ISO/TS TECHNICAL SPECIFICATION 18867 Firstedition 2015-09-15 Microbiology of the food chain Polymerase chain reaction (PCR) for the detection of food-borne pathogens Detection of pathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis Microbiologie de la chaine alimentaire - Réaction de polymérisation en chaine (PCR) pour la détection de micro-organismes pathogenes dans les aliments - Détection des Yersinia enterocolitica et Yersinia pseudotuberculosis pathogenes Reference number IS0/TS 18867:2015(E) SO International Organization for Standardization @ IS0 2015 lardization5956617 ed without license from IHS IS0/TS 18867:2015(E) COPYRIGHTPROTECTEDDOCUMENT IS0 2015, Published in Switzerland 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 PrganizationforStandardization etworking permitted without license from IHS IS0/TS 18867:2015(E) Contents Page Foreword ..V Introduction. ...vi 1 Scope. 2 Normative references 3 Terms and definitions 4 Principles ..1 4.1 General .1 4.2 Microbial enrichment. .2 4.3 Nucleic acid extraction. .2 4.4 Amplification and detection 4.5 Isolation 2 Reagents. .2 5 5.1 General .2 5.2 Culture media. 2 5.2.1 General. .2 5.2.2 Diluent .2 5.2.3 Enrichment media .2 5.2.4 Selective solid medium .4 5.2.5 Potassium hydroxide in saline solution, KOH .5 5.3 Nucleic acid extraction. .5 5.4 Reagents for PCR .5 5.5 Primers and probes. .5 Apparatus and equipment ..5 6.1 General ..5 6.2 Equipment for sample preparation priorto enrichment 6.3 Equipment for microbial enrichment. ..6 6.4 Equipment for nucleic acid extraction. ..6 6.5 Equipment for real-time PCR . 6 7 Sampling. ..6 8 Procedure .6 8.1 Sample preparation prior to enrichment. .6 8.1.1 General. ..6 8.1.2 Preparation of the sample ..6 8.2 Microbial enrichment. .7 8.2.1 Pathogenic Y. enterocolitica .7 8.2.2 Y. pseudotuberculosis. 8.2.3 Pathogenic Y. enterocolitica and Y.pseudotuberculosis ..7 8.3 Isolation of colonies, optional. 8.3.1 Pathogenic Y. enterocolitica .7 8.3.2 Y. pseudotuberculosis 8.3.3 Process controls .8 8.4 Nucleic acid extraction .8 8.5 PCR amplification .8 8.5.1 General .8 8.5.2 PCR controls. 8.6 Confirmation of the PCR product ..8 8.6.1 General. 8.6.2 Interpretation of the PCR result ..8 9 Test report .9 Annex A (normative) PCR detection and isolation of pathogenic Y. enterocolitica (see iii icenseeZhejang Institute of Standardization 5956617 ed without license from IHS