1、| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | BRITISH STANDARD BS EN 12686:1998 The Euro
2、pean Standard EN 12686:1998 has the status of a British Standard ICS 07.080; 07.100.01 NO COPYING WITHOUT BSI PERMISSION EXCEPT AS PERMITTED BY COPYRIGHT LAW Biotechnology Modified organisms for application in the environment Guidance for the sampling strategies for deliberate releases of geneticall
3、y modified micro-organisms, including virusesBS EN 12686:1998 This British Standard, having been prepared under the direction of the Sector Committee for Materials and Chemicals, was published under the authority of the Standards Committee and comes into effect on 15 December 1998 BSI 1998 ISBN 0 58
4、0 30179 6 Amendments issued since publication Amd. No. Date Text affected National foreword This British Standard is the English language version of EN 12686:1998. The UK participation in its preparation was entrusted to Technical Committee CII/58, Biotechnology, which has the responsibility to: aid
5、 enquirers to understand the text; present to the responsible European committee any enquiries on the interpretation, or proposals for change, and keep the UK interests informed; monitor related international and European developments and promulgate them in the UK. A list of organizations represente
6、d on this committee can be obtained on request to its secretary. Cross-references The British Standards which implement international or European publications referred to in this document may be found in the BSI Standards Catalogue under the section entitled “International Standards Correspondence I
7、ndex”, or by using the “Find” facility of the BSI Standards Electronic Catalogue. A British Standard does not purport to include all the necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself
8、 confer immunity from legal obligations. Summary of pages This document comprises a front cover, an inside front cover, the EN title page, pages 2 to 8, an inside back cover and a back cover.CEN European Committee for Standardization Comite Europe en de Normalisation Europa isches Komitee fu r Normu
9、ng Central Secretariat: rue de Stassart 36, B-1050 Brussels 1998 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 12686:1998 E EUROPEAN STANDARD EN 12686 NORME EUROPE ENNE EUROPA ISCHE NORM July 1998 ICS 07.080;100.01 Descriptors: b
10、iotechnology, genetics, modified organisms, environments, environmental protection, sampling, experimental design English version Biotechnology Modified organisms for application in the environment Guidance for the sampling strategies for deliberate releases of genetically modified micro-organisms,
11、including viruses Biotechnologie Organismes modifie s disse mine s dans lenvironnement Guide des strate gies de chantillonnage pour les disse minations volontaires de micro-organismes ge ne tiquement modifie s, y compris de virus Biotechnik Vera nderte Organismen zum Einsatz in der Umwelt Leitfaden
12、fu r Probenahmestrategien bei der absichtlichen Freisetzung gentechnisch vera nderter Mikroorganismen einschlielich Viren This European Standard was approved by CEN on 1 July 1998. CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving thi
13、s European Standard the status 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 Central Secretariat or to any CEN member. This European Standard exists in three official versions (E
14、nglish, French, 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 Central Secretariat has the same status as the official versions. CEN members are the national standards bodies of Austria, Belgium, Czech R
15、epublic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom.Page 2 EN 12686:1998 BSI 1998 Foreword This European Standard has been prepared by Technical Committee CEN/TC 233, Biotechnology, the
16、Secretariat of which is held by AFNOR. 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 January 1999, and conflicting national standards shall be withdrawn at the latest by January 1999. This Europ
17、ean Standard has been prepared under a mandate given to CEN by the European Commission and the European Free Trade Association. According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, B
18、elgium, Czech Republic, Denmark, Finland, France, Germany, Greece, Iceland, Ireland, Italy, Luxembourg, Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Contents Page Foreword 2 Introduction 3 1 Scope 3 2 Normative references 3 3 Definitions 3 4 General consideration
19、s 4 5 Sampling strategy 4 Annex A (informative) Relationship between a sampling and monitoring valid strategy 7 Annex B (informative) Bibliography 8Page 3 EN 12686:1998 BSI 1998 Introduction When genetically modified micro-organisms including viruses (GMMs) are subject to an experimental release int
20、o the environment, it is important to ensure the validity of sampling strategies used to monitor the release. The sampling strategy and the statistical analysis used will vary with the predicted frequency of occurrence and spatial distribution of the relevant GMMs or genes in the area studied. Since
21、 there are many different techniques available for the detection and the identification of GMMs, this European Standard is formulated as a recommendation to the experimenter to design a sampling strategy appropriate to the purpose of the field experiment, to the micro-organisms and to the particular
22、 phenotypic and genotypic properties of the GMMs being used. This European Standard gives the experimenter a list of points that should be considered in determining the validity of a sampling strategy comprising valid design, review, execution and documentation of a sampling protocol. 1 Scope This E
23、uropean Standard provides guidance concerning the procedures for setting up a valid sampling strategy to meet the objectives of a monitoring strategy for GMMs released into the environment. Since monitoring methods of micro-organisms in environmental samples usually require pre-treatment of the samp
24、les, for example the extraction and isolation of GMMs and/or their nucleic acid, this is included in the scope of this European Standard. The sampling is to provide material to which subsequent analytical or biological methods for monitoring of GMMs can be applied (see EN 12685). This European Stand
25、ard is intended to address sampling of micro-organisms, including viruses (and their relevant hosts), or their nucleic acid. This European Standard does not cover: the sampling of virus-like entities or similar agents; the sampling of GMMs in food, human health and veterinary applications. NOTE Atte
26、ntion is drawn to national, European and international regulations, and relevant standards covering the sampling of GMMs in food, human health and veterinary applications. This European Standard can be applied to sampling of GMMs in all habitats and micro-environments as required to meet the defined
