1、December 2015 English price group 12No 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!%Jm“2399674www.din.de
2、DIN EN ISO 18743Microbiology of the food chain Detection of Trichinella larvae in meat by artificial digestion method (ISO 18743:2015);English version EN ISO 18743:2015,English translation of DIN EN ISO 18743:2015-12Mikrobiologie der Lebensmittelkette Nachweis von Trichinella-Larven in Fleisch mit k
3、nstlichem Verdauungsverfahren (ISO 18743:2015);Englische Fassung EN ISO 18743:2015,Englische bersetzung von DIN EN ISO 18743:2015-12Microbiologie de la chane alimentaire Recherche des larves de Trichinella dans la viande par une mthode de digestion artificielle (ISO 18743:2015);Version anglaise EN I
4、SO 18743:2015,Traduction anglaise de DIN EN ISO 18743:2015-12www.beuth.deDocument comprises 22 pagesDTranslation by DIN-Sprachendienst.In case of doubt, the German-language original shall be considered authoritative.12.15DIN EN ISO 18743:2015-12 2 A comma is used as the decimal marker. National fore
5、word This document (EN ISO 18743:2015) has been prepared by Technical Committee ISO/TC 34 “Food products” in collaboration with Technical Committee CEN/TC 275 “Food analysis Horizontal methods” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was DIN-Normenausschu
6、ss Lebensmittel und landwirtschaftliche Produkte (DIN Standards Committee Food and Agricultural Products), Working Committee NA 057-01-06 AA Mikrobiologie der Lebensmittelkette. The DIN Standard corresponding to the International Standard referred to in this document is as follows: ISO 7218 DIN EN I
7、SO 7218 National Annex NA (informative) Bibliography DIN EN ISO 7218, Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinations EUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 18743 September 2015 ICS 07.100.30 English Version Microbio
8、logy of the food chain - Detection of Trichinella larvae in meat by artificial digestion method (ISO 18743:2015) Microbiologie de la chane alimentaire - Recherche des larves de Trichinella dans la viande par une mthode de digestion artificielle (ISO 18743:2015) Mikrobiologie der Lebensmittelkette -
9、Nachweis von Trichinella-Larven in Fleisch mit knstlichem Verdauungsverfahren (ISO 18743:2015) This European Standard was approved by CEN on 24 July 2015. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this European Standard the st
10、atus 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,
11、German). 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, Cro
12、atia, 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 andU
13、nited Kingdom. EUROPEAN COMMITTEE FOR STANDARDIZATION COMIT EUROPEN DE NORMALISATION EUROPISCHES KOMITEE FR NORMUNG CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels 2015 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No.
14、EN ISO 18743:2015 EContents Page European foreword . 3 Foreword .4Introduction 51 Scope . 62 Normative references 63 Terms and definitions . 64 Principle 74.1 General . 74.2 Sample size . 74.3 Blending/grinding of the muscle sample . 74.4 Preparation of the digest fluid . 74.5 Digestion of the chopp
15、ed meat . 74.6 Filtration of the digest fluid . 84.7 Sedimentation of the digest fluid 84.8 Microscopic examination . 84.9 Verification of findings . 85 Reagents 86 Apparatus . 97 Sampling, labelling, and transport . 108 Sample preparation 109 Procedure 109.1 General 109.2 Blending/grinding 109.3 Pr
16、eparation of the digest fluid 119.4 Digestion of the chopped meat in the glass beaker 119.5 Filtration of the digest fluid 119.6 Sedimentation of the digest fluid in the separatory funnel . 129.7 Collection of the primary and secondary sediment . 129.8 Microscopic examination 1210 Documentation 1311
17、 Expression of the results . 1312 Safety measures 13Annex A (normative) Sample collection . 14Annex B (normative) Frozen samples .16Annex C (informative) Artificial digestion magnetic stirrer method 17Annex D (informative) Example of a laboratory worksheet for recording data when testing pooled samp
18、les by digestion assay .19Bibliography .202 EN ISO 18743:2015 (E) DIN EN ISO 18743:2015-12European foreword This document (EN ISO 18743:2015) has been prepared by Technical Committee ISO/TC 34 “Food products“ in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” t
19、he 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 March 2016, and conflicting national standards shall be withdrawn at the latest by March 2016. Attention is
20、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 Regulations, the national standards organizations of the foll
21、owing 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, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands,
22、Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom. Endorsement notice The text of ISO 18743:2015 has been approved by CEN as EN ISO 18743:2015 without any modification. 3EN ISO 18743:2015 (E)DIN EN ISO 18743:2015-12ForewordISO (the Inter
23、national 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 technical committees. Each member body interested in a subject for which a technical committee has
24、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 with the International Electrotechnical Commission (IEC) on all matters of electrotechnical st
25、andardization.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 for the different types of ISO documents should be noted. This document was drafted in accor
26、dance 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 rights. ISO shall not be held responsible for identifying any or all such patent rights. Details
27、 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 document is information given for the convenience of users and does not constitute an endor
28、sement.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 Trade (TBT) see the following URL: Foreword - Supplementary informationThe committee respons
29、ible for this document is ISO/TC 34, Food products, Subcommittee SC 9, Microbiology.4 EN ISO 18743:2015 (E) DIN EN ISO 18743:2015-12IntroductionTrichinella spp. are the causative agents of human trichinellosis, a disease which is a public health hazard and, as a result, also represents an economic p
