1、BSI Standards PublicationBS EN ISO 18743:2015Microbiology of the food chain Detection of Trichinellalarvae in meat by artificialdigestion methodBS EN ISO 18743:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of EN ISO 18743:2015.The UK participation in its prepar
2、ation was entrusted to Technical Committee AW/9, Microbiology.A list of organizations represented on this committee can be obtained on request to its secretary.This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application.
3、The British Standards Institution 2015. Published by BSI Standards Limited 2015ISBN 978 0 580 81675 8 ICS 07.100.30 Compliance with a British Standard cannot confer immunity from legal obligations.This British Standard was published under the authority of the Standards Policy and Strategy Committee
4、on 31 October 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dEUROPEAN STANDARD NORME EUROPENNE EUROPISCHE NORM EN ISO 18743 September 2015 ICS 07.100.30 English Version Microbiology of the food chain - Detection of Trichinella larvae in meat by artificial digestion me
5、thod (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 von Lebensmitteln und Futtermitteln - Nachweis von Trichinellenlarven aus Fleischproben - Physikalisches Verdauungs
6、verfahren (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 status of a national standard without any alteration. Up-to-date lists
7、 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 translation under t
8、he 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, Finland, Former Yugo
9、slav 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 STANDARDIZATION COMIT EUROPEN
10、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. EN ISO 18743:2015 EBS EN ISO 18743:2015EN ISO 18743:2015 (E) 3 Europ
11、ean 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” the secretariat of which is held by DIN. This European Standard shall be given the status of a n
12、ational 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 drawn to the possibility that some of the elements of this document may be the subject of paten
13、t 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, Bulgaria, Croa
14、tia, 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 and t
15、he United Kingdom. Endorsement notice The text of ISO 18743:2015 has been approved by CEN as EN ISO 18743:2015 without any modification. BS EN ISO 18743:2015ISO 18743:2015(E)Foreword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 14 Principle 24.1 General . 24.2 Sample
16、size . 24.3 Blending/grinding of the muscle sample . 24.4 Preparation of the digest fluid . 24.5 Digestion of the chopped meat . 24.6 Filtration of the digest fluid . 34.7 Sedimentation of the digest fluid 34.8 Microscopic examination . 34.9 Verification of findings . 35 Reagents 36 Apparatus . 47 S
17、ampling, labelling, and transport 58 Sample preparation . 59 Procedure. 59.1 General . 59.2 Blending/grinding . 59.3 Preparation of the digest fluid . 69.4 Digestion of the chopped meat in the glass beaker . 69.5 Filtration of the digest fluid . 69.6 Sedimentation of the digest fluid in the separato
18、ry funnel 79.7 Collection of the primary and secondary sediment 79.8 Microscopic examination . 710 Documentation . 811 Expression of the results 812 Safety measures . 8Annex A (normative) Sample collection . 9Annex B (normative) Frozen samples .11Annex C (informative) Artificial digestion magnetic s
19、tirrer method 12Annex D (informative) Example of a laboratory worksheet for recording data when testing pooled samples by digestion assay .14Bibliography .15 ISO 2015 All rights reserved iiiContents PageBS EN ISO 18743:2015ISO 18743:2015(E)ForewordISO (the International Organization for Standardizat
20、ion) 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 r
21、epresented 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.The procedures used to d
22、evelop 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 accordance with the editorial rules of the
23、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 of any patent rights identified durin
24、g 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 endorsement.For an explanation on the meani
25、ng 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 responsible for this document is ISO/TC 34, F
26、ood products, Subcommittee SC 9, Microbiology.iv ISO 2015 All rights reservedBS EN ISO 18743:2015ISO 18743:2015(E)IntroductionTrichinella spp. are the causative agents of human trichinellosis, a disease which is a public health hazard and, as a result, also represents an economic problem in porcine
27、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 Trichinella larvae in muscl
28、e 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 detect infected anima
29、ls with very small numbers of larvae in muscle samples, that can pose a risk for subclinical infections in humans. ISO 2015 All rights reserved vBS EN ISO 18743:2015BS EN ISO 18743:2015Microbiology of the food chain Detection of Trichinella larvae in meat by artificial digestion methodWARNING Person
30、s 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 to establish appropriate safety and health practices and t
31、o ensure compliance with any national regulatory conditions.1 ScopeThis International Standard specifies a method 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
32、sylvatic animal species, which can be infected by nematodes of the genus Trichinella.This method does not allow 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 S
33、tandard is intended to be used in conjunction with the guidelines in the OIE Manual of Diagnostic Tests and 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
34、other means that the animal was not at risk for exposure to Trichinella.The artificial digestion/magnetic stirrer 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 th
35、is International Standard can be documented, alternative methods can be used for analysis.2 Normative referencesThe 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
36、 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 microbiological examinationsInternational Commission on Trichinellosis (ICT), “Q
37、uality Assurance in Digestion Testing Programs for Trichinella”, Recommendations and Guidelines. 2012World Organisation 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 th
38、is document, the following terms and definitions apply.3.1Trichinella muscle larvaeMLfirst larval stage (L1) 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 m
39、m in length and 0,03 mm in width.INTERNATIONAL STANDARD ISO 18743:2015(E) ISO 2015 All rights reserved 1BS EN ISO 18743:2015ISO 18743:2015(E)3.2digestion assaymethod to detect Trichinella larvae in muscle tissue by an enzymatic digestion step to release larvae from muscle tissues followed by filtrat
40、ion and sedimentation steps, and detection of recovered larvae by microscopy3.3larvae per gramlpgnumber of Trichinella larvae present in one gram of muscle tissue4 Principle4.1 GeneralThe artificial digestion method, which is accepted as the international reference method (see OIE, Terrestrial Manua
41、l 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 series of sedimentation and washing steps; equivalent validated methods can also be used. Test performance is greatly influenced by the
42、 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 selected muscle, by the method used, and by the skills of the technician performing the test. A sensitivity of at least one to thre
43、e 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 target animal. There are no internal quality controls that can be used while performing the method. Some key elements in the principle
44、 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 from predilection sites (see Annex A) and the sample size shall be risk-based, as determined by a competent authority, but not less
45、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 degradation. The blending or grinding procedure shall be adjusted to maximize digestion efficiency.NOTE Too little blending or grinding can re
46、sult 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 powder, granular, and liquid forms. The activity of the pepsin shall be certified and pepsin shall be stored according to the manufac
47、turers 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 therefor
48、e can be recovered free from muscle tissue. Dead Trichinella larvae can be destroyed by artificial digestion.To facilitate an efficient and rapid digestion, a maximum ratio of 1:20, meat to digest fluid, and a temperature of 45 C 2 C, shall be maintained throughout the process. The time required for
49、 digestion shall be at least 30 min, but in the case of muscle samples which are less digestible, such as from wildlife 2 ISO 2015 All rights reservedBS EN ISO 18743:2015ISO 18743:2015(E)carcasses, or the tongue of many species (see Annex A), the digestion time should be increased but, unless otherwise validated for a particular sample matrix, shall not exceed 60 min in total.NOTE If the time-temperature conditions are less than the required values, there might be an incomplete digestion of the muscle tissue. Conversely, elevated temperature (ov
