ISO INTERNATIONAL STANDARD 21286 First edition 2019-03 Soil quality Identification of ecotoxicological test species by DNA barcoding Qualite du sol - Identification des especes par code-bare ADN dans les essais d'écotoxicologie Reference number ISO 21286:2019(E) so @ IS0 2019 IS0 21286:2019(E) COPYRIGHTPROTECTEDDOCUMENT @ ISO 2019 All rights reserved. Unless otherwise specified, or required in the context of its implementation, 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 below or Iso's member body in the country of the requester. ISO copyright office CP 401 : Ch. de Blandonnet 8 CH-1214 Vernier, Geneva Phone: +4122 749 0111 Fax: +41 22 749 09 47 Email: [email protected] Website: www.iso.org Published in Switzerland ii @ IS0 2019 - All rights reserved IS0 21286:2019(E) Contents Page Foreword ..iv Introduction. ..V 1 Scope. ..1 2 Normative references 3 Terms and definitions 4 Principle ..2 5 Reagents and material .3 5.1 Biological material .3 5.2 Enzyme. .3 5.3 Oligonucleotide PCR primers 3 5.4 Reagents. .3 6 Apparatus. .4 7 General requirements .5 7.1 Experimental precaution and contamination avoidance .5 7.2 Safety precautions... .5 7.2.1 Chemical hazards .5 7.2.2 Physical hazards. .6 8 Procedure 8.1 DNAisolation 6 8.2 Quantification 7 8.3 PCR 7 8.3.1 Target genomic region 7 8.3.2 Primer design 7 8.3.3 Primer synthesis 7 8.3.4 PCR 8.4 Checking the amplicon size. 9 8.5 Purification .9 8.6 Sequencing .9 8.7 Bioinformatics ..9 8.7.1 General 9 8.7.2 Electropherogram or raw sequence quality checking 10 8.7.3 Trimming of low-quality regions and primers sequences ..10 8.7.4 Sequence overlapping ..10 8.7.5 Sequence verification. ..11 8.7.6 Reviewing the edited sequence .11 8.7.7 Species assignment.. ..11 8.7.8 Qualityofthereferencedatabases .12 9 Calculation and expression of results .13 10 Validity of the test ..13 11 Test report ..14 Annex A (informative) Eisenia Barcoding Initiative: A ring test to evaluate the applicability of DNA barcoding for the identification of Eisenia species. ..15 Bibliography ..18 @ IS0 2019 - All rights reserved ii