1、BSI Standards PublicationPD CEN ISO/TS 17919:2013Microbiology of the food chain Polymerase chain reaction(PCR) for the detection of food-borne pathogens Detectionof botulinum type A, B, E and Fneurotoxin-producing clostridia(ISO/TS 17919:2013)PD CEN ISO/TS 17919:2013 PUBLISHED DOCUMENTNational forew
2、ordThis Published Document is the UK implementation of CEN ISO/TS17919:2013.The UK participation in its preparation was entrusted to TechnicalCommittee AW/9, Microbiology.A list of organizations represented on this committee can beobtained on request to its secretary.This publication does not purpor
3、t to include all the necessaryprovisions of a contract. Users are responsible for its correctapplication. The British Standards Institution 2013. Published by BSI StandardsLimited 2013ISBN 978 0 580 78549 8ICS 07.100.30Compliance with a British Standard cannot confer immunity fromlegal obligations.T
4、his Published Document was published under the authority of theStandards Policy and Strategy Committee on 30 November 2013.Amendments issued since publicationDate Text affectedTECHNICAL SPECIFICATION SPCIFICATION TECHNIQUE TECHNISCHE SPEZIFIKATION CEN ISO/TS 17919 November 2013 ICS 07.100.30 English
5、 Version Microbiology of the food chain - Polymerase chain reaction (PCR) for the detection of food-borne pathogens - Detection of botulinum type A, B, E and F neurotoxin-producing clostridia (ISO/TS 17919:2013) Microbiologie de la chane alimentaire - Raction de polymrisation en chane (PCR) pour la
6、dtection de micro-organismes pathognes dans les aliments - Dtection des clostridies productrices de neurotoxine botulique de type A, B, E et F (ISO/TS 17919:2013) Mikrobiologie von Lebensmitteln, Futtermitteln und Umgebungsproben - Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorg
7、anismen in Lebensmitteln - Nachweis von Botulinum-Neurotoxin-Typ A, B, E und F produzierenden Clostridien (ISO/TS 17919:2013) This Technical Specification (CEN/TS) was approved by CEN on 16 April 2013 for provisional application. The period of validity of this CEN/TS is limited initially to three ye
8、ars. After two years the members of CEN will be requested to submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard. CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS availab
9、le promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached. CEN members are the national standards bodies of Austria, Be
10、lgium, 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, Slovakia, Slovenia, Spain, Sweden, Swit
11、zerland, 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 2013 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN nation
12、al Members. Ref. No. CEN ISO/TS 17919:2013 EPD CEN ISO/TS 17919:2013CEN ISO/TS 17919:2013 (E) 3 Foreword This document (CEN ISO/TS 17919:2013) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”
13、the secretariat of which is held by DIN. 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. According to the CEN-CENELEC Internal Regulat
14、ions, the national standards organizations of the following countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland,
15、 Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO/TS 17919:2013 has been approved by CEN as CEN ISO/TS 17919:2013 without any modification. PD
16、 CEN ISO/TS 17919:2013ISO/TS 17919:2013(E) ISO 2013 All rights reserved iiiContents PageForeword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Symbols and abbreviated terms . 14.1 Symbols . 14.2 Abbreviated terms . 25 Principle 25.1 General . 25.2 Microbial enrichme
17、nt 25.3 Nucleic acid extraction . 25.4 Amplification by PCR . 25.5 Detection of PCR products 25.6 Confirmation . 26 Reagents 36.1 General . 36.2 Culture media . 36.3 Nucleic acid extraction . 46.4 Reagents for PCR . 57 Apparatus and equipment 57.1 General . 57.2 Equipment for sample preparation prio
18、r to enrichment .57.3 Equipment for microbial enrichment 57.4 Equipment used for nucleic acid extraction 67.5 Equipment used for PCR . 67.6 Equipment used for the detection of the PCR product .68 Sampling 79 Procedure. 79.1 Sample preparation prior to enrichment . 79.2 Microbial enrichment 79.3 Nucl
19、eic acid preparation. 89.4 PCR amplification . 99.5 Confirmation of a positive PCR result . 9Annex A (normative) Flow-diagram of the procedure 10Annex B (informative) Multiplex PCR assays for detection of genes encoding botulinum neurotoxin types A, B, E, and F using agarose gel electrophoresis 11An
20、nex C (informative) Assays for detection of genes encoding botulinum neurotoxin types A, B, E, and F using real-time PCR 29Annex D (informative) Preparation of C. botulinum spores.40Bibliography .46PD CEN ISO/TS 17919:2013ISO/TS 17919:2013(E)ForewordISO (the International Organization for Standardiz
21、ation) is a worldwide federation of national standards bodies (ISO member bodies). The 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
22、 represented on that committee. International organizations, governmental and non-governmental, 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.International Standard
23、s are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.The main task of technical 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 Internat
24、ional Standard requires approval by at least 75 % of the member bodies casting a vote.In other circumstances, particularly when there is an urgent market requirement for such documents, a technical committee may decide to publish other types of document: an ISO Publicly Available Specification (ISO/
25、PAS) represents an agreement between technical experts in an ISO working group and is accepted for publication if it is approved by more than 50 % of the members of the parent committee casting a vote; an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical
26、 committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting a vote.An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further three years, revised to become an International Standard, or withdrawn.
