SIST EN ISO 15216-1:2017
(Main)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)
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)
This document specifies a method for the quantification of levels of HAV and norovirus genogroup I
(GI) and II (GII) RNA, from test samples of foodstuffs (soft fruit, leaf, stem and bulb vegetables, bottled
water, BMS) 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 method is not validated for detection of the target viruses in other foodstuffs (including multicomponent
foodstuffs), or any other matrices, nor for the detection of other viruses in foodstuffs, food
surfaces or other matrices.
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zur Bestimmung von Hepatitis A-Virus und Norovirus mittels Real-time-RT-PCR - Teil 1: Verfahren zur Quantifizierung (ISO 15216-1:2017)
In diesem Teil von ISO 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 (Weichobst, Salat, in Flaschen abgefülltes Trinkwasser, zweischalige Weichtiere (Muscheln)) 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 ist weder für den Nachweis der Zielviren in anderen Lebensmitteln (einschließlich zusammengesetzter Lebensmittel) oder anderen Matrices noch für den Nachweis anderer Viren in Lebensmitteln, auf Lebensmitteloberflächen oder anderen Matrices validiert.
Microbiologie dans la chaine alimentaire - Méthode horizontale pour la recherche des virus de l'hépatite A et norovirus par la technique RT-PCR en temps réel - Partie 1: Méthode de quantification (ISO 15216-1:2017)
ISO 15216-1:2017 décrit une méthode de quantification des niveaux d'ARN de VHA et norovirus des génogroupes I (GI) et II (GII) présents dans des échantillons pour essai d'aliments (fruits tendres, légumes feuilles, tiges et bulbes, eau embouteillée, MBV) 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 méthode n'est pas validée pour la détection des virus ciblés dans d'autres aliments (y compris les aliments à plusieurs composants) ou d'autres matrices, ni pour la détection d'autres virus dans les aliments, sur les surfaces alimentaires ou dans d'autres matrices.
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje virusa hepatitisa A in norovirusov z RT-PCR v realnem času - 1. del: Metoda za kvantifikacijo (ISO 15216-1:2017)
Ta dokument določa kvantitativno metodo za določevanje ravni genske skupine virusa hepatitisa A in norovirusa I (GI) in II (GII) RNK v vzorcih ali živilih (mehkem sadju, listnati in stebelni zelenjavi, čebulnicah, ustekleničeni vodi, BMS) 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 metoda ni potrjena za odkrivanje ciljnih virusov v drugih živilih (vključno z večkomponentnimi živili) ali drugih matricah ali odkrivanje drugih virusov v živilih, na površinah živil ali drugih matricah.
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 15216-1:2017
01-junij-2017
1DGRPHãþD
SIST-TS CEN ISO/TS 15216-1:2013
0LNURELRORJLMDYSUHKUDQVNLYHULJL+RUL]RQWDOQDPHWRGD]DXJRWDYOMDQMHYLUXVD
KHSDWLWLVD$LQQRURYLUXVRY]573&5YUHDOQHPþDVXGHO 0HWRGD]D
NYDQWLILNDFLMR ,62
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)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zur Bestimmung von
Hepatitis A-Virus und Norovirus mittels Real-time-RT-PCR - Teil 1: Verfahren zur
Quantifizierung (ISO 15216-1:2017)
Microbiologie dans la chaine alimentaire - Méthode horizontale pour la recherche des
virus de l'hépatite A et norovirus par la technique RT-PCR en temps réel - Partie 1:
Méthode de quantification (ISO 15216-1:2017)
Ta slovenski standard je istoveten z: EN ISO 15216-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 15216-1:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 15216-1:2017
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SIST EN ISO 15216-1:2017
EN ISO 15216-1
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2017
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes CEN ISO/TS 15216-1:2013
English Version
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)
Microbiologie dans la chaine alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales
horizontale pour la recherche des virus de l'hépatite A Verfahren zur Bestimmung von Hepatitis A-Virus und
et norovirus par la technique RT-PCR en temps réel - Norovirus mittels Real-time-RT-PCR - Teil 1: Verfahren
Partie 1: Méthode de quantification (ISO 15216- zur Quantifizierung (ISO 15216-1:2017)
1:2017)
This European Standard was approved by CEN on 23 February 2017.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 15216-1:2017 E
worldwide for CEN national Members.
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SIST EN ISO 15216-1:2017
EN ISO 15216-1:2017 (E)
Contents Page
European foreword . 3
2
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SIST EN ISO 15216-1:2017
EN ISO 15216-1:2017 (E)
European foreword
This document (EN ISO 15216-1:2017) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN, in collaboration with Technical
Committee ISO/TC 34 "Food products".
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by September 2017 and conflicting national standards
shall be withdrawn at the latest by September 2017.
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.
This document supersedes CEN ISO/TS 15216-1:2013.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the
following countries are bound to implement this European Standard: 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, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
the United Kingdom.
Endorsement notice
The text of ISO 15216-1:2017 has been approved by CEN as EN ISO 15216-1:2017 without any
modification.
3
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SIST EN ISO 15216-1:2017
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SIST EN ISO 15216-1:2017
INTERNATIONAL ISO
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 chaîne alimentaire — Méthode horizontale
pour la recherche des virus de l’hépatite A et norovirus par la
technique RT-PCR en temps réel —
Partie 1: Méthode de quantification
Reference number
ISO 15216-1:2017(E)
©
ISO 2017
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, 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.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
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
4.4.1 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
5.2 Reagents used as supplied . 5
5.3 Prepared reagents . 6
6 Equipment and consumables . 7
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 . 9
8.2.5 Bottled water .10
8.2.6 Bivalve molluscan shellfish .11
8.3 RNA extraction .11
8.4 Real-time RT-PCR .12
8.4.1 General requirements .12
8.4.2 Real-time RT-PCR analysis .12
9 Interpretation of results .14
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
© ISO 2017 – All rights reserved iii
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
Annex D (informative) Real-time RT-PCR primers and hydrolysis probes for the detection of
HAV, norovirus GI and GII and mengo virus (process control) .24
Annex E (informative) Growth of mengo virus strain MC for use as a process control .27
0
®
Annex F (informative) RNA extraction using the NucliSENS system .28
Annex G (informative) Generation of dsDNA control stocks .30
Annex H (informative) Generation of EC RNA stocks .33
Annex I (informative) Typical optical plate layout.35
Annex J (informative) Method validation studies and performance characteristics .37
Bibliography .48
iv © ISO 2017 – All rights reserved
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: w w w . i s o .org/ iso/ foreword .html.
This document was prepared by the European Committee for Standardization (CEN) Technical
Committee CEN/TC 275, in collaboration with ISO Technical Committee ISO/TC 34, Food products,
Subcommittee SC 9, Microbiology, in accordance with the agreement on technical cooperation between
ISO and CEN (Vienna Agreement).
This first edition cancels and replaces ISO/TS 15216-1:2013, which has been technically revised with
the following changes:
— use of linear dsDNA molecules for quantification prescribed;
— use of a suitable buffer for dilution of control materials prescribed;
— change to the method for generating process control virus RNA for the standard curve;
— addition of breakpoints with defined temperature and time parameters in the extraction methods;
— change in terminology from amplification efficiency to RT-PCR inhibition;
— addition of extra real-time RT-PCR reactions for negative controls;
— addition of precision data and results of interlaboratory study.
A list of all parts in the ISO 15216 series can be found on the ISO website.
© ISO 2017 – All rights reserved v
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
Introduction
Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness. No
routine methods exist for culture of norovirus, and HAV culture methods are not appropriate for routine
application to food matrices. Detection is therefore reliant on molecular methods using the reverse-
transcriptase polymerase chain reaction (RT-PCR). As many food matrices contain substances that
are inhibitory to RT-PCR, it is necessary to use an extraction method that produces highly clean RNA
preparations that are fit for purpose. For food surfaces, viruses are removed by swabbing. For soft fruit,
leaf, stem and bulb vegetables, virus extraction is by elution with agitation followed by precipitation
with PEG/NaCl. For bottled water, adsorption and elution using positively charged membranes followed
by concentration by ultrafiltration is used and for bivalve molluscan shellfish (BMS), viruses are
extracted from the tissues of the digestive glands using treatment with a proteinase K solution. For all
matrices that are not covered by this document, it is necessary to validate this method. All matrices
share a common RNA extraction method based on virus capsid disruption with chaotropic reagents
followed by adsorption of RNA to silica particles. Real-time RT-PCR monitors amplification throughout
the real-time RT-PCR cycle by measuring the excitation of fluorescently labelled molecules. In real-
time RT-PCR with hydrolysis probes, the fluorescent label is attached to a sequence-specific nucleotide
probe that also enables simultaneous confirmation of target template. These modifications increase
the sensitivity and specificity of the real-time RT-PCR method, and obviate the need for additional
amplification product confirmation steps post real-time RT-PCR. Due to the complexity of the method,
it is necessary to include a comprehensive suite of controls. The method described in this document
enables quantification of levels of virus RNA in the test sample. A schematic diagram of the testing
procedure is shown in Annex A.
