1、December 2016 English price group 14No 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!%h9“2596922www.din.de
2、DIN EN ISO 18744Microbiology of the food chain Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berry fruits (ISO 18744:2016);English version EN ISO 18744:2016,English translation of DIN EN ISO 18744:2016-12Mikrobiologie der Lebensmittelkette Nachweis und
3、Zhlung von Cryptosporidium und Giardia in frischem grnem Blattgemse und Beeren (ISO 18744:2016);Englische Fassung EN ISO 18744:2016,Englische bersetzung von DIN EN ISO 18744:2016-12Microbiologie de la chane alimentaire Recherche et dnombrement des Cryptosporidium et Giardia dans les lgumes verts fra
4、is feuilles et les fruits baies (ISO 18744:2016);Version anglaise EN ISO 18744:2016,Traduction anglaise de DIN EN ISO 18744:2016-12www.beuth.deDocument comprises 28 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.12.16 DIN EN I
5、SO 18744:2016-12 2 A comma is used as the decimal marker. National foreword This document (EN ISO 18744:2016) has been prepared by Technical Committee ISO/TC 34 “Food products” (Secretariat: AFNOR, France), Subcommittee SC 9 “Microbiology” (Secretariat: AFNOR, France) in collaboration with Technical
6、 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 parallel voting, Technical Committee CEN/TC 275 decided to adopt ISO 18744:2016 as a European Standard. The responsible German body invol
7、ved in its preparation was DIN-Normenausschuss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-01-06 AA Mikrobiologie der Lebensmittelkette. The DIN Standards corresponding to the International Standards referred to in
8、this document are as follows: ISO 7218 DIN EN ISO 7218 ISO 16140 DIN EN ISO 16140 National Annex NA (informative) Bibliography DIN EN ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations DIN EN ISO 16140, Microbiology of food and
9、 animal feeding stuffs Protocol for the validation of alternative EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 18744 April 2016 ICS 07.100.30 English Version Microbiology of the food chain - Detection and enumeration of Cryptosporidium and Giardia in fresh leafy green vegetables and berr
10、y fruits (ISO 18744:2016) Microbiologie de la chane alimentaire - Recherche et dnombrement des Cryptosporidium et Giardia dans les lgumes verts frais feuilles et les fruits baies (ISO 18744:2016) Mikrobiologie der Lebensmittelkette - Nachweis und Zhlung von Cryptosporidium und Giardia in frischem gr
11、nem Blattgemse und Beeren (ISO 18744:2016) This European Standard was approved by CEN on 11 March 2016. 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.
12、 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 language made by tr
13、anslation 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, Denmark, Estonia, Fin
14、land, 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, Switzerland, Turkey andUnited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATI
15、ON COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2016 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN ISO 18744:2016 EEuropean foreword .3Introduction
16、 51 Scope . 62 Normative references 63 Terms and definitions . 64 Principle 75 Reagents 76 Apparatus . 87 Sampling and transport 97.1 Sampling . 97.2 Transport . 97.3 Receipt of samples . 97.4 Storage . 97.5 Preparation of test sample 98 Procedure 108.1 Removal of parasites from leafy green vegetabl
17、es 108.2 Removal of parasites from berry fruits . 118.3 Immunomagnetic separation (IMS) . 128.4 Sample staining . 128.5 Microscopy 138.5.1 General comments . 138.5.2 Examination of sample preparations using epifluorescence microscopy . 148.5.3 Examination of sample preparations using DIC microscopy
18、169 Quality control procedures.179.1 General 179.2 Inclusion and interpretation of controls 179.3 Equipment cleaning . 1810 Reporting of results 1810.1 Expression of results . 1810.2 Test report 18Annex A (normative) Preparation of reagents 20Annex B (normative) Preparation of positive process contr
19、ol suspensions 22Annex C (informative) Percentage recoveries achieved by the methods .24Bibliography .25Contents PageDIN EN ISO 18744:2016-12 EN ISO 18744:2016 (E)Foreword .42 European foreword This document (EN ISO 18744:2016) has been prepared by Technical Committee ISO/TC 34 “Food products” in co
20、llaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is held by DIN. 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 october 2016, and conf
21、licting national standards shall be withdrawn at the latest by october 2016. 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
22、 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, Former Yugoslav Republic of Macedonia, France, Germany, Gre
23、ece, Hungary, Iceland, Ireland, 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 18744:2016 has been approved by CEN as EN ISO 18744:2016 with
24、out any modification. DIN EN ISO 18744:2016-12 EN ISO 18744:2016 (E)3 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 technic
25、al 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 wi
26、th 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
27、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 possibility that some of the elements of this document may be the subject of patent righ
28、ts. 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 this
29、 document is information given for the convenience of users and does not constitute an endorsement.For an explanation on the meaning of ISO specific terms and expressions related to conformity assessment, as well as information about ISOs adherence to the WTO principles in the Technical Barriers to
