1、BSI Standards PublicationBS ISO 13495:2013Foodstuffs Principlesof selection and criteriaof validation for varietalidentification methods usingspecific nucleic acidBS ISO 13495:2013 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 13495:2013.The UK participation
2、in its preparation was entrusted to TechnicalCommittee AW/275, Food analysis - Horizontal methods.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 respons
3、ible for its correctapplication. The British Standards Institution 2013. Published by BSI StandardsLimited 2013ISBN 978 0 580 68034 2ICS 67.050Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Polic
4、y and Strategy Committee on 30 November 2013.Amendments issued since publicationDate Text affectedBS ISO 13495:2013 ISO 2013Foodstuffs Principles of selection and criteria of validation for varietal identification methods using specific nucleic acidProduits alimentaires Principes de slection et crit
5、res de validation des mthodes didentification varitale utilisant des acides nucliques spcifiquesINTERNATIONAL STANDARDISO13495First edition2013-11-01Reference numberISO 13495:2013(E)BS ISO 13495:2013ISO 13495:2013(E)ii ISO 2013 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2013All rights reser
6、ved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission. Permission can be requested from either ISO
7、 at the address below or ISOs member body in the country of the requester.ISO copyright officeCase postale 56 CH-1211 Geneva 20Tel. + 41 22 749 01 11Fax + 41 22 749 09 47E-mail copyrightiso.orgWeb www.iso.orgPublished in SwitzerlandBS ISO 13495:2013ISO 13495:2013(E) ISO 2013 All rights reserved iiiC
8、ontents PageForeword ivIntroduction v1 Scope . 12 Normative references 13 Terms and definitions . 13.1 Terms related to variety 13.2 Terms related to DNA/RNA extraction and purification . 23.3 Terms related to PCR amplification of nucleic acids 23.4 Terms related to detection 33.5 Terms related to c
9、ontrols . 33.6 Terms related to markers 44 Quality assurance on the test results 45 Selection of methods 66 Markers selection 66.1 General criteria . 66.2 Marker set selection . 66.3 SSR markers. 66.4 Single nucleotide polymorphism (SNP) 77 Laboratory samples . 77.1 Sample 77.2 Sample size . 77.3 Re
10、ference material . 78 Laboratory validation . 88.1 General . 88.2 Intralaboratory validation criteria . 88.3 Interlaboratory validation criteria . 99 Interpretation and expression of the results .109.1 General 109.2 Data annotation system 109.3 Analysis of results 119.4 Expression of results . 1110
11、Test report 11Bibliography .13BS ISO 13495:2013ISO 13495:2013(E)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 technical com
12、mittees. 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 with the
13、 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 for th
14、e 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. www.iso.org/directivesAttention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shal
15、l 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. www.iso.org/patentsAny trade name used in this document is infor
16、mation given for the convenience of users and does not constitute an endorsement.The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 16, Horizontal methods for molecular biomarker analysis.iv ISO 2013 All rights reservedBS ISO 13495:2013ISO 13495:2013(E)Introduct
17、ionThis International Standard outlines guidelines designed to support decision-making and validation on the protocols used to produce high-quality molecular data for varietal identification.Varietal identification testing requires high-quality markers, which are able to provide reproducible data us
18、ing a variety of equipment, chemistries and reagents. Accordingly, this International Standard only addresses specific amplification methods.The aims of this International Standard are to ensure that the methods of analysis are compatible with customer requests, to list the different steps towards m
19、ethod validation, and to define acceptance criteria. It also guarantees that the general principles employed in performing these analyses will be the same across all laboratories (reference material, sample size, laboratory sample, test portion, extraction, results analysis and interpretation, certi
20、ficate of analysis).Finally, this International Standard plays a role in standardizing the results obtained by different laboratories. ISO 2013 All rights reserved vBS ISO 13495:2013BS ISO 13495:2013Foodstuffs Principles of selection and criteria of validation for varietal identification methods usi