The main changes, listed in the Foreword, introduced in this document compared to ISO/TS 15216-
1:2013 are considered as minor (see ISO 17468).
vi © ISO 2017 – All rights reserved
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SIST EN ISO 15216-1:2017
INTERNATIONAL STANDARD ISO 15216-1:2017(E)
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
1 Scope
This document specifies a method for the quantification of levels of HAV and norovirus genogroup I
(GI) and II (GII) RNA, from test samples of foodstuffs (soft fruit, leaf, stem and bulb vegetables, bottled
water, BMS) 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 method is not validated for detection of the target viruses in other foodstuffs (including multi-
component foodstuffs), or any other matrices, nor for the detection of other viruses in foodstuffs, food
surfaces or other matrices.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 20838, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — Requirements for amplification and detection for qualitative methods
ISO 22119, Microbiology of food and animal feeding stuffs — Real-time polymerase chain reaction (PCR) for
the detection of food-borne pathogens — General requirements and definitions
ISO 22174, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — General requirements and definitions
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 22174, ISO 22119 and
ISO 20838 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
foodstuff
substance used or prepared for use as food
Note 1 to entry: For the purposes of this document, this definition includes bottled water.
© ISO 2017 – All rights reserved 1
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
3.2
food surface
surface of food, food preparation surface or food contact surface
3.3
soft fruit
small edible stoneless fruit
EXAMPLE Strawberries, raspberries or currants
3.4
leaf, stem and bulb vegetables
leaves, stems and bulbs of plants, eaten as a vegetable
3.5
hepatitis A virus
HAV
member of the Picornaviridae family responsible for infectious hepatitis
Note 1 to entry: Genetically, HAV can be subdivided into six genotypes on the basis of the VP1/2A region
(genotypes 1, 2, and 3 have been found in humans, while genotypes 4, 5, and 6 are of simian origin). There is only
one serotype.
Note 2 to entry: Transmission occurs via the faecal-oral route by person-to-person contact, through the
consumption of contaminated foodstuffs, contact with contaminated water or food surfaces, or contact with
contaminated fomites. HAV is classified as a group 2 biological agent by the European Union and as a risk group 2
human aetiological agent by the United States National Institutes of Health.
3.6
norovirus
member of the Caliciviridae family responsible for sporadic cases and outbreaks of acute gastroenteritis
Note 1 to entry: Genetically, norovirus can be subdivided into seven separate genogroups. Three of these
genogroups, GI, GII and GIV have been implicated in human gastrointestinal disease. GI and GII are responsible
for the vast majority of clinical cases.
Note 2 to entry: Transmission occurs via the faecal-oral route by person-to-person contact, through the
consumption of contaminated foodstuffs or through contact with contaminated water or food surfaces or contact
with contaminated fomites. GI and GII noroviruses are classified as group 2 biological agents by the European
Union and as risk group 2 human aetiological agents by the United States National Institutes of Health.
3.7
quantification of HAV
estimation of number of copies of HAV RNA in a predetermined mass or volume of foodstuff, or area of
food surface
3.8
quantification of norovirus
estimation of number of copies of norovirus RNA in a predetermined mass or volume of foodstuff, or
area of food surface
3.9
process control virus
virus added to the sample portion at the earliest opportunity prior to virus extraction to control for
extraction efficiency
3.10
process control virus RNA
RNA extracted from the process control virus in order to produce standard curve data for the estimation
of extraction efficiency
2 © ISO 2017 – All rights reserved
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
3.11
negative RNA extraction control
control free of target RNA carried through all steps of the RNA extraction and detection procedure to
monitor any contamination events
3.12
negative process control
target pathogen-free sample of the food matrix, or target pathogen-free non-matrix sample, that is run
through all stages of the analytical process
3.13
hydrolysis probe
fluorescent probe coupled with a fluorescent reporter molecule and a quencher molecule, which are
sterically separated by the 5′-3′-exonuclease activity of the enzyme during the amplification process
3.14
negative real-time RT-PCR control
aliquot of highly pure water used in a real-time RT-PCR reaction to control for contamination in the
real-time RT-PCR reagents
3.15
external control RNA
EC RNA
reference RNA that can be used to assess inhibition of amplification for the real-time RT-PCR assay of
relevance by being added in a defined amount to an aliquot of sample RNA in a separate reaction
EXAMPLE RNA synthesized by in-vitro transcription from a plasmid carrying a copy of the target gene
3.16
C value
q
quantification cycle; the cycle at which the target is quantified in a given real-time RT-PCR reaction
Note 1 to entry: This corresponds to the poin
...
