DIN EN ISO 22119-2011 Microbiology of food and animal feeding stuffs - Real-time polymerase chain reaction (PCR) for the detection of food-borne pathogens - General requirements an.pdf

1、November 2011 Translation by DIN-Sprachendienst.English price group 11No 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).I

2、CS 07.100.30!$x6j“1851971www.din.deDDIN EN ISO 22119Microbiology of food and animal feeding stuffs Real-time polymerase chain reaction (PCR) for the detection offood-borne pathogens General requirements and definitions (ISO 22119:2011)English translation of DIN EN ISO 22119:2011-11Mikrobiologie von

3、Lebensmitteln und Futtermitteln Real-time-Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenenMikroorganismen in Lebensmitteln Allgemeine Anforderungen und Begriffe (ISO 22119:2011)Englische bersetzung von DIN EN ISO 22119:2011-11Microbiologie des aliments Raction de polymrisation en chane (P

4、CR) en temps rel pour la dtection desmicro-organismes pathognes dans les aliments Exigences gnrales et dfinitions (ISO 22119:2011)Traduction anglaise de DIN EN ISO 22119:2011-11www.beuth.deIn case of doubt, the German-language original shall be considered authoritative.Document comprises 18 pages11.

5、11 DIN EN ISO 22119:2011-11 2 A comma is used as the decimal marker. National foreword This standard has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France/ABNT, Brazil), Subcommittee SC 9 “Microbiology” (Secretariat: AFNOR, France), in collaboration with Tech

6、nical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany), Working Group WG 6 “Microbiology of the food chain”. Based on the results of the parallel voting procedure carried out in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna Agre

7、ement), ISO 22119:2011 has been adopted as a European Standard. The responsible German body involved in its preparation was the Normenausschuss Lebensmittel und landwirtschaftliche Produkte (Food and Agricultural Products Standards Committee), Working Committee NA 057-01-06 AA Mikrobiologische Leben

8、smitteluntersuchung einschlielich Schnellverfahren, NA 57-01-06-01 AK Polymerase Kettenreaktion zum Nachweis von Mikroorganismen. The DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO/TS 20836 DIN ISO/TS 20836 ISO 20837 DIN EN ISO 20837 ISO 2

9、0838 DIN EN ISO 20838 ISO 22118 DIN EN ISO 22118 ISO 22174 DIN EN ISO 22174 National Annex NA (informative) Bibliography DIN ISO/TS 20836, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens Performance testing of thermal cyclers D

10、IN EN 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 qualitative detection DIN EN ISO 20838, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for

11、 the detection of food-borne pathogens Requirements for amplification and detection for qualitative methods DIN EN ISO 22118, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detection of food-borne pathogens Performance characteristics DIN EN ISO 22174, Microbi

12、ology 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 22119 July 2011 ICS 07.100.30 English Version Microbiology of food and animal feeding stuffs

13、 - 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 pathognes dans les aliments - Exigenc

14、es 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 European Standard was approved by CEN on 1

15、4 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 concerning such national standards may be

16、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 into its own language and notified to th

17、e 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, Ireland, Italy, Latvia, Lithuania, Luxembo

18、urg, 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, B-1000 Brussels 2011 CEN All rights

19、 of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 22119:2011: EEN ISO 22119:2011 (E) 2 Contents PageForeword3Introduction .41 Scope 52 Normative references 53 Terms and definitions .54 Principle94.1 General94.2 Probes for real-time PCR 95 Gene

20、ral laboratory requirements.106 Reagents and materials 107 Apparatus .118 Laboratory sample.129 Procedure .129.1 Sample preparation .129.2 Amplification 129.3 Controls 139.4 Analysis of the fluorescence data1310 Evaluation and documentation 15Bibliography 16DIN EN ISO 22119:2011-11 EN ISO 22119:2011

21、 (E) 3 Foreword This document (EN ISO 22119:2011) 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

22、a national standard, either by publication of an identical text or by endorsement, at the latest by January 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 o

23、f patent rights. CEN and/or CENELEC shall not be held responsible for identifying any or all such patent 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, Bulgari

24、a, Croatia, 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 the United Kingdom. DIN EN ISO 22119:2011

25、-11 EN ISO 22119:2011 (E) 4 Introduction The 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.

26、The principle relies on the excitation of 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

27、20836, Microbiology of food and animal feeding 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

28、Requirements for sample preparation for qualitative 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 a

29、nimal feeding stuffs Polymerase chain reaction (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 requirem

30、ents and definitions ISO 22174, Microbiology 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

31、 stuffs Horizontal method for the detection 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 DIN EN ISO 22119:2011-11 EN ISO 22119:2011 (E) 5 1 Scope This International Standard

32、defines terms for the detection of food-borne pathogens in foodstuffs, and isolates obtained from them, using the polymerase chain reaction (PCR). This International Standard also specifies requirements for the amplification and detection of nucleic acid sequences (DNA or RNA after reverse transcrip

33、tion) by real-time PCR. The minimum requirements laid down in this International Standard provide the basis for comparable and reproducible results within individual and between different laboratories. This International Standard is also applicable, for example, to the detection of food-borne pathog

34、ens in environmental samples and in animal feeding stuffs. NOTE Because of the rapid progress in this field, the 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

35、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 20838, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) for the detectio

36、n of food-borne pathogens Requirements for amplification and detection for qualitative methods ISO 22174:2005, Microbiology 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

37、 purposes of this document, the following terms and definitions apply. 3.1 real-time polymerase chain reaction real-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 t

38、he detection of specific PCR products during the amplification process NOTE 1 Generally, the amplification reaction 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

39、 the target nucleic acid sequence and excitation with light of a definite wavelength. DIN EN ISO 22119:2011-11 EN ISO 22119:2011 (E) 6 NOTE 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 amplifi

40、ed by PCR ISO 22174:2005, 3.4.5 3.3 fluorescence resonance energy transfer FRET food-borne pathogen 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 radi

41、ation of a definite wavelength NOTE Taken from Reference 2. 3.4 reporter food-borne pathogen 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

42、by PCR fluorescent molecule serving as an energy acceptor and thus quenching the fluorescence 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

43、5-3-exonuclease activity ability of an enzyme, e.g. a nucleic acid polymerase, to cleave a hybridized 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-

44、and Tfl-polymerase. 3.8 fluorescent probe oligonucleotide or oligonucleotide analogon of defined 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 specif

45、ic equipment can be used as a fluorescent probe. 3.9 hydrolysis probe fluorescent probe coupled 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 Figu

46、re 1. DIN EN ISO 22119:2011-11 EN ISO 22119:2011 (E) 7 a) Unhybridized probe 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

47、a hydrolysis probe 3.10 hybridization probe system of two fluorescent probes 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) H

48、ybridized probes resulting in acceptor fluorescence Key 1 DNA substrate 2 acceptor molecule 3 donor molecule Figure 2 Principle of a hybridization probe DIN EN ISO 22119:2011-11 EN ISO 22119:2011 (E) 8 3.11 molecular beacon fluorescent probe consisting of three different parts: a central part comple

49、mentary to the target nucleic acid sequence, plus a 5-part 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 reporter fluorescence Key 1 DNA substrate 2 fluorescent molecule (reporter) 3 quenching molecule Figure 3 Princi