27、 experimental objectives. The mode of sampling and the nature of the samples are dependent on the particular purpose. Therefore this European Standard provides the experimenter with a list of guiding parameters that should be considered in determining the validity of the proposed strategy for sampli
28、ng. 2 Normative references This European Standard incorporates by dated or undated reference, provisions from other publications. These normative references are cited at the appropriate places in the text and the publications are listed hereafter. For dated references, subsequent amendments to or re
29、visions of any of these publications apply to this European Standard only when incorporated in it by amendment or revision. For undated references the latest edition of the publication referred to applies. EN 12685, Biotechnology Modified organisms for application in the environment Guidance for the
30、 monitoring strategies for deliberate releases of genetically modified micro-organisms, including viruses. 3 Definitions For the purposes of this standard, the following definitions apply: 3.1 control preparation of known characteristics used to standardize an analysis 3.2 genetically modified micro
31、-organism micro-organism in which the genetic material has been altered in a way that does not occur naturally by mating and/or natural recombination NOTE Within the terms of this definition genetic modification occurs at least through the use of the techniques listed in the Directive 90/219/EEC or
32、its appropriate annexes (see Annex B 1). 3.3 genotype genetic constitution of an organism NOTE The genotype can be described with respect to particular genes. 3.4 host target species for virus replication as defined in the experimental design 3.5 micro-environment defined location in the environment
33、 potentially occupied by an organism NOTE This “micro-environment” can impart a degree of confinement on the dispersal of the organism. 3.6 monitoring regular or continuous observation or collection of data with respect to an organism, process or procedure NOTE In this standard, monitoring applies t
34、o the progress of a released genetically modified micro-organism.Page 4 EN 12686:1998 BSI 1998 3.7 monitoring strategy procedure for designing, reviewing, executing and documenting a monitoring protocol 3.8 phenotype sum of the traits of an organism NOTE 1 The phenotype can be described with respect
35、 to one or more traits under a given set of conditions. NOTE 2 In the case of a virus, the phenotype can be described by one or more traits manifested in the infected host. 3.9 release site defined area which contains one or more experimental fields NOTE Several different trials can occur within a r
36、elease site. 3.10 sample materials collected for analysis 3.11 sampling protocol written document describing the manner in which samples are collected, transported, stored and their pre-treatment 3.12 sampling strategy procedure for designing, reviewing, executing and documenting a sampling protocol
37、 4 General considerations In order to evaluate the validity of a proposed sampling design, the following considerations should be taken into account. The availability and use of adequate controls is essential for the validity of the results. The design of most field trials with GMMs follows the same
38、 principles as applied to field trials with micro-organisms which are not genetically modified. Statistical analysis, required for quantitative evaluation, determines the sampling design and hence the validity of the results. The purpose of the experiment should be clearly defined in order to prepar
39、e a statistically adequate protocol for sampling. Specific requirements of any subsequent detection and identification method, should be considered and can influence the utility of the results for monitoring purposes (see prEN 12685). NOTE The development of a monitoring and sampling valid strategy
40、for deliberate releases of genetically modified micro-organisms in the environment is summarized in Figure A.1. 5 Sampling strategy 5.1 General It is first necessary to determine the objectives of the sampling strategy. The main steps in the development of the sampling strategy are: a) statement of
41、experimental objectives; b) design and review of sampling protocol; c) execution of the sampling protocol; d) appropriate record keeping. Responsibilities should be assigned for each step to a specific authority, organization or person. The sampling strategy should be reviewed regularly, for example
42、 in the light of the inspections of the release site and monitoring results, to ensure its continuing validity (see EN 12685). 5.2 Design criteria The design of the sampling protocol should meet defined objectives as determined by the experimenter. The design can vary widely, given distinctive requi
43、rements for, for example, trials to study phenotypic properties of GMMs, gene transfer, or dispersal and/or persistence of GMMs in the environment. Depending on the objectives for which the sampling is carried out, the nature and composition of the samples which are collected, can be different. Samp
44、ling can involve host material, soil and water, but also air (e.g. dispersal of spores), animals or plant parts (e.g. roots harbouring symbiotic micro-organisms). The protocol for sampling should be part of the preparation and design of the experiment, keeping the flexibility that could be required
45、by unexpected observations, and should therefore be supported by a clear design of the experiment. Parameters such as starting date or period, location(s) and site size, number of genotypes, are important in the development of the design. When designing the sampling protocol a very important factor
46、concerns the pre-treatment of the samples which is required to allow subsequent detection and/or identification of GMMs. The choice of the pre-treatment procedure is affected by the nature and composition of the samples and also by the demands of the detection and/or identification technique used fo
47、r monitoring and/or characterization of the GMM. Many of these techniques require extraction of micro-organisms, nucleic acid and/or gene products from the sample. Examples of approaches for detection and isolation of GMMs from environmental samples are: bioassay; filtration techniques; decanting an
48、d sieving; centrifugation and gradient centrifugation; immunological techniques; affinity techniques; culturing and propagation techniques.Page 5 EN 12686:1998 BSI 1998 For the extraction of macromolecules, such as nucleic acids and proteins, the above techniques and the following are also appropria
49、te: lysis by freeze-thawing; extraction with organic solvent(s); lysis using lytic enzymes; cellular disruption techniques (e.g. disruption using glass-beads, disruption by sonication, disruption by a French Press); homogenization. Alternatively, macromolecules can be extracted directly from the sample without prior isolation of a GMM. Each of the above listed treatments generates losses of the target GMM or macromolecules. In case a combination of techniques is utilized, these losses are c
