1、raising standards worldwideNO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAWBSI Standards PublicationBS EN ISO 22119:2011Microbiology of food andanimal feeding stuffs Real-time polymerase chain reaction(PCR) for the detection of food-borne pathogens Generalrequirements and defin
2、itions(ISO 22119:2011)BS EN ISO 22119:2011 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 22119:2011.The UK participation in its preparation was entrusted to Technical Committee AW/9, Microbiology.A list of organizations represented on this committee can be
3、 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. BSI 2011 ISBN 978 0 580 60377 8 ICS 07.100.30Compliance with a British Standard cannot confer immunity from legal obligati
4、ons.This British Standard was published under the authority of the Standards Policy and Strategy Committee on 31 August 2011.Amendments issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 22119 July 2011 ICS 07.100.30 English Version Microbiol
5、ogy of food and animal feeding stuffs - Real-time polymerase chain reaction (PCR) for the detection of food-borne pathogens - General requirements and definitions (ISO 22119:2011) Microbiologie des aliments - Raction de polymrisation en chane (PCR) en temps rel pour la dtection des micro-organismes
6、pathognes dans les aliments - Exigences gnrales et dfinitions (ISO 22119:2011) Mikrobiologie von Lebensmitteln und Futtermitteln - Real-time-Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Allgemeine Anforderungen und Begriffe (ISO 22119:2011)This Europ
7、ean Standard was approved by CEN on 14 July 2011. 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 conce
8、rning 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 language made by translation under the responsibility of a CEN member int
9、o 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, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Irela
10、nd, 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 KOMITEE FR NORMUNG Management Centre: Avenue Marnix 17
11、, B-1000 Brussels 2011 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 22119:2011: EBS EN ISO 22119:2011EN ISO 22119:2011 (E) 3 Foreword This document (EN ISO 22119:2011) has been prepared by Technical Committee CEN/TC 275 “Foo
12、d 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, either by publication of an identical text or by endorsement, at the latest by Janu
13、ary 2012, and conflicting national standards shall be withdrawn at the latest by January 2012. 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
14、 rights. 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, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland
15、, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. BS EN ISO 22119:2011ISO 22119:2011(E) ISO 2011 All rights reserved iiiContents Foreword iv Introduction.v 1 Scope1 2 Normativ
16、e references1 3 Terms and definitions .1 4 Principle5 4.1 General .5 4.2 Probes for real-time PCR5 5 General laboratory requirements.6 6 Reagents and materials 6 7 Apparatus.7 8 Laboratory sample 8 9 Procedure.8 9.1 Sample preparation .8 9.2 Amplification8 9.3 Controls9 9.4 Analysis of the fluoresce
17、nce data .9 10 Evaluation and documentation 11 Bibliography12 BS EN ISO 22119:2011ISO 22119:2011(E) iv ISO 2011 All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing Inter
18、national 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-governmental, in liaison wit
19、h 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. The main task of technica
20、l 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. Attention is drawn to
21、 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 22119 was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 275, Food analysis H
22、orizontal 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). BS EN ISO 22119:2011ISO 22119:2011(E) ISO 2011 All rights reserved vIntroduction T
23、he polymerase chain reaction (PCR) has been shown to be a fast, sensitive, and specific method for detection of food-borne pathogens. Further developments of the technology allow the detection of specific PCR products generated by the amplification process. The principle relies on the excitation of
24、fluorescent markers during the PCR process. This International Standard is part of a series of documents under the general title Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens: ISO/TS 20836, Microbiology of food and animal fee
25、ding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens Performance testing for thermal cyclers ISO 20837, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens Requirements for sample preparation for qu
26、alitative detection 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 ISO 22118, Microbiology of food and animal feeding stuffs Polymerase chain reac
27、tion (PCR) for the detection and quantification of food-borne pathogens Performance characteristics ISO 22119, Microbiology of food and animal feeding stuffs Real-time polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitions ISO 22174, Microbiolo
28、gy of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitions The following Technical Specification is in preparation: ISO/TS 13136, Microbiology of food and animal feeding stuffs Horizontal method for the detectio
29、n of Shiga toxin-producing Escherichia coli (STEC) belonging to O157, O111, O26, O103 and O145 serogroups Qualitative real-time polymerase chain reaction (PCR)-based method BS EN ISO 22119:2011BS EN ISO 22119:2011INTERNATIONAL STANDARD ISO 22119:2011(E) ISO 2011 All rights reserved 1Microbiology of
