1、BSI Standards PublicationBS ISO 18074:2015Textiles Identification ofsome animal fibres by DNAanalysis method Cashmere,wool, yak and their blendsBS ISO 18074:2015 BRITISH STANDARDNational forewordThis British Standard is the UK implementation of ISO 18074:2015.The UK participation in its preparation
2、was entrusted to TechnicalCommittee TCI/80, Chemical testing of textiles.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 for its correctappli
3、cation. The British Standards Institution 2015.Published by BSI Standards Limited 2015ISBN 978 0 580 78914 4ICS 59.080.01Compliance with a British Standard cannot confer immunity fromlegal obligations.This British Standard was published under the authority of theStandards Policy and Strategy Committ
4、ee on 30 November 2015.Amendments/corrigenda issued since publicationDate T e x t a f f e c t e dBS ISO 18074:2015 ISO 2015Textiles Identification of some animal fibres by DNA analysis method Cashmere, wool, yak and their blendsTextiles Identification de certaines fibres animales par la mthode danal
5、yse de lADN Cachemire, laine, ya ck et leurs mlangesINTERNATIONAL STANDARDISO18074First edition2015-12-01Reference numberISO 18074:2015(E)BS ISO 18074:2015ISO 18074:2015(E)ii ISO 2015 All rights reservedCOPYRIGHT PROTECTED DOCUMENT ISO 2015, Published in SwitzerlandAll rights reserved. Unless otherw
6、ise 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 at the address be
7、low or ISOs member body in the country of the requester.ISO copyright officeCh. de Blandonnet 8 CP 401CH-1214 Vernier, Geneva, SwitzerlandTel. +41 22 749 01 11Fax +41 22 749 09 47copyrightiso.orgwww.iso.orgBS ISO 18074:2015ISO 18074:2015(E)Foreword ivIntroduction v1 Scope . 12 Caution 13 Normative r
8、eferences 14 Terms and definitions . 15 Principle 26 Apparatus and equipment 37 Reagents 48 Sampling 69 Test methods . 69.1 General . 69.2 Chipping sample 69.3 DNA extraction 69.4 DNA purification 79.5 DNA amplification 89.5.1 Composition of the reaction solution . 89.5.2 Condition of PCR amplificat
9、ion instrument for DNA amplification . 89.5.3 DNA Amplification test method 89.6 Detection and confirmation of DNA amplification . 99.6.1 Preparation . 99.6.2 Electrophoretic migration test . 910 Verification . 910.1 General . 910.2 No amplification verification after extraction process (negative ve
10、rification) . 910.3 Amplification verification after extraction process (positive verification) .1010.4 Amplification verification after PCR process . 1010.5 No amplification verification after PCR process .1010.6 Amplification verification of PCR reaction solution 1011 Assessment 1112 Precision 111
11、3 Test report 11Annex A (informative) Amplification of the constant length DNA fragment by PCR method .13Annex B (informative) DNA additional purification 15Annex C (informative) Repeatability and reproducibility 17Annex D (informative) Practical application to various textile products21Bibliography
12、 .22 ISO 2015 All rights reserved iiiContents PageBS ISO 18074:2015ISO 18074:2015(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 throu
13、gh 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 liaison with ISO, also take part in the work. ISO collabora
14、tes 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 Directives, Part 1. In particular the different approval cr
15、iteria 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 some of the elements of this document may be the subject
16、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 declarations received (see www.iso.org/patents).Any trade nam
17、e 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 about ISOs adherence to the WTO principles in the Technica
18、l Barriers to Trade (TBT), see the following URL: Foreword Supplementary information .The committee responsible for this document is ISO/TC 38, Textiles.iv ISO 2015 All rights reservedBS ISO 18074:2015ISO 18074:2015(E)IntroductionThe composition of fibres in textile products is one of the most impor
19、tant properties. Labelling of composition of textile products is required globally by legislation or by voluntary regulation for fair trade.The testing method to determine the composition of some animal fibres in the textile products has been developed as ISO 177513. This is only one method to deter
20、mine the animal fibre composition currently available. In this method, animal fibres are observed by microscope and identified from the shape of scales by experienced examiners. Many samples can be tested with a high degree of efficiency using this method. However, even experienced examiners have di
21、fficulties in identifying fibres, because textile products have a broad variety of colours and finishings, and there are many blends in animal fibres.Given this situation, several testing methods to obtain the more accurate results have been investigated and developed. Among those methods, the DNA (
22、deoxyribonucleic acid) analysis method has been found to be a practical and feasible method to identify the inherent type of animal fibres.As it is well known, DNA is specific for animals. The DNA-PCR (polymerase chain reaction) method has recently been developed with high accuracy. A very trivial q
23、uantity of the DNA extracted from animal fibres is amplified by PCR to yield a huge quantity of copy DNA. Mitochondrial DNA is used for this analysis because it provides greater numbers than nuclear DNA. ISO 2015 All rights reserved vBS ISO 18074:2015BS ISO 18074:2015Textiles Identification of some
24、animal fibres by DNA analysis method Cashmere, wool, yak and their blendsWARNING The use of this International Standard can involve hazardous materials, operations, and equipment. This International Standard does not purport to address all of the safety problems associated with its use. It is the re
25、sponsibility of the user of this International Standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations and suppliers requirement for safety prior to use.1 ScopeThis International Standard specifies a testing method for DNA analysis of s
26、ome animal fibres to identify cashmere, wool, yak, and their blends by using extraction, amplification by the polymerase chain reaction (PCR) method and DNA detection processes.This International Standard is applicable to cashmere, yak, and wool and their blends as a qualitative method.2 CautionTest
27、 results for fibre identification by the DNA analysis method can be obtained with a high accuracy for the above-mentioned textile products which were processed at lower dye concentration levels or dyed in light colours.However, when such textile products were processed under severe conditions or hig
28、h temperatures, the mitochondrial DNA could have been damaged. In such cases, identification can be difficult because amplification of DNA by PCR cannot take place. If textile products were contaminated by using products from another species, such as cashmere grease on wool fibres, this situation ma
29、y be solved by checking using microscopy techniques.3 Normative referencesThe following documents, in whole or in part, are normatively referenced in this document and are indispensable for its application. For dated references, only the edition cited applies. For undated references, the latest edit
30、ion of the referenced document (including any amendments) applies.ISO 3696, Water for analytical laboratory use Specification and test methodsISO 8655-2, Piston-operated volumetric apparatus Part 2: Piston pipettes4 Terms and definitionsFor the purposes of this document, the following terms and defi
31、nitions apply.4.1DNAdeoxyribonucleic acid, that exists in nuclei and in mitochondria of animal fibre cells and is composed of a linear array of 4 bases (Adenine: A, Thymine: T, Guanine: G and Cytosine: C)Note 1 to entry: The DNA sequence is identical and intrinsic for each animal fibre.4.2animal fib
32、rescashmere, wool, or yak fibresINTERNATIONAL STANDARD ISO 18074:2015(E) ISO 2015 All rights reserved 1BS ISO 18074:2015ISO 18074:2015(E)4.3buffer solutionsolution used to maintain pH of reaction solution at required value4.4reducing agentagent that degrades animal fibres through reductive cleavage
33、of S-S bonds in the fibres4.5DNA amplificationamplification of the specific fragment of DNA by the PCR method4.6PCR methodmethod of the polymerase chain reactionNote 1 to entry: The amplification process of the DNA fragment with a constant length is explained in Annex A.4.7DNA polymerase for PCR met
34、hodheat-stable DNA polymerase that is used for PCR and has no proof reading activity4.8primershort length fragment of a single strand DNA which is a reaction initiator and designed as the identical sequence of 18-30 bases to DNA of the animal fibre4.9primer setset of primer with the reaction directi
35、on of forward and reverse 4.10primer for cashmereprimer with an identical base sequence of mitochondrial DNA of cashmereNote 1 to entry: The sequence of base for primers will be submitted to the public database.4.11primer for woolprimer with an identical base sequence to mitochondrial DNA of wool4.1
36、2primer for yakprimer DNA with an identical base sequence to mitochondrial DNA of yakNote 1 to entry: Information concerning the primers may be obtained from ISO/TC 38 secretariat.4.13gel electrophoresis migrationmethod to detect the amplified constant length DNA fragments5 PrincipleMitochondrial DN
37、A is extracted from animal fibre samples by using a chemical and enzyme reaction. The extracted DNA is purified by using a precipitation method and centrifuge. The purified DNA is applied for the amplification reaction of PCR method. In the PCR method, primers for cashmere, yak and wool are respecti
38、vely tested. If the sample is cashmere, only cashmere primer can amplify the constant length of DNA fragments. Then, the constant length of DNA fragments is detected by the electrophoretic migration method.2 ISO 2015 All rights reservedBS ISO 18074:2015ISO 18074:2015(E)The sample fibres are identifi
39、ed by knowing whether amplification was observed or not for the tests using all primers respectively.6 Apparatus and equipment6.1 Pipettes, capable of measuring and taking (0 to 20) l (0,20 l), (20 to 200) l (1,60 l), (200 to 1 000) l (8 l) within systematic errors defined in ISO 8655-2.6.2 Micro tu
40、be, capable of withstanding the centrifugation of 14 000g and autoclave.The capacity is 2 ml for purification and 0,2 ml for PCR method. A tube of 0,2 ml for PCR method should follow the manufacturers recommendation of the PCR instrument.6.3 Cap lock, use for micro tube.6.4 Heat block, with mounting
41、 holes for micro tubes and capable of heating up to about 80 C (1,0 C).6.5 Shaking agitator, capable of heating up to 50 C and maintaining at the temperature of 50 C and shaking micro tube at around 500 r/min or higher.6.6 Shaking machine, capable of mounting micro tubes and shaking at around 500 ti
42、mes/min or higher.This machine can be replaced by an equivalent instrument such as microtube rotator which is possible to rotate at 30 r/min or higher.6.7 Centrifuge, capable of centrifuging of 14 000g or higher, setting up temperature from 0 C to room temperature and mounting micro tubes or units o
43、f centrifugal ultrafiltration.6.8 Unit of centrifugal ultrafiltration1), capable of capturing molecules with the molecular weight of 100 kDa (Dalton) or more.6.9 PCR instrument2), capable of programing for temperature and time.6.10 UV illuminator, UV irradiator.6.11 Photo booth.6.12 Generic plastic
44、box with a resealable lid.6.13 Comb, used for making wells in the agarose gel of the gel electrophoresis migration test.6.14 Mixer mill, used for mixing and homogenizing animal fibres.6.15 Erlenmeyer flask, with capacity 200 ml.1) Amicon Ultra is an example of a suitable product available commercial
45、ly. This information is given for the convenience of users of this document and does not constitute an endorsement by ISO of this product.2) Life Technologies Corporation is a provider of a suitable product available commercially. This information is given for the convenience of users of this docume
46、nt and does not constitute an endorsement by ISO of this product. Equivalent products may be used if they can be shown to lead to the same results. ISO 2015 All rights reserved 3BS ISO 18074:2015ISO 18074:2015(E)7 Reagents7.1 Pure water.Use pure water as defined in ISO 3696 with the purity of Grade
47、1. This water should not have DNase (DNA digesting) activity. It should not contain a significant amount of DNA which can be amplified by primers and should not show inhibitory effects for this testing method.7.2 Chloroform/isoamyl alcohol reagent.100 % concentration of the highest grade. Chloroform
48、 9,6 ml Isoamyl alcohol 400 l7.3 Sodium perchlorate solution. Sodium perchlorate 6,1 gMake the solution up to 10 ml by adding pure water.7.4 1 mol/l Tris HCl tris(hydroxymethyl)aminomethane.Dissolve 12,1 g of tris(hydroxymethyl)aminomethane in 800 ml of pure water, then adjust the pH to 8,0 by addin
49、g HCl and using a pH-meter. Then, make it up to 1 000 ml by adding pure water.7.5 500 mmol/l EDTA (ethylenediaminetetraacetic acid).Dissolve 186,1 g of EDTANa22H2O (disodium salt-dihydrate of EDTA) in 800 ml pure water, then adjust pH to 8,0 by adding NaOH and using a pH-meter. Then, make it up to 1 000 ml by adding pure water.7.6 Buffer solution A. 1 mol/l Tris-HCL (7.4) 5 ml 500 mmol/l EDTA (7.5) 2 ml Sodium lauryl sulfate (SLS) (C12H25SO4Na) 0,2 g Sucrose 1,2 g Sodium chloride 170 mg Dithiothreit
