ISO/TS TECHNICAL SPECIFICATION 12869 Second edition 2019-04 Water quality - Detection and quantification of Legionella spp. and/or Legionella pneumophila by concentration and genic amplification by quantitative polymerase chain reaction (qPCR) Qualité de I'eau - Détection et quantification de Legionella spp. et/ou Legionella pneumophila par concentration et amplification genique par réaction de polymérisation en chaine quantitative (qPCR) Reference number IS0/TS 12869:2019(E) os1 @ IS0 2019 IS0/TS 12869:2019(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/TS 12869:2019(E) Contents Page Foreword ..V Introduction.. ...vi 1 Scope. ...1 2 Normative references 3 Terms, definitions, symbols and abbreviated terms. 3.1 Terms and definitions. ..1 3.2 Symbols and abbreviated terms. 4 Principle. ..4 5 Sampling. ..4 6 General testing conditions .5 6.1 General. .5 6.2 Staff. .5 6.3 Premises 6.4 Apparatus and consumables (excluding reagents) 6.4.1 Apparatus. .6 6.4.2 Consumables ..6 6.4.3 Concentration .6 6.4.4 Extraction and PCR (detection and quantification) ..6 6.5 Reagents. .7 6.5.1 General. .7 6.5.2 PCR reagents .7 6.5.3 Other reagents .7 6.6 Decontamination of equipment and premises 6.7 Treatment and elimination of waste ..8 7 Procedure. ..8 7.1 Concentration. 7.2 DNA extraction. 8 7.2.1 General. ..8 7.2.2 Protocols .8 7.2.3 Stability of DNA extracts ..9 7.3 DNA amplification by PCR .9 7.3.1 General. .9 7.3.2 Target sequences, primers and probes. .9 7.3.3 Amplification mix preparation .11 7.4 Quantitative detection ..12 7.4.1 General. ..12 7.4.2 PCR protocol ..13 7.5 Qualitative detection 14 8 Expression of the results. ..14 9 Technical protocol for the characterization and the validation of the method ..16 9.1 General. ..16 9.2 Inclusivity and exclusivity of probes and primers ..16 9.3 Verification of the calibration function of the quantitative PCR phase ..17 9.3.1 General. ..17 9.3.2 Calibration curve verification principle. ..17 9.3.3 Calibration curve evaluation protocol ..18 9.3.4 Analysis of the results .19 9.3.5 Use of the calibration curve 21 9.4 Verification of the PCR limit of quantification, LQqPCR .22 9.4.1 Principle .22 9.4.2 Experimental design. 22 @ IS0 2019 - All rights reserved ii