27、If the ISO/PAS or ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an International Standard or be withdrawn.ISO/TS 17919 was prepared by the European Committee for Standardization (CEN) Technical Committee CEN/TC 275 Food analys
28、is Horizontal 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).iv ISO 2013 All rights reservedPD CEN ISO/TS 17919:2013ISO/TS 17919:2013(E)Intr
29、oductionBotulinum neurotoxin-producing clostridia are ubiquitous in the environment. Botulism is a severe neuroparalytic disease resulting from the action of botulinum neurotoxins (BoNTs). Seven different serotypes of BoNTs (type A to G) and a number of subtypes have been identified to date.BoNT typ
30、e A (BoNT/A), type B (BoNT/B), type E (BoNT/E) and type F (BoNT/F) are mainly responsible for botulism in humans and the genes encoding these toxins are the targets of this Technical Specification. BoNT type A, B, E, and F-producing clostridia exist in four physiologically distinct groups (Group I C
31、lostridium botulinum, Group II C. botulinum, C. baratii, C. butyricum).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 patents.ISO take no position concerning the evidence, validity and
32、scope of this patent right.The holder of this patent right has assured ISO that they are willing to negotiate licences under reasonable and non-discriminatory terms and conditions with applicants throughout the world. In this respect, the statement of the holder of this patent right is registered wi
33、th ISO. Information may be obtained from:Applied Biosystems, LLC Scott Miller, Legal Department 5781 Van Allen Way CARLSBAD CA 92008 USATel: +1 760 476 4387 Fax: +1 760 476 6048 e-mail: SAttention is drawn to the possibility that some of the elements of this document may be the subject of patent rig
34、hts other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights.ISO (www.iso.org/patents) maintains an on-line database of patents relevant to its documents. Users are encouraged to consult the databases for the most up-to-date information conce
35、rning patents. ISO 2013 All rights reserved vPD CEN ISO/TS 17919:2013PD CEN ISO/TS 17919:2013Microbiology of the food chain Polymerase chain reaction (PCR) for the detection of food-borne pathogens Detection of botulinum type A, B, E and F neurotoxin-producing clostridia1 ScopeThis Technical Specifi
36、cation specifies a horizontal method for the molecular detection of clostridia carrying botulinum neurotoxin A, B, E, and F genes by a PCR method. This method detects the genes and not the toxins, therefore a positive result does not necessarily mean the presence of these toxins in the sample invest
37、igated. This Technical Specification is applicable to products for human consumption, animal feed, and environmental samples.The PCR assays for detection of genetic sequences encoding specific toxin types are described in Annexes B and C.2 Normative referencesThe following referenced documents are i
38、ndispensable for 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 6887-1, Microbiology of food and animal feeding stuffs Preparation of test samples, in
39、itial suspension and decimal dilutions for microbiological examination Part 1: General rules for the preparation of the initial suspension and decimal dilutionsISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinationsISO 11133, Microb
40、iology of food, animal feed and water Preparation, production, storage and performance testing of culture mediaISO 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 detec
41、tionISO 20838:2006, 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 22174, Microbiology of food and animal feeding stuffs Polymerase chain reaction (PCR) f
42、or the detection of food-borne pathogens General requirements and definitions3 Terms and definitionsFor the purpose of this document, the terms and definitions given in ISO 22174 apply.4 Symbols and abbreviated terms4.1 Symbolsc substance concentrationTECHNICAL SPECIFICATION ISO/TS 17919:2013(E) ISO
43、 2013 All rights reserved 1PD CEN ISO/TS 17919:2013ISO/TS 17919:2013(E) mass concentration volume fractionw mass fraction4.2 Abbreviated termsBoNT botulinum neurotoxin5 Principle5.1 GeneralThe method comprises the following consecutive steps:a) microbial enrichment (see 5.2);b) nucleic acid extracti
44、on (see 5.3);c) amplification (see 5.4);d) detection of PCR products (see 5.5);e) confirmation (see 5.6).NOTE Real-time-PCR combines steps c) to e).5.2 Microbial enrichmentThe number of BoNT-producing clostridia (spores or vegetative cells) to be detected is increased by encouraging their germinatio
45、n and growth in non-selective liquid nutrient medium tryptonepeptoseglucoseyeast extract broth under anaerobic conditions.5.3 Nucleic acid extractionBacterial cells are separated from the nutrient medium, lysed and the nucleic acids are extracted for use in the PCR reaction.5.4 Amplification by PCRT
46、he extracted nucleic acid is transferred to the PCR mix and the amplification is carried out in a thermal cycler.5.5 Detection of PCR productsPCR products are detected by gel electrophoresis or an appropriate alternative.5.6 ConfirmationThe identity of the PCR products shall be confirmed by any appr
47、opriate method, e.g. sequencing, hybridization or restriction analysis.2 ISO 2013 All rights reservedPD CEN ISO/TS 17919:2013ISO/TS 17919:2013(E)6 Reagents6.1 GeneralFor all stages 5.1 b) to e), use only reagents of recognized analytical grade and consumables suitable for molecular biology applicati
48、ons as specified in ISO 20837 and ISO 20838.Reagent requirements specified in ISO 20838:2006, Clause 5, apply.6.2 Culture media6.2.1 GeneralFollow ISO 11133 for the preparation, production and performance testing of culture media.6.2.2 DiluentFollow ISO 6887-1 and the relevant part of ISO 6887913dea
49、ling with the product to be examined.6.2.3 Nonselective enrichment culture medium, tryptonepeptoneglucoseyeast extract broth (TPGY broth) (Reference 7)6.2.3.1 GeneralOther approved non-selective enrichment culture media can be used provided equivalent performance is shown.6.2.3.2 Composition and pHTryptone 50 gPeptone 5 gYeast extract 20 gd-Glucose 4 gSodium thioglycolate, HSCH2COONa 1 gWater to 1 000 mlpH 7,0 0,26.2.3.3 PreparationDissolve the components in the water by boiling.