30、food and animal feeding stuffs Real-time polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitions 1 Scope This International Standard defines terms for the detection of food-borne pathogens in foodstuffs, and isolates obtained from them, using th
31、e polymerase chain reaction (PCR). This International Standard also specifies requirements for the amplification and detection of nucleic acid sequences (DNA or RNA after reverse transcription) by real-time PCR. The minimum requirements laid down in this International Standard provide the basis for
32、comparable and reproducible results within individual and between different laboratories. This International Standard is also applicable, for example, to the detection of food-borne pathogens in environmental samples and in animal feeding stuffs. NOTE Because of the rapid progress in this field, the
33、 examples given are those most frequently in use at the time of development of this International Standard. 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,
34、the latest edition of the referenced document (including any amendments) applies. 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 ISO 22174:2005, M
35、icrobiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens General requirements and definitions 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 real-time polymerase chain reaction r
36、eal-time PCR enzymatic procedure which combines the in vitro amplification of specific DNA segments by a process of denaturation, annealing of specific primers, and synthesis of DNA with the detection of specific PCR products during the amplification process NOTE 1 Generally, the amplification react
37、ion mixture contains one or more specific DNA probes coupled with one or more fluorescent dyes. Using this technology, the signal is generated after specific hybridization of the probes to the target nucleic acid sequence and excitation with light of a definite wavelength. BS EN ISO 22119:2011ISO 22
38、119:2011(E) 2 ISO 2011 All rights reservedNOTE 2 The use of non-specific DNA-binding fluorescent dyes can be applied if positive results are verified in accordance with ISO 20838. 3.2 PCR product DNA amplified by PCR ISO 22174:2005, 3.4.5 3.3 fluorescence resonance energy transfer FRET food-borne pa
39、thogen detection by PCR distance-dependent energy transfer from a donor molecule to an acceptor molecule resulting in enhanced fluorescence of the acceptor molecule after excitation with electromagnetic radiation of a definite wavelength NOTE Taken from Reference 2. 3.4 reporter food-borne pathogen
40、detection by PCR fluorescent molecule used to detect the hybridization of specific probes by excitation with electromagnetic radiation of an appropriate wavelength 3.5 quencher food-borne pathogen detection by PCR fluorescent molecule serving as an energy acceptor and thus quenching the fluorescence
41、 signal of the reporter (donor) 3.6 dark quencher molecule serving as an acceptor, which does not emit energy in a spectral range detected by the optical detection system of the real-time PCR instrument 3.7 5-3-exonuclease activity ability of an enzyme, e.g. a nucleic acid polymerase, to cleave a hy
42、bridized nucleic acid molecule in the 5-3-direction NOTE The activity of 5-3-exonuclease is double stranded DNA specific. It is dependent on the type of enzyme and can be present, for example, in Taq-, Tth- and Tfl-polymerase. 3.8 fluorescent probe oligonucleotide or oligonucleotide analogon of defi
43、ned sequence coupled with one or more fluorescent molecules NOTE Any system emitting a fluorescence signal after specific hybridization to the target nucleic acid sequence which can be detected by the specific equipment can be used as a fluorescent probe. 3.9 hydrolysis probe fluorescent probe coupl
44、ed with two fluorescent molecules which are sterically separated by the 5-3-exonuclease activity of the enzyme during the amplification process NOTE The principle of a hydrolysis probe is illustrated in Figure 1. BS EN ISO 22119:2011ISO 22119:2011(E) ISO 2011 All rights reserved 3a) Unhybridized pro
45、be in solution b) Cleavage of the hybridized probe c) Cleaved probe resulting in reporter fluorescence after excitation Key 1 DNA substrate 2 fluorescent molecule (reporter) 3 quenching molecule 4 enzyme Figure 1 Principle of a hydrolysis probe 3.10 hybridization probe system of two fluorescent prob
46、es coupled with one fluorescent molecule each, where one molecule serves as donor and the other serves as acceptor NOTE The principle of a hybridization probe is illustrated in Figure 2. a) Unhybridized probes in solution b) Hybridized probes resulting in acceptor fluorescence Key 1 DNA substrate 2
47、acceptor molecule 3 donor molecule Figure 2 Principle of a hybridization probe BS EN ISO 22119:2011ISO 22119:2011(E) 4 ISO 2011 All rights reserved3.11 molecular beacon fluorescent probe consisting of three different parts: a central part complementary to the target nucleic acid sequence, plus a 5-p
48、art and a 3-part which are complementary; the reporter is attached to one arm of the molecule, while the end of the other carries a quencher NOTE The principle of a molecular beacon is illustrated in Figure 3. a) Unhybridized molecular beacon in solution b) Hybridized molecular beacon resulting in r
49、eporter fluorescence Key 1 DNA substrate 2 fluorescent molecule (reporter) 3 quenching molecule Figure 3 Principle of a molecular beacon 3.12 probe for detection of a specific pathogen DNA sequence probe with a sequence complementary to the DNA of a pathogen with a reporter emitting a signal of a definite wavelength which can be detected by the optical detection system 3.13 probe for detection of an internal control nucleic acid sequence probe with a reporter desig
