CEN ISO/TS 15216-1:2013

Microbiology of food and animal feed - Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR - Part 1: Method for quantification (ISO/TS 15216-1:2013, Corrected Version 2013-05-01)

ISO/TS 15216-1:2013 describes a method for quantification of levels of HAV and NoV genogroup I (GI) and II (GII) RNA, from test samples of foodstuffs or food surfaces. Following liberation of viruses from the test sample, viral RNA is then extracted by lysis with guanidine thiocyanate and adsorption on silica. Target sequences within the viral RNA are amplified and detected by real-time RT-PCR.
This approach is also relevant for detection of the target viruses on fomites, or of other human viruses in foodstuffs, on food surfaces or on fomites following appropriate validation and using target-specific primer and probe sets.

Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zur Bestimmung des Hepatitis A-Virus und Norovirus in Lebensmitteln mittels Real-time-RT-PCR - Teil 1: Verfahren zur Quantifizierung (ISO/TS 15216-1:2013)

In diesem Teil von ISO/TS 15216 wird ein Verfahren zur quantitativen Bestimmung des Gehalts an RNS von HAV und NoV der Genogruppen I (GI) und II (GII) in Untersuchungsproben von Lebensmitteln oder Lebensmitteloberflächen beschrieben. Nach der Freisetzung der Viren aus der Untersuchungsprobe wird die virale RNS durch Aufschluss des Viruskapsids mit Guanidinthiocyanat und Adsorption an Silica(-Partikel) extrahiert. Die Zielsequenzen innerhalb der viralen RNS werden amplifiziert und mittels Real-time-RT-PCR nachgewiesen.
Dieses Verfahren gilt nach einer entsprechenden Validierung und bei Anwendung zielmolekülspezifischer Primer- und Sondensätze auch für den Nachweis der Zielviren auf Virusträgern sowie für den Nachweis von anderen humanen Viren in Lebensmitteln, auf Lebensmitteloberflächen oder Virusträgern.

Microbiologie des aliments - Méthode horizontale pour la recherche des virus de l'hépatite A et norovirus dans les aliments par la technique RT-PCR en temps réel - Partie 1: Méthode de quantification (ISO/TS 15216-1:2013, Version Corrigée 2013-05-01)

L'ISO/TS 15216-1:2013 décrit une méthode de quantification des niveaux de VHA et NoV des génogroupes I (GI) et II (GII) présents dans des échantillons pour essai d'aliments ou sur des surfaces alimentaires. Après libération des virus contenus dans l'échantillon pour essai, l'ARN viral est extrait par lyse à l'aide de thiocyanate de guanidine et par adsorption sur silice. Les séquences cibles de l'ARN viral sont amplifiées et détectées par la technique RT-PCR en temps réel.
Cette approche est aussi applicable pour la détection desdits virus sur des matières contaminées, ou d'autres virus humains dans les aliments, sur les surfaces alimentaires ou sur les matières contaminées en suivant une validation appropriée et en utilisant des jeux d'amorces et de sondes propres à la cible.

Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje virusa hepatitisa A in norovirusov v živilih z RT-PCR v realnem času - Kvantitativna metoda (ISO/TS 15216-1:2013, popravljena verzija 2013-05-01)

Ta del ISO/TS 15216 opisuje kvantitativno metodo za določevanje ravni genske skupine virusa hepatitisa A in norovirusa I (GI) in II (GII) RNK v vzorcih ali živilih oz. na površinah živil. Po sprostitvi virusov iz vzorca se nato virusni RNK odstrani z lizo z gvanidinijevim tiocianatom in adsorpcijo na silicijev oksid. Ciljna zaporedja v virusni RNK poudari in zazna RT-PCR v realnem času. Ta pristop je pomemben tudi za odkrivanje ciljnih virusov na predmetih oz. drugih človeških virusov v živilih, na površini živil ali na predmetih, in sicer po ustrezni validaciji ter uporabi ciljno specifičnega primerja in kompletov sond.

General Information

• Status: Withdrawn
• Publication Date: 07-May-2013
• Withdrawal Date: 28-Mar-2017
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 275 - Food analysis - Horizontal methods
• Drafting Committee: CEN/TC 275/WG 6 - Microbiological contamination
• Current Stage: 9960 - Withdrawal effective - Withdrawal
• Completion Date: 29-Mar-2017
• Ref Project: SIST-TS CEN ISO/TS 15216-1:2013 - Microbiology of food and animal feed - Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR - Part 1: Method for quantification (ISO/TS 15216-1:2013, Corrected Version 2013-05-01)

Relations

• Replaced By: EN ISO 15216-1:2017 - 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 (ISO 15216-1:2017)
• Effective Date: 15-Jan-2014

Standards Content (Sample)

SLOVENSKI STANDARD
01-september-2013
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Microbiology of food and animal feed - Horizontal method for determination of hepatitis A
virus and norovirus in food using real-time RT-PCR - Part 1: Method for quantification
(ISO/TS 15216-1:2013, Corrected Version 2013-05-01)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zur
Bestimmung des Hepatitis A-Virus und Norovirus in Lebensmitteln mittels Real-time-RT-
PCR - Teil 1: Verfahren zur Quantifizierung (ISO/TS 15216-1:2013)
Microbiologie des aliments - Méthode horizontale pour la recherche des virus de
l'hépatite A et norovirus dans les aliments par la technique RT-PCR en temps réel -
Partie 1: Méthode de quantification (ISO/TS 15216-1:2013, Version Corrigée 2013-05-
01)
Ta slovenski standard je istoveten z: CEN ISO/TS 15216-1:2013
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

TECHNICAL SPECIFICATION
CEN ISO/TS 15216-1
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
May 2013
ICS 07.100.30
English Version
Microbiology of food and animal feed - Horizontal method for
determination of hepatitis A virus and norovirus in food using
real-time RT-PCR - Part 1: Method for quantification (ISO/TS
15216-1:2013, Corrected Version 2013-05-01)
Microbiologie des aliments - Méthode horizontale pour la Mikrobiologie von Lebensmitteln und Futtermitteln -
recherche des virus de l'hépatite A et norovirus dans les Horizontales Verfahren zum Nachweis von Hepatitis A-
aliments par la technique RT-PCR en temps réel - Partie 1: Viren und Noroviren in Lebensmitteln mittels Real time
Méthode de quantification (ISO/TS 15216-1:2013, Version PCR - Teil 1: Verfahren zur quantitativen Bestimmung
Corrigée 2013-05-01) (ISO/TS 15216-1:2013, korrigierten Fassung von 2013-05-
01)
This Technical Specification (CEN/TS) was approved by CEN on 8 March 2013 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2013 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 15216-1:2013: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
This document (CEN ISO/TS 15216-1:2013) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the
secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus,
Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany,
Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
Endorsement notice
The text of ISO/TS 15216-1:2013, Corrected Version 2013-05-01 has been approved by CEN as CEN ISO/TS
15216-1:2013 without any modification.

TECHNICAL ISO/TS
SPECIFICATION 15216-1
First edition
2013-03-15
Corrected version
2013-05-01
Microbiology of food and animal feed —
Horizontal method for determination
of hepatitis A virus and norovirus in
food using real-time RT-PCR —
Part 1:
Method for quantification
Microbiologie des aliments — Méthode horizontale pour la recherche
des virus de l’hépatite A et norovirus dans les aliments par la
technique RT-PCR en temps réel —
Partie 1: Méthode de quantification
Reference number
ISO/TS 15216-1:2013(E)
©
ISO 2013
ISO/TS 15216-1:2013(E)
© ISO 2013
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
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Tel. + 41 22 749 01 11
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Published in Switzerland
ii © ISO 2013 – All rights reserved

ISO/TS 15216-1:2013(E)
Contents Page
Foreword .iv
Introduction .vii
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 3
4.1 Virus extraction . 3
4.2 RNA extraction . 3
4.3 Real-time reverse transcription polymerase chain reaction (real time RT-PCR) . 4
4.4 Control materials . 4
4.5 Test results. 5
5 Reagents . 5
5.1 General . 5
5.2 Reagents used as supplied . 5
5.3 Prepared reagents . 6
6 Apparatus and materials. 7
7 Sampling . 8
8 Procedure. 8
8.1 General laboratory requirements . 8
8.2 Virus extraction . 9
8.3 RNA extraction .11
8.4 Real-time RT-PCR .11
9 Interpretation of results .13
9.1 General .13
9.2 Construction of standard curves .13
9.3 Calculation of amplification efficiency .13
9.4 Calculation of extraction efficiency .14
9.5 Sample quantification .14
9.6 Theoretical limit of detection .15
10 Expression of results .15
11 Test report .15
Annex A (normative) Diagram of procedure .17
Annex B (informative) Real-time RT-PCR mastermixes and cycling parameters .18
Annex C (informative) Real-time RT-PCR primers and hydrolysis probes for the detection of HAV,
norovirus GI and GII and mengo virus (process control) .19
Annex D (informative) Growth of mengo virus strain MC for use as a process control .21 ®
Annex E (informative) RNA extraction using the BioMerieux NucliSens system .22
Annex F (normative) Composition and preparation of reagents and buffers .24
Annex G (informative) Generation of double-stranded DNA (dsDNA) control stocks .26
Annex H (informative) Generation of external control RNA (EC RNA) stocks .28
Annex I (informative) Typical optical plate layout.30
Bibliography .31
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