ISO INTERNATIONAL STANDARD 17601 First edition 2016-01-15 Soil quality Estimation of abundance of selected microbial gene sequences by quantitative PcR from DNA directly extracted from soil Qualité du sol -Estimation de I'abondance de séquences de genes microbiens par amplification par reaction de polymérisation en chaine (PCR) quantitative a partir d'ADN directement extrait du sol Reference number ISO 17601:2016(E) SO International Organization for Standardization @ IS0 2016 HEJIANG INST OF STANDARDIZATION C1 5956617 vided by IHS under ed without license from IHS IS0 17601:2016(E) COPYRIGHTPROTECTEDDOCUMENT IS02016,PublishedinSwitzerland All rights reserved. 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 at the address below or ISO's member body in the country of the requester. ISOcopyrightoffice Ch. de Blandonnet 8 . CP 401 CH-1214 Vernier, Geneva, Switzerland Tel. +41 22 749 01 11 Fax +41 22 749 09 47 [email protected] www.iso.org Intenationaibr PrganizationforStandardization ensee-ZHEJIANG INST OF STANDARDIZRoISQ ,6 - All rights reserved Not for Resale, 2016/3/29 02:54:16 etworking permitted without license from IHS IS0 17601:2016(E) Contents Page Foreword ..iv Introduction. ..V 1 Scope. ..1 2 Normative references ..1 3 Terms and definitions 4 Principle ..2 5 Test materials .4 5.1 DNA .4 5.2 Bacteria. ..4 5.3 Plasmid .4 5.4 Enzyme. ..4 5.5 Chemicals. 4 5.6 Product for bacterial culture medium .5 5.7 Buffer and reagents. .5 6 Apparatus ..6 7 Procedure .6 7.1 qPCR standard preparation and calibration of qPCR assay (task 1) ..6 7.1.1 General .6 7.1.2 Amplicon design (task 1, step 1) .6 7.1.3 qPCR standard preparation (task 1, step 2) .7 7.1.4 Isolate DNA, environmental DNA, artificial DNA ..7 7.1.5 Calibration of the qPCR (task 1, step 3) .9 7.2 Preparation of soil DNA template and inhibition test (task 2) .10 7.2.1 General 10 7.2.2 Soil DNA preparation (task 2, step 4) .10 7.2.3 Inhibition test (task 2, step 5) .10 7.2.4 Dilution of DNA template .12 7.3 qPCR assay (task 3) 12 7.3.1 General. 12 7.3.2 qPCR 12 7.4 Validation and analysis of qPCR assay (task 4) ..12 7.4.1 General .12 7.4.2 Validation of the qPCR assay 12 7.4.3 Calculation of the copy number of the gene of interest in the soil DNA extract....13 8 Examination of the critical steps of the qPCR assay .14 9 Expression of the results of the qPCR assay ..14 10 International ring test .14 11 Test report .14 Annex A (informative) Description of principal steps of TaqMan? qPCR assay ..15 Annex B (informative) International ring-test for evaluating qPCR to quantify the abundance of selected microbial gene sequences from DNA directly extracted from soil ..17 Bibliography .30 iii ee=ZHEJIANG INSTOFSTANDARDIZATION C15956617 vided by IHS under I ed without license from IHS