21、ng specific nucleic acid1 ScopeThis International Standard specifies molecular tools for generating molecular profiles of varieties of plant species, enabling varietal identification, i.e. confirmation of identity in relation to one or more references.This International Standard is applicable to var
22、ious matrices, seeds, leaves, roots, industrial products composed of only one variety. Matrices presented in the form of mixtures of varieties (such as purees, compotes, flours) are excluded from the scope of this document.This International Standard does not deal with genetic purity.2 Normative ref
23、erencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable to its application. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.IS
24、O/IEC 17025:2005, General requirements for the competence of testing and calibration laboratories3 Terms and definitionsFor the purposes of this document, the following terms and definitions apply.3.1 Terms related to variety3.1.1cultivargroup of cultivated plants which may be clearly defined by mor
25、phological, physical, cytological, chemical or other characteristics and which, after sexual or asexual reproduction, keeps its distinct characterSOURCE: ISO 7563:1998, definition 1.12Note 1 to entry: The concept of “cultivar” is essentially different from the concept of the botanical variety “varie
26、tas”, in that “cultivar” is an infraspecific division resulting from controlled selection, even if empirical; “varietas” is an infraspecific division resulting from natural selection. The terms “cultivar” and “variety” (in the sense of cultivated variety) are equivalent. In translations or adaptatio
27、ns of botanical nomenclature for particular uses, the terms “cultivar” or “variety” (or their equivalents in other languages) may be used in text.Note 2 to entry: The names of botanical varieties and species are always in Latin form and are governed by botanical nomenclature.3.1.2speciesgroup of org
28、anisms that have a high level of genetic (DNA) similarity and are capable of interbreeding: often containing subspecies, varieties or racesNote 1 to entry: A species is designated in italics by the genus name followed by the specific name, e.g. Ananas comosus.INTERNATIONAL STANDARD ISO 13495:2013(E)
29、 ISO 2013 All rights reserved 1BS ISO 13495:2013ISO 13495:2013(E)3.1.3varietyunique and uniform member of a species of plant (except for hybrid species) that retains its characteristics from generation to generation through its natural mode of reproductionSOURCE: ISO 5527:, definition 2.1.7, modifie
30、d the other preferred term “cultivar” has been deleted.3.2 Terms related to DNA/RNA extraction and purification3.2.1nucleic acid extractionsample treatment for the liberation of target nucleic acidNote 1 to entry: The nucleic acid extraction procedure is used for isolating nucleic acids from other c
31、ellular components, such as protein, lipids, carbohydrates and other impurities in a test sample.SOURCE: ISO 22174:2005, definition 3.2.1, modified Note 1 has been added.3.2.2nucleic acid purificationmethod resulting in a more purified DNANote 1 to entry: A procedure or process involving sequential
32、steps used to separate DNA and/or RNA from other components in a sample. A highly purified DNA or RNA sample contains negligible observable or measurable effects attributable to inhibitors of the polymerase chain reaction.Note 2 to entry: In this context, purity refers to the reduction of observable
33、 and measurable effects of PCR inhibitors.SOURCE: ISO 22174:2005, definition 3.2.2, modified Note 1 has been added; Note 2 has been modified.3.3 Terms related to PCR amplification of nucleic acids3.3.1ampliconspecific DNA fragment produced by a DNA-amplification technology, such as the polymerase ch
34、ain reaction (PCR)3.3.2hybridizationspecific binding of complementary nucleic acid sequences under suitable reaction conditionsSOURCE: ISO 22174:2005, definition 3.6.33.3.3multiplex PCRPCR that uses multiple pairs of primers in different loci combined within a single reaction mixture to produce mult
35、iple amplicons simultaneouslySOURCE: ISO 22174:2005, definition 3.4.11, modified the phrase following “primers” has been added.3.3.4polymerase chain reactionPCRenzymatic procedure which allows in vitro amplification of DNASOURCE: ISO 22174:2005, definition 3.4.12 ISO 2013 All rights reservedBS ISO 1
36、3495:2013ISO 13495:2013(E)3.3.5primeroligonucleotide of defined length and sequence complementary to a segment of an analytically relevant DNA sequenceSOURCE: ISO 22174:2005, definition 3.4.123.3.6probelabelled nucleic acid molecule with a defined sequence used to detect target DNA by hybridizationS
37、OURCE: ISO 22174:2005, definition 3.6.1, modified the term was originally “DNA probe”.3.3.7specificityproperty of a method to respond exclusively to the characteristic or analyte under investigationNote 1 to entry: It describes the ability to specifically recognize the nucleic acid sequence to be de
38、tected by distinguishing it from other nucleic acid sequences, and the tendency for a primer or probe to hybridize with its intended target and not hybridize with other non-target sequences.SOURCE: ISO 24276:2006, definition 3.1.4, modified Note 1 has been added.3.3.8thermocyclerautomated laboratory
39、 apparatus used to repeatedly raise and lower the temperature of a sample by cycling through a series of discrete, pre-programmed stepsNote 1 to entry: This cycling of temperatures drives the PCR process.3.4 Terms related to detection3.4.1electrophoresismethod of separating electrically-charged part
40、icles by their differential migration under an electric fieldNote 1 to entry: PCR products can be separated by various types of electrophoresis.3.5 Terms related to controls3.5.1reference samplereference materialmaterial or substance, one or more of whose property values are sufficiently homogeneous
41、 and well established to be used for the calibration of an apparatus, the assessment of a measurement method, or for assigning values to materialsNote 1 to entry: The reference material may be either provided by the customer, internal to the laboratory, or an officially-designated reference.SOURCE:
42、ISO Guide 30SOURCE: ISO 24276:2006, definition 3.5.1, modified Note 1 has been added.3.5.2test controlone or more samples that have undergone all or part of the analytical process designed for the target samples and which can reveal known alleles of the markers used, thereby signalling any process e
43、rrors and providing reference alleles which can facilitate the reading of results ISO 2013 All rights reserved 3BS ISO 13495:2013ISO 13495:2013(E)3.6 Terms related to markers3.6.1allele competitionpreferential amplification of one allele over another in a heterozygote or a mixture3.6.2allele frequen
44、cymeasure of how common an allele is in a population; the proportion or percentage of all of the occurrences of a locus that is occupied by a given allele3.6.3markergenetic marker that typically applies to DNA fragments matching a given locus that gives information on the genotype of the carrier or
45、on the genotype of neighbouring loci3.6.4null allelesequence variant that precludes PCR amplification of a particular target, resulting in the absence of detectable PCR product3.6.5repeat regiongenomic region in which a particular DNA or RNA sequence occurs as multiple copies3.6.6simple sequence rep
46、eatSSRregion of DNA consisting of a short (1 bp to 6 bp) sequence (repeat unit) that is tandemly repeated many (typically five to 50) timesNote 1 to entry: SSRs are commonly known as microsatellites.Note 2 to entry: The number of repeat units present at a specified SSR, and thus the overall length o
47、f the SSR, often varies among individuals.3.6.7single nucleotide polymorphismSNPsingle nucleotide variation in a genetic sequence that occurs at appreciable frequency in the populationNote 1 to entry: SNP is often pronounced “snip”.SOURCE: ISO 25720:2009, definition 4.23, modified Note 1 has been ad
48、ded.4 Quality assurance on the test resultsThe requirements and guidelines set out in Table 1 are coded as follows: C: compulsory; R: Recommended.4 ISO 2013 All rights reservedBS ISO 13495:2013ISO 13495:2013(E)Table 1 Requirements and guidelines on the test resultsSTAFFAll staff carrying out specifi
49、c tasks shall be qualified based on an appropriate level of education, training, expe-rience and/or proven competency, according to the task skills required.Criteria Requirement (C or R)Training in use of the equipment CHandling chemicals CStaff responsible for issuing opinions and inter-pretations shall be fully conversant with the various genetic structures and seed production systems involved, in order to provide customers with advice and/or suggest ways to interpret the results.CEQUIPMENTM
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