprehensive suite of controls. The method described in this document enables quantification of levels of virus
41、 RNA in the test sample. A schematic diagram of the testing procedure is shown in Annex A.The main changes, listed in the Foreword, introduced in this document compared to ISO/TS 15216-1:2013 are considered as minor (see ISO 17468).vi ISO 2017 All rights reservedBS EN ISO 15216-1:2017Microbiology of
42、 the food chain Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR Part 1: Method for quantification1 ScopeThis document specifies a method for the quantification of levels of HAV and norovirus genogroup I (GI) and II (GII) RNA, from test samples of foodstu
43、ffs (soft fruit, leaf, stem and bulb vegetables, bottled water, BMS) or food surfaces. Following liberation of viruses from the test sample, viral RNA is then extracted by lysis with guanidine thiocyanate and adsorption on silica. Target sequences within the viral RNA are amplified and detected by r
44、eal-time RT-PCR.This method is not validated for detection of the target viruses in other foodstuffs (including multi-component foodstuffs), or any other matrices, nor for the detection of other viruses in foodstuffs, food surfaces or other matrices.2 Normative referencesThe following documents are
45、referred to in the text in such a way that some or all of their content constitutes requirements 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 7218, Microbiology of
46、food and animal feeding stuffs General requirements and guidance for microbiological examinationsISO 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 methodsISO
47、 22119, Microbiology of food and animal feeding stuffs Real-time polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitionsISO 22174, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pat
48、hogens General requirements and definitions3 Terms and definitionsFor the purposes of this document, the terms and definitions given in ISO 22174, ISO 22119 and ISO 20838 and the following apply.ISO and IEC maintain terminological databases for use in standardization at the following addresses: IEC
49、Electropedia: available at h t t p :/ www .electropedia .org/ ISO Online browsing platform: available at h t t p :/ www .iso .org/ obp3.1foodstuffsubstance used or prepared for use as foodNote 1 to entry: For the purposes of this document, this definition includes bottled water.INTERNATIONAL STANDARD ISO 15216-1:2017(E) ISO 2017 All rights reserved 1BS EN ISO 15216-1:2017ISO 15216-1:2017(E)3.2food surfacesurface of food, food preparation surface or food contact surface3.3soft fruitsmall edible stoneless fruitEXAMPLE Strawberrie