30、Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. DIN EN ISO 18744:2016-12 EN ISO 18744:2016 (E)4 IntroductionCryptosporidium spp. and Giardia duodenalis (syn. G. lamblia, G
31、. intestinalis) are protozoan parasites that can cause enteric illness in humans. Both organisms are characterized by a robust transmission stage, the Cryptosporidium oocyst and the Giardia cyst, which can survive in moist environments for prolonged periods. These transmission stages are hereafter r
32、eferred to collectively as (oo)cysts. Cryptosporidium oocysts in particular are highly resistant to chlorine at the concentrations used in the treatment of drinking water, and chemical disinfection of leafy green vegetables and berry fruits, where performed during processing, may also be ineffective
33、. Consequently, the absence of vegetative bacteria on fresh produce as indicators of faecal contamination does not necessarily indicate the absence of (oo)cysts. No practical method exists to culture Cryptosporidium spp. and Giardia duodenalis for the purpose of detection, and therefore, in order to
34、 detect contamination with these parasites, direct removal of the (oo)cysts from the food sample must be performed, followed by visualization of the (oo)cysts by microscopy. The methods described in this International Standard are for determining whether Cryptosporidium and/or Giardia (oo)cysts are
35、present on the surfaces of fresh produce and for their enumeration. This International Standard is based on published methods that have been tested in a multicentre collaborative trial. Alternative methods can be used following a demonstration of their equivalence with this International Standard fo
36、llowing the protocol described in ISO 16140.1DIN EN ISO 18744:2016-12 EN ISO 18744:2016 (E)5 WARNING Persons using this International Standard should be familiar with normal laboratory practice. This International Standard does not purport to address all of the safety problems, if any, associated wi
37、th its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions.1 ScopeThis International Standard specifies a method that is applicable for the detection and enumeration of Cryptosporidium oocysts
38、 and Giardia cysts on or in food products that are described herein as fresh leafy green vegetables and berry fruits. With suitable controls, it may also be applicable for the examination of other fresh produce.The microscopy descriptions are for Cryptosporidium spp. oocysts and Giardia duodenalis c
39、ysts of size ranges which include those species (Cryptosporidium) or assemblages (Giardia) known to be pathogenic to humans.This method does not include any molecular analysis and therefore is not suitable for the determination of the species or genotypes/assemblages of Cryptosporidium oocysts and G
40、iardia cysts. The method will detect all species and genotypes/assemblages that are known to be pathogenic for humans and also others that are not. For further identification, molecular typing assays are required. However, these cannot be reliably performed if process positive controls have been spi
41、ked into the samples, as the result of molecular typing assays will be obfuscated.This method does not allow the determination of viability or infectivity of any Cryptosporidium oocysts and Giardia cysts which may be present.2 Normative referencesThe following documents, in whole or in part, are nor
42、matively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.ISO 7218:2007, Microbiology of food and animal feeding stuffs
43、 General requirements and guidance for microbiological examinations3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1Cryptosporidium oocysttransmission stage of Cryptosporidium spp.Note 1 to entry: Its detection is based on reaction with specific
44、anti-Cryptosporidium antibodies and morphological characteristics as described in Clause 8.DIN EN ISO 18744:2016-12 EN ISO 18744:2016 (E)6 3.2Giardia cyststransmission stage of Giardia spp.Note 1 to entry: Its detection is based on reaction with specific anti-Giardia antibodies and typical morpholog
45、ical characteristics as described in Clause 8.3.3fresh leafy green vegetableplant leaves eaten as a vegetable, which have not been subjected to any process, except perhaps cutting and washing3.4fresh berry fruitsmall, round or oblong, fleshy and juicy fruit, which has not been subjected to any proce
46、ss except perhaps cutting and washing3.5internal extraction control(oo)cysts labelled with specific fluorogenic reporters that may be added in defined numbers to the sample prior to processing to assure that the method is operating properly3.6positive controlsample to which (oo)cysts have been added
47、 in defined numbers prior to extraction to verify that the method after the elution step is operating efficiently3.7negative controlsample having an equivalent quantity of material to the tested sample, which is considered to be free of (oo)cysts and processed in the same manner as the tested sample
48、4 PrincipleThe principle of the method is based on removal of the (oo)cysts from the sample by elution procedures, followed by concentration in the eluate by centrifugation and isolation by immunomagnetic separation (IMS). Detection of the (oo)cysts is performed by microscopy after labelling with specific monoclonal antibodies (mAbs) conjugated to a fluorochrome.5 Reagents