30、roblem in porcine animal production. Due to the zoonotic importance of this infection in many countries, the main efforts have focused on control and/or eradication of Trichinella from domestic pigs, the most important source of human infection worldwide. Digestion methods for detection of Trichinel
31、la larvae in muscle samples from pigs and other susceptible animal species intended for human consumption (e.g. horses, wild boars, walruses, and bears), are effective for preventing clinical trichinellosis in humans. Due to the limits of sensitivity of digestion methods, these methods might not det
32、ect infected animals with very small numbers of larvae in muscle samples, that can pose a risk for subclinical infections in humans.5EN ISO 18743:2015 (E)DIN EN ISO 18743:2015-12WARNING Persons using this International Standard should be familiar with normal laboratory practice. This International S
33、tandard does not purport to address all of the safety problems, if any, associated with 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 m
34、ethod of detection of Trichinella spp. muscle stage larvae in meat of individual animal carcasses intended for human consumption. It is applicable to the examination of meat from domestic and sylvatic animal species, which can be infected by nematodes of the genus Trichinella.This method does not al
35、low the identification of the species or genotype of detected parasites; species or genotype identification can be carried out by molecular methods.The method described in this International Standard is intended to be used in conjunction with the guidelines in the OIE Manual of Diagnostic Tests and
36、Vaccines and by the International Commission on Trichinellosis (ICT) for Trichinella testing and the inspection of carcasses intended for human consumption, unless it has been demonstrated by other means that the animal was not at risk for exposure to Trichinella.The artificial digestion/magnetic st
37、irrer method is considered to be the standard method because it has proven to give the most reliable results in validation studies.NOTE Provided equivalence with the method described within this International Standard can be documented, alternative methods can be used for analysis.2 Normative refere
38、ncesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable 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 amendme
39、nts) applies.ISO 7218:2007,Microbiology of food and animal feeding stuffs General requirements and guidance for microbiological examinationsInternational Commission on Trichinellosis (ICT), “Quality Assurance in Digestion Testing Programs for Trichinella”, Recommendations and Guidelines. 2012World O
40、rganisation for Animal Health (OIE), Chapter 2.1.16 “Trichinellosis”, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals. 7th ed. 20123 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1Trichinella muscle larvaeMLfirst larval stage (L1)
41、 of nematodes belonging to the genus Trichinella, which is located in striated muscles of animals worldwide and can infect humansNote 1 to entry: These larvae are approximately 0,7 mm to 1,1 mm in length and 0,03 mm in width.6 EN ISO 18743:2015 (E) DIN EN ISO 18743:2015-123.2digestion assaymethod to
42、 detect Trichinella larvae in muscle tissue by an enzymatic digestion step to release larvae from muscle tissues followed by filtration and sedimentation steps, and detection of recovered larvae by microscopy3.3larvae per gramlpgnumber of Trichinella larvae present in one gram of muscle tissue4 Prin
43、ciple4.1 GeneralThe artificial digestion method, which is accepted as the international reference method (see OIE, Terrestrial Manual 2012, Chapter 2.1.16 Trichinellosis, page 307), relies on enzymatic degradation of muscle fibres in a fluid composed of pepsin and hydrochloric acid followed by a ser
44、ies of sedimentation and washing steps; equivalent validated methods can also be used. Test performance is greatly influenced by the sample size (1 g samples will enable the reliable detection of infections of 3 lpg, and 3 g to 5 g samples will enable reliable detection of 1 lpg), by the type of the
45、 selected muscle, by the method used, and by the skills of the technician performing the test. A sensitivity of at least one to three larvae per gram shall be achieved to protect human health; consequently, an appropriate size of muscle sample should be collected from a predilection muscle of the ta
46、rget animal. There are no internal quality controls that can be used while performing the method. Some key elements in the principle of its use are as follows (see 4.2 to 4.9).4.2 Sample sizeFor inspection of individual food animal carcasses for public health purposes, the sample shall be collected
47、from predilection sites (see Annex A) and the sample size shall be risk-based, as determined by a competent authority, but not less than 1 g per carcass.4.3 Blending/grinding of the muscle sampleMeat samples are chopped using a blender or grinder to increase the surface area for enzymatic degradatio
48、n. The blending or grinding procedure shall be adjusted to maximize digestion efficiency.NOTE Too little blending or grinding can result in poor digestion, while too much blending or grinding can damage or disrupt muscle larvae.4.4 Preparation of the digest fluidPepsin is commercially available in p
49、owder, granular, and liquid forms. The activity of the pepsin shall be certified and pepsin shall be stored according to the manufacturers recommendation.NOTE The use of a liquid pepsin formulation can be advantageous as it could reduce the risk of occupational hazard, such as allergic reaction in laboratory staff.4.5 Digestion of the chopped meatViable Trichinella larvae are resistant to the pepsin-HCl digest fluid and therefore can be recovered free from muscle tissue. Dead Trichinella larvae can be destroyed by artificial digestion.To facilitate an ef
