SIST-TS CEN ISO/TS 15216-2:2013

SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 15216-2:2013
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 2: Method for qualitative
detection (ISO/TS 15216-2:2013, Corrected Version 2013-05-01)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zur
Bestimmung von Hepatitis A-Viren und Noroviren in Lebensmitteln mittels Real-time-RT-
PCR - Teil 2: Verfahren für den qualitativen Nachweis (ISO/TS 15216-2:2013)
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 2: Méthode de détection qualitative (ISO/TS 15216-2:2013, Version Corrigée 2013
-05-01)
Ta slovenski standard je istoveten z: CEN ISO/TS 15216-2:2013
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST-TS CEN ISO/TS 15216-2:2013 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN ISO/TS 15216-2:2013

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SIST-TS CEN ISