1、BSI Standards PublicationBS EN ISO 18744:2016Microbiology of the food chain Detection and enumerationof Cryptosporidium and Giardiain fresh leafy green vegetablesand berry fruitsBS EN ISO 18744:2016 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO18744:2016.T
2、he 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 purport to include all the necessaryprovisions of a contract. Users are responsible
3、 for its correctapplication. The British Standards Institution 2016.Published by BSI Standards Limited 2016ISBN 978 0 580 81676 5ICS 07.100.30Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy
4、 and Strategy Committee on 30 April 2016.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN 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
5、 Giardia in fresh leafy green vegetables and berry 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 Zhlu
6、ng von Cryptosporidium und Giardia in frischem grnem 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 stat
7、us 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 versions (English, French, Ge
8、rman). 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 Austria, Belgium, Bulgaria, Croat
9、ia, 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, Switzerland, Turkey andUni
10、ted Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION 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
11、 ISO 18744:2016 EBS EN ISO 18744:2016EN ISO 18744:2016 (E) 3 European foreword This document (EN ISO 18744:2016) has been prepared by Technical Committee ISO/TC 34 “Food products” in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the secretariat of which is he
12、ld 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 conflicting national standards shall be withdrawn at the latest by october 2016. Attention is drawn to the possibility
13、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 Regulations, the national standards organizations of the following countries are bound
14、 to implement this European Standard: Austria, 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,
15、 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 without any modification. BS EN ISO 18744:2016ISO 18744:2016(E)Foreword ivIntroduction v1 Scope . 12 Normative referenc
16、es 13 Terms and definitions . 14 Principle 25 Reagents 26 Apparatus . 37 Sampling and transport 47.1 Sampling . 47.2 Transport . 47.3 Receipt of samples . 47.4 Storage . 47.5 Preparation of test sample 48 Procedure. 58.1 Removal of parasites from leafy green vegetables . 58.2 Removal of parasites fr
17、om berry fruits 68.3 Immunomagnetic separation (IMS) 78.4 Sample staining 78.5 Microscopy . 88.5.1 General comments 88.5.2 Examination of sample preparations using epifluorescence microscopy 98.5.3 Examination of sample preparations using DIC microscopy 119 Quality control procedures.129.1 General 1
18、29.2 Inclusion and interpretation of controls 129.3 Equipment cleaning . 1310 Reporting of results 1310.1 Expression of results . 1310.2 Test report 13Annex A (normative) Preparation of reagents 15Annex B (normative) Preparation of positive process control suspensions 17Annex C (informative) Percent
19、age recoveries achieved by the methods .19Bibliography .20 ISO 2016 All rights reserved iiiContents PageBS EN ISO 18744:2016ISO 18744:2016(E)ForewordISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of prepar
20、ing 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-governmental, in li
21、aison 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 the ISO/IEC Di
22、rectives, 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 possibility that
23、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 list of patent decl
24、arations 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 meaning of ISO specific terms and expressions related to conformity assessment, as well as information a
25、bout ISOs adherence to the WTO principles in the Technical Barriers to Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9, Microbiology. iv ISO 2016 All rights reservedBS EN ISO 18744:2016I
26、SO 18744:2016(E)IntroductionCryptosporidium spp. and Giardia duodenalis (syn. G. lamblia, G. 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 sur
27、vive in moist environments for prolonged periods. These transmission stages are hereafter referred 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 gr
28、een vegetables and berry fruits, where performed during processing, may also be ineffective. 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 Cryptospo
29、ridium spp. and Giardia duodenalis for the purpose of detection, and therefore, in order to 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 Inter
30、national Standard are for determining whether Cryptosporidium and/or Giardia (oo)cysts are 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
31、 be used following a demonstration of their equivalence with this International Standard following the protocol described in ISO 16140.1 ISO 2016 All rights reserved vBS EN ISO 18744:2016BS EN ISO 18744:2016Microbiology of the food chain Detection and enumeration of Cryptosporidium and Giardia in fr
32、esh leafy green vegetables and berry fruitsWARNING 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 with its use. It is the responsibility of the user t
33、o 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 and Giardia cysts on or in food products that are
34、 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 cysts of size ranges which include those species (C
35、ryptosporidium) 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 Giardia cysts. The method will detect all species a
36、nd 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 spiked into the samples, as the result of molecular t
37、yping 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 normatively referenced in this document and are indis
38、pensable 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 General requirements and guidance for microbiolog
39、ical 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 anti-Cryptosporidium antibodies and morphological
40、characteristics as described in Clause 8.INTERNATIONAL STANDARD ISO 18744:2016(E) ISO 2016 All rights reserved 1BS EN ISO 18744:2016ISO 18744:2016(E)3.2Giardia cyststransmission stage of Giardia spp.Note 1 to entry: Its detection is based on reaction with specific anti-Giardia antibodies and typical
41、 morphological 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
42、 any process 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
43、been added 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 tes
44、ted sample4 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 labelli
45、ng with specific monoclonal antibodies (mAbs) conjugated to a fluorochrome.5 Reagents5.1 Reagents required for eluting (oo)cysts from leafy green vegetables and berry fruits5.1.1 Glycine buffer, pH 5,5 for leafy green vegetables (A.2.1).5.1.2 Glycine buffer, pH 3,5 for berry fruits (A.2.2).5.2 Reage
46、nts required for concentrating, fixing, staining, detection, and quality control5.2.1 Methanol, analytical grade.5.2.2 Paramagnetic beads, coupled with antibodies specific to the walls of Cryptosporidium oocysts and/or Giardia cysts.5.2.3 Hydrochloric acid (HCl), 0,1 mol/l (A.3.1).2 ISO 2016 All rig
47、hts reservedBS EN ISO 18744:2016ISO 18744:2016(E)5.2.4 Sodium hydroxide (NaOH), 1 mol/l (A.3.2).5.2.5 Fluorescently-labelled monoclonal antibodies (mAbs), against Cryptosporidium oocysts and/or Giardia cysts.5.2.6 Immunofluorescence mounting medium (A.3.3).5.2.7 4,6-diamidino-2-phenylindole dihydroc
48、hloride dihydrate (DAPI), freeze-dried reagent.5.2.8 DAPI stock solution (A.3.4).5.2.9 DAPI working solution (A.3.5).5.2.10 Phosphate buffered saline (PBS), pH 7,3 (A.1.1).5.2.11 Non-fluorescing immersion oil.5.2.12 Stock suspensions of Cryptosporidium oocysts and Giardia cysts (Annex B).5.2.13 Susp
49、ensions of pre-labelled and enumerated non-viable Cryptosporidium oocysts and Giardia cysts.The fluorochrome label shall be a different colour than that which is used for the detection of the target organism.5.2.14 Parasite storage medium (A.3.6; A.3.7).5.2.15 Demineralized and filtered water (ultrapure water type 1).5.2.16 Fingernail varnish (for sealing coverslips onto slides as necessary).6 ApparatusThis method requires common microbiological laboratory equipment (refer to ISO 7218) and, in particular, the following.6.1 Tweezers, for handlin
