ISO/TS TECHNICAL SPECIFICATION 17919 First edition 2013-11-01 Microbiology of the food chain Polymerase chain reaction (PCR) for the detection of food-borne pathogens Detection of botulinum type A, B, E and F neurotoxin-producing clostridia Microbiologie de la chaine alimentaire - Reaction de polymérisation en chaine (PCR) pour la détection de micro-organismes pathogenes dans les aliments - Détection des clostridies productrices de neurotoxine botulique de type A, B, E et F Reference number ISO/TS 17919:2013(E) ISO IS02013 Not for Resale IS0/TS 17919:2013(E) COPYRIGHTPROTECTEDDOCUMENT @IS02013 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. ISO copyright office Case postale 56 : CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail copyright@iso.org Web www.iso.org Published in Switzerland @ IS0 2013 - All rights reserved Not for Resale IS0/TS 17919:2013(E) Contents Page Foreword ..iv Introduction. .. 1 Scope. ..1 2 Normative references 3 Terms and definitions 4 Symbols and abbreviated terms ..1 4.1 Symbols .1 4.2 Abbreviated terms .2 5 Principle .2 5.1 General .2 5.2 Microbial enrichment. .2 5.3 Nucleic acid extraction. .2 5.4 Amplification by PCR 2 5.5 Detection of PCR products 2 5.6 Confirmation .2 6 Reagents .3 6.1 General. .3 6.2 Culture media. .3 6.3 Nucleic acid extraction 4 6.4 Reagents for PCR .5 7 Apparatus and equipment. .5 7.1 General. .5 7.2 Equipment for sample preparation prior to enrichment 5 7.3 Equipment for microbial enrichment. 5 7.4 Equipment used for nucleic acid extraction, .6 7.5 EquipmentusedforPCR ..6 7.6 Equipment used for the detection of the PCR product .6 8 Sampling. ..7 9 Procedure .7 9.1 Sample preparation prior to enrichment 9.2 Microbial enrichment 7 9.3 Nucleic acid preparation ..8 9.4 PCR amplification 9 9.5 Confirmation of a positive PCR result 9 Annex A (normative) Flow-diagram of the procedure ..10 Annex B (informative) Multiplex PCR assays for detection of genes encoding botulinum neurotoxin types A, B, E, and F using agarose gel electrophoresis 11 Annex C (informative) Assays for detection of genes encoding botulinum neurotoxin types A, B, E, and F using real-time PCR. .29 Annex D (informative) Preparation of C. botulinum spores. ..40 Bibliography ..46 ii se with ISt ed without license from IHS Not for Resale