ISO INTERNATIONAL STANDARD 15216-1 First edition 2017-03 Microbiology of the food chain Horizontal method for determination of hepatitis A virus and norovirus using real-time RT-PCR - Part 1: Method for quantification Microbiologie dans la chaine alimentaire - Methode horizontale pour la recherche des virus de I'hépatite A et norovirus par la technique RT-PCR en temps reel - Partie 1: Méthode de quantification Reference number ISO 15216-1:2017(E) rso International Organization for Standardization @ IS0 2017 HEJIANG INST OF STANDARDIZATION C15956617 ed without license from IHS IS0 15216-1:2017(E) COPYRIGHTPROTECTEDDOCUMENT IS02017,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. ISO copyright office Ch. de Blandonnet 8 . CP 401 CH-1214 Vernier, Geneva, Switzerland Tel. +41 22 749 01 11 Fax+412274909 47 [email protected] www.iso.org Intenationai br PrganizationforStandardization icensee-ZHEJIANG INST OF STANDARDIZRoISQ s1.7 - All rights reserved I by IHS unde etworking permitted without license from IHS IS0 15216-1:2017(E) Contents Page Foreword ..V Introduction. ...vi 1 Scope. 2 Normative references 3 Terms and definitions 4 Principle .3 4.1 Virus extraction .3 4.2 RNA extraction. ..4 4.3 Real-time RT-PCR .4 4.4 Control materials 4.4.1 Process control virus. 4 4.4.2 Double-stranded DNA (dsDNA) control ..4 4.4.3 EC RNA control. 4 4.5 Test results. ..5 5 Reagents ..5 5.1 General. 5.2 Reagents used as supplied ..5 5.3 Prepared reagents. .6 6 Equipment and consumables .1 7 Sampling. ..9 8 Procedure. .9 8.1 General laboratory requirements .9 8.2 Virus extraction .9 8.2.1 Process control virus material .9 8.2.2 Negative process control. .9 8.2.3 Food surfaces .9 8.2.4 Soft fruit, leaf, stem and bulb vegetables 8.2.5 Bottledwater .10 8.2.6 Bivalve molluscan shellfish 11 8.3 RNA extraction .11 8.4 Real-time RT-PCR .12 8.4.1 Generalrequirements ..12 8.4.2 Real-time RT-PCR analysis ..12 Interpretation of results. ..14 9 9.1 General .14 9.2 Construction of standard curves ..14 9.3 Calculation of RT-PCR inhibition .15 9.4 Calculation of extraction efficiency .15 9.5 Sample quantification. ..16 10 Expression of results ..17 11 Precision .17 11.1 Interlaboratory study .17 11.2 Repeatability .17 11.3 Reproducibility limit. ..18 12 Test report .18 Annex A (normative) Diagram of procedure ..19 Annex B (normative) Composition and preparation of reagents and buffers .20 Annex C (informative) Real-time RT-PCR mastermixes and cycling parameters .23 iii icenseeZHEJIANG INST OF STANDARDIZATION C1 5956617 Not for Resale, 2017/5/11 02:03:43 ed without license from IHS