SLOVENSKI STANDARD
oSIST prEN ISO 15216-1:2015
01-oktober-2015
0LNURELRORJLMDYSUHKUDQVNLYHULJL+RUL]RQWDOQDPHWRGD]DXJRWDYOMDQMHYLUXVD
KHSDWLWLVD$LQQRURYLUXVRYYåLYLOLK]573&5YUHDOQHPþDVXGHO 0HWRGD]D
NYDQWLILNDFLMR ,62',6
Microbiology of the food chain - Horizontal method for determination of hepatitis A virus
and norovirus in food using real-time RT-PCR - Part 1: Method for quantification
(ISO/DIS 15216-1:2015)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zur bestimmung von
Hepatitis A-Virus und Norovirus in Lebensmitteln mittels Real-time-RT-PCR - Teil 1:
Verfahren zur Quantifizierung (ISO/DIS 15216-1:2015)
Microbiologie dans la chaine alimentaire - 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/DIS 15216-1:2015)
Ta slovenski standard je istoveten z: prEN ISO 15216-1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 15216-1:2015 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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oSIST prEN ISO 15216-1:2015
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oSIST prEN ISO 15216-1:2015
DRAFT INTERNATIONAL STANDARD
ISO/DIS 15216-1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2015-08-06 2015-11-06
Microbiology of the food chain — Horizontal method for
determination of hepatitis A virus and norovirus in food
using real-time RT-PCR —
Part 1:
Method for quantification
Microbiologie dans la chaine alimentaire — 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
ICS: 07.100.30
ISO/CEN PARALLEL PROCESSING
This draft has been developed within the European Committee for Standardization
(CEN), and processed under the CEN lead mode of collaboration as defined in the
Vienna Agreement.
This draft is hereby submitted to the ISO member bodies and to the CEN member
bodies for a parallel five month enquiry.
Should this draft be accepted, a final draft, established on the basis of comments
received, will be submitted to a parallel two-month approval vote in ISO and
THIS DOCUMENT IS A DRAFT CIRCULATED
formal vote in CEN.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
To expedite distribution, this document is circulated as received from the
IN ADDITION TO THEIR EVALUATION AS
committee secretariat. ISO Central Secretariat work of editing and text
BEING ACCEPTABLE FOR INDUSTRIAL,
composition will be undertaken at publication stage.
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
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ISO/DIS 15216-1:2015(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2015
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oSIST prEN ISO 15216-1:2015
ISO/DIS 15216-1:2015(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 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.
ISO copyright office
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CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
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Contents Page
Foreword vi
Introduction vii
1 Scope .1
2 Normative references .1
3 Terms and definitions .1
4 Principle .3
5 Reagents .5
6 Apparatus and materials .7
7 Sampling .8
8 Procedure .9
9 Interpretation of results . 14
10 Expression of results . 16
11 Precision . 17
12 Test report . 18
Annex A (normative) Diagram of procedure . 19
Annex B (informative) Real-time RT-PCR mastermixes and cycling parameters . 20
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) . 21
Annex D (informative) Growth of mengo virus strain MC for use as a process control . 23
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Annex E (informative) RNA extraction using the BioMerieux NucliSens system . 24
Annex F (normative) Composition and preparation of reagents and buffers . 26
Annex G (informative) Generation of double-stranded DNA (dsDNA) control stocks . 29
Annex H (informative) Generation of external control RNA (EC RNA) stocks . 32
Annex I (informative) Typical optical plate layout . 34
Annex J (informative) Results of interlaboratory study . 35
Bibliography . 46
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Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national
standards bodies (ISO member bodies). The work of preparing International Standards is normally
carried out through ISO technical committees. Each member body interested in a subject for which a
technical committee has been established has the right to be represented on that committee.
International organizations, governmental and non-governmental, in liaison with ISO, also take part in
the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all
matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International
Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies
casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 15216-1 was prepared by the European Committee for Standardization (CEN), in collaboration with
Technical committee ISO/TC 34, Food products, Subcommittee SC 9 Microbiology in accordance with the
Agreement on technical cooperation between ISO and CEN (Vienna Agreement).
ISO 15216 consists of the following parts, under the general title Microbiology of the food chain —
Horizontal method for determination of hepatitis A virus and norovirus in food using real-time RT-PCR:
— Part 1: Method for quantification
— Part 2: Method for qualitative detection (Technical Specification)
This edition cancels and replaces ISO/TS 15216-1:2013, which has been technically revised.
Main changes in this edition:
Use of linear dsDNA molecules for quantification prescribed;
Use of a suitable buffer for dilution of control materials prescribed;
Change to the method for generating process control virus RNA for the standard curve;
Addition of breakpoints in the extraction methods;
Change in terminology from amplification efficiency to RT-PCR inhibition;
Addition of extra real-time RT-PCR reactions for negative controls;
Precision data and results of interlaboratory study.
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Introduction
Hepatitis A virus (HAV) and norovirus (NoV) are important agents of food-borne human viral illness. No
routine methods exist to culture these viruses from food matrices. Detection is therefore reliant on
molecular methods using the reverse-transcriptase polymerase chain reaction (RT-PCR). As many food
matrices contain substances that are inhibitory to RT-PCR, it is necessary to use an extraction method
that produces highly clean RNA preparations that are fit for purpose. For food surfaces, viruses are
removed by swabbing. For soft fruit and salad vegetables, virus extraction is by elution with agitation
followed by precipitation with PEG/NaCl. For bottled water, adsorption and elution using positively
charged membranes followed by concentration by ultrafiltration is used and for bivalve molluscan
shellfish, viruses are extracted from the tissues of the digestive glands using treatment with a
proteinase K solution. For all matrices which are not covered by this Technical Specification, it is
necessary to validate this method. All matrices share a common RNA extraction method based on virus
capsid disruption with chaotropic reagents followed by adsorption of RNA to silica particles. Real-time
RT-PCR monitors amplification throughout the real-time RT-PCR cycle by measuring the excitation of
fluorescently labelled molecules. In the 5’ fluorogenic nuclease real-time RT-PCR assay, the fluorescent
labels are attached to a sequence-specific nucleotide probe (hydrolysis probe) that also enables
simultaneous confirmation of target template. These modifications increase the sensitivity and
specificity of the real-time RT-PCR method, and obviate the need for additional amplification product
confirmation steps post real-time RT-PCR. Due to the complexity of the method, it is necessary to
include a comprehensive suite of controls. The method described in this part of ISO 15216 enables
quantification of levels of virus RNA in the test sample. A schematic diagram of the testing procedure is
shown in Annex A.
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Microbiology of the food chain — Horizontal method for
determination of hepatitis A virus and norovirus in food using
real-time RT-PCR — Part 1: Method for quantification
1 Scope
This part of ISO 15216 describes a method for quantification of levels of HAV and NoV genogroup I (GI)
and II (GII) RNA, from test samples of foodstuffs (soft fruit, salad vegetables, bottled water, bivalve
molluscan shellfish) 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 method is not validated for detection of the target viruses in other foodstuffs (including multi-
component foodstuffs), or any other matrices, nor for the detection of other viruses in foodstuffs, food
surfaces or other matrices.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated
references, only the edition cited applies. For undated references, the latest edition of the referenced
document (including any amendments) applies.
ISO 22174, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — General requirements and definitions
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 22174 and the following
apply.
3.1
foodstuff
substance used or prepared for use as food
NOTE For the purposes of this part of ISO 15216, this definition includes bottled water.
3.2
food surface
surface of food, food preparation surface or food contact surface
3.3
hepatitis A virus
HAV
member of the Picornaviridae family responsible for infectious hepatitis
NOTE 1 Genetically, HAV can be subdivided into six genotypes on the basis of the VP1/2A region (genotypes 1,
2, and 3 have been found in humans, while genotypes 4, 5, and 6 are of simian origin). There is only one serotype.
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NOTE 2 Transmission occurs via the faecal-oral route by person-to-person contact, through the consumption of
contaminated foodstuffs, contact with contaminated water or food surfaces, or contact with contaminated fomites.
Hepatitis A virus is classified as a group 2 biological agent by the European Union and as a risk group 2 human
aetiological agent by the United States National Institutes of Health.
3.4
norovirus
member of the Caliciviridae family responsible for sporadic cases and outbreaks of acute gastroenteritis
NOTE 1 Genetically, norovirus can be subdivided into seven separate genogroups.
NOTE 2 Three of these genogroups, GI, GII and GIV have been implicated in human gastrointestinal disease. GI
and GII are responsible for the vast majority of clinical cases. Transmission occurs via the faecal-oral route by
person-to-person contact, through the consumption of contaminated foodstuffs or through contact with
contaminated water or food surfaces or contact with contaminated fomites. Genogroup I and II noroviruses are
classified as group 2 biological agents by the European Union and as risk group 2 human aetiological agents by the
United States National Institutes of Health.
3.5
quantification of hepatitis A virus
estimation of number of copies of HAV RNA in a predetermined mass or volume of foodstuff, or area of
food surface
3.6
quantification of norovirus
estimation of number of copies of norovirus RNA in a predetermined mass or volume of foodstuff, or
area of food surface
3.7
process control virus
virus added to the sample portion at the earliest opportunity prior to virus extraction to control for
extraction efficiency
3.8
process control virus RNA
RNA extracted from the process control virus in order to produce standard curve data for the
estimation of extraction efficiency
3.9
negative RNA extraction control
control free of target RNA carried through all steps of the RNA extraction and detection procedure to
monitor any contamination events
3.10
negative process control
target pathogen-free sample of the food matrix which is run through all stages of the analytical process
3.11
hydrolysis probe
fluorescent probe coupled with two fluorescent molecules which are sterically separated by the 5′-3′-
exonuclease activity of the enzyme during the amplification process
3.12
negative real-time RT-PCR control
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aliquot of highly pure water used in a real-time RT-PCR reaction to control for contamination in the
real-time RT-PCR reagents
3.13
external control RNA
reference RNA that can serve as target for the real-time RT-PCR assay of relevance, e.g. RNA synthesized
by in-vitro transcription from a plasmid carrying a copy of the target gene, which is added to an aliquot
of sample RNA in a defined amount to serve as a control for amplification in a separate reaction
3.14
C value
q
quantification cycle; the cycle at which the target is quantified in a given real-time RT-PCR reaction
NOTE This corresponds to the point at which reaction fluorescence rises above a threshold level.
3.15
limit of detection
LOD
lowest concentration of target in a test sample that can be reproducibly detected (95 % confidence
interval) under the experimental conditions specified in the method, as demonstrated by a collaborative
trial or other validation
NOTE The LOD is related to the test portion and the quality of the template RNA.
3.16
limit of quantification
LOQ
lowest concentration of target in a test sample that can be quantitatively determined with acceptable
level of precision and accuracy under the experimental conditions specified in the method, as
demonstrated by a collaborative trial or other validation
NOTE The LOQ is related to the test portion and the quality of the template RNA.
4 Principle
4.1 Virus extraction
The foodstuffs and food surfaces covered by this part of ISO 15216 are often highly complex matrices
and the target viruses can be present at low concentrations. It is therefore necessary to carry out
matrix-specific virus extraction and/or concentration in order to provide a substrate for subsequent
common parts of the process. The choice of method depends upon the matrix.
4.2 RNA extraction
It is necessary to extract RNA using a method that yields clean RNA preparations to reduce the effect of
RT-PCR inhibitors. In this part of ISO 15216 the chaotropic agent guanidine thiocyanate is used to
disrupt the viral capsid. RNA is then adsorbed to silica to assist purification through several washing
stages. Purified viral RNA is released from the silica into a buffer prior to real-time RT-PCR.
4.3 Real-time reverse transcription polymerase chain reaction (real time RT-PCR)
This part of ISO 15216 uses one step real-time RT-PCR using hydrolysis probes. In one step real-time
RT-PCR, reverse transcription and PCR amplification are carried out consecutively in the same tube.
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Real-time RT-PCR using hydrolysis probes utilizes a short DNA probe with a fluorescent label and a
fluorescence quencher attached at opposite ends. The assay chemistry ensures that as the quantity of
amplified product increases, the probe is broken down and the fluorescent signal from the label
increases proportionately. Fluorescence can be measured at each stage throughout the cycle. The first
point in the real-time RT-PCR cycle at which amplification can be detected for any reaction is
proportional to the quantity of template, therefore analysis of the fluorescence plots enables
determination of the concentration of target sequence in the sample.
Due to the low levels of virus template often present in foodstuffs and the strain diversity in the target
viruses, selection of fit-for-purpose one step real-time RT-PCR reagents and PCR primers and hydrolysis
probes for the target viruses is important. Guidelines for their selection are given in 5.2.18 and 5.2.19.
Illustrative details of reagents, primers, and probes (used in the development of this part of ISO 15216)
are provided in Annexes B and C.
4.4 Control materials
4.4.1 Process control virus
Losses of target virus can occur at several stages during sample virus extraction and RNA extraction. To
control for these losses, samples are spiked prior to processing with a defined amount of a process
control virus. The level of recovery of the process control virus shall be determined for each sample.
The virus selected for use as a process control shall be a culturable non-enveloped positive-sense
ssRNA virus of a similar size to the target viruses to provide a good morphological and physicochemical
model. The process control virus shall exhibit similar persistence in the environment to the targets. The
virus shall be sufficiently distinct genetically from the target viruses that real-time RT-PCR assays for
the target and process control viruses do not cross-react, and shall not normally be expected to occur
naturally in the foodstuffs under test.
An example of the preparation of process control virus (used in the development of this part of
ISO 15216) is provided in Annex D.
4.4.2 Double-stranded DNA (dsDNA) control
For quantification of a target virus, results shall be related to a standard of known concentration. A
dilution series of linear double-stranded DNA carrying the target sequence of interest (5.3.11) and
quantified using an appropriate method, e.g. spectrophotometry, fluorimetry, digital PCR etc. shall be
used to produce a standard curve in template copies per microlitre. Reference to the standard curve
enables quantification of the sample in detectable virus genome copies per microlitre.
4.4.3 External amplification control (EC) RNA control
Many foodstuffs contain substances inhibitory to RT-PCR, and there is also a possibility of carryover of
further inhibitory substances from upstream processing. In order to control for RT-PCR inhibition in
individual samples, external control (EC) RNA (an RNA species carrying the target sequence of interest,
5.3.12) is added to an aliquot of sample RNA and tested using the real-time RT-PCR method.
Comparison of the results of this with the results of EC RNA in the absence of sample RNA enables
determination of the level of RT-PCR inhibition in each sample under test.
Alternative approaches for RT-PCR inhibition control that provide equivalent stringency are permitted.
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4.5 Test results
This method provides a result expressed in detectable virus genome copies per millilitre, per gram or
per square centimetre. In samples where virus is not detected, results shall be reported as “not
detected;
is the limit of detection (LOD) for the sample.
5 Reagents
5.1 General
Use only reagents of recognized analytical grade, unless otherwise specified.
[1]
For current laboratory practice, see ISO 7218.
5.2 Reagents used as supplied
5.2.1 Molecular biology grade water.
5.2.2 Polyethylene glycol (PEG), mean relative molecular mass 8 000.
5.2.3 Sodium chloride (NaCl).
5.2.4 Potassium chloride (KCl).
5.2.5 Disodium hydrogenphosphate (Na HPO ).
2 4
5.2.6 Potassium dihydrogenphosphate (KH PO ).
2 4
5.2.7 Tris base.
5.2.8 Glycine.
5.2.9 Beef extract powder.
5.2.10 Proteinase K (30 U/mg).
5.2.11 Pectinase from Aspergillus niger or A. aculeatus.
5.2.12 Chloroform.
5.2.13 Butanol.
5.2.14 Sodium hydroxide (NaOH).
5.2.15 Hydrochloric acid (HCl).
5.2.16 Ethylenediaminetetraacetic acid (EDTA).
5.2.17 Silica, lysis, wash, and elution buffers for extraction of viral RNA. Reagents shall enable
processing of 500 μl of extracted virus, using lysis with a chaotropic buffer containing guanidine
thiocyanate (Reference [4]) and using silica as the RNA-binding matrix. Following treatment of silica-
bound RNA with wash buffer(s) to remove impurities, RNA shall be eluted in 100 μl elution buffer.
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The RNA preparation shall be of a quality and concentration suitable for the intended purpose. See
Annex E for illustrative details of RNA extraction reagents (used in the development of the method
described in this part of ISO 15216).
5.2.18 Reagents for one step real-time RT-PCR. Reagents shall allow processing of 5 μl RNA in 25 μl
total volume. They shall be suitable for one step real-time RT-PCR using hydrolysis probes (the DNA
polymerase used shall possess 5'-3' exonuclease activity) and sufficiently sensitive for the detection of
levels of virus RNA as typically found in virus-contaminated foodstuffs. See Annex B for illustrative
details of one step real-time RT-PCR reagents (used in the development of this part of ISO 15216).
5.2.19 Primers and hydrolysis probes for detection of HAV and norovirus GI and GII. Primer and
hydrolysis probe sequences shall be published in a peer-reviewed journal and be verified for use
against a broad range of strains of target virus. Primers for detection of HAV shall target the 5’ non-
coding region of the genome. Primers for detection of norovirus GI and GII shall target the ORF1/ORF2
junction of the genome. See Annex C for illustrative details of primers and hydrolysis probes (used in
the development of this part of ISO 15216).
5.2.20 Primers and hydrolysis probes for detection of the process control virus. Primer and
hydrolysis probe sequences shall be published in a peer-reviewed journal and be verified for use
against the strain of process virus used. They shall demonstrate no cross-reactivity with the target
virus.
5.3 Prepared reagents
Because of the large number of reagents requiring individual preparation, details of composition and
preparation are given in Annex F.
5.3.1 5 × PEG/NaCl solution (500 g/l PEG 8 000, 1,5 mol/l NaCl). See F.1.
5.3.2 Chloroform/butanol mixture. See F.2.
5.3.3 Proteinase K solution. See F.3.
5.3.4 Phosphate-buffered saline (PBS). See F.4.
5.3.5 Tris/glycine/beef extract (TGBE) buffer. See F.5.
5.3.6 1M Tris solution. See F.6.
5.3.7 0.5M EDTA solution. See F.7.
5.3.8 Tris EDTA (TE) buffer. See F.8.
5.3.9 Process control virus material. Process control virus stock shall be diluted by a minimum
factor of 10 in a suitable buffer, e.g. PBS (5.3.4). This dilution shall allow for inhibition-free detection of
the process control virus genome using real-time RT-PCR, but still be sufficiently concentrated to allow
reproducible determination of the lowest dilution used for the process control virus RNA standard
curve (8.4.2.2). Split the diluted process control virus material into single use aliquots and store at -
15 °C or below. See Annex D for illustrative details of the preparation of process control virus (used in
the development of the method described in this part of ISO 15216).
5.3.10 Real-time RT-PCR mastermixes for target and process control virus. Reagents shall be
added in quantities as specified by the manufacturers (5.2.18) to allow 20 μl mastermix per reaction in
a 25 μl total volume. Optimal primer and probe concentrations shall be used after determination
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following the recommendations of the reagent manufacturers. See Annex B for illustrative details of
real-time RT-PCR mastermixes (used in the development of this part of ISO 15216).
5.3.11 Double-stranded DNA (dsDNA) control material. Purified linear DNA molecules carrying the
target sequence for each target virus shall be used. The sequence of the DNA molecules shall be verified
prior to use. The preparations shall not cause RT-PCR inhibition. The concentrations of each dsDNA
stock in template copies per microlitre shall be determined then the stock shall be diluted in a suitable
4 5
buffer e.g. TE buffer (5.3.8), to a concentration of 1 × 10 to 1 × 10 template copies per microlitre. As
EDTA can act as an inhibitor of RT-PCR, buffers used to dilute dsDNA shall not contain concentrations of
EDTA greater than 1mM. Split the diluted dsDNA preparation (dsDNA control material) into single use
aliquots and store frozen at −15 °C or below. See Annex G for illustrative details of the preparation of
dsDNA (used in the development of this part of ISO 15216).
5.3.12 External control (EC) RNA control material. Purified ssRNA carrying the target sequence for
each target virus shall be used. They shall contain levels of c
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