ISO/TS TECHNICAL SPECIFICATION 13136 First edition 2012-11-15 Microbiology of food and animal feed Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens Horizontal method for the detection of Shiga toxin- producing Escherichia coli (STEC) and the determination of 0157, O0111, 026, O103 and 0145 serogroups Microbiologie des aliments -Methode basée sur la reaction de polymerisation en chaine (PCR) en temps réel pour la détection des micro-organismes pathogenes dans les aliments Méthode horizontale pour la détection des Escherichia coli producteurs de Shigatoxines (STEC) et la détermination des serogroupes O157, O111, O26, O103 et 0145 Reference number ISO/TS 13136:2012(E) TSO @ ISO 2012 nsefromIHS Not for Resale ISO/TS13136:2012(E) COPYRIGHTPROTECTEDDOCUMENT ISO2012 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO's member body in the country of the requester. ISO copyright office Casepostale56·CH-1211Geneva 20 Tel. + 41 22 749 01 11 Fax + 4122 749 09 47 E-mail [email protected] Web www.iso.org Published in Switzerland @ ISO 2012 -All rights reserved No reproductic etworking permitted without license from IHS Not for Resale ISO/TS13136:2012(E) Contents Page Foreword ..iv Introduction 1 Scope 2 Normative references 3 Terms and definitions 2 4 Principle 2 4.1 General 2 4.2 Microbial enrichment 4.3 Nucleic acid extraction. 4.4 Targetgenes 3 4.5 Detection 4.6 Isolation 3 5 Diluents, culture media and reagents 3 5.1 Culture media .. 5.2 Reagents for nucleic acid extraction 5 5.3 Reagents for PCR. 6 Equipment 5 7 Sampling. 6 8 Preparation of test sample.. 6 9 Procedure... 6 9.1 Testportionand initial suspension. 6 9.2 Enrichment.... 9.3 Nucleic acid extraction .. 9.4 PCR amplification (forreal-timePCR) 9.5 Strain isolation. 8 10 Expression of results 8 11 Performance data. .8 Annex A (normative) Flow diagram of the screening procedure. 12 Annex B (normative) Flow diagram of the isolation and confirmation procedure . .13 Annex C (informative) Identification of Shiga toxin-producing Escherichia coli (STEC) by multiplex PcR amplification of virulence genes and detection of PcR products by agarose gel electrophoresis .. 14 Annex D (informative) Internal amplification control. ..18 Annex E (informative) Primers and probes for the PcR assays. .19 AnnexF(normative)Isolation of STEC strains 21 Bibliography .22 ili Not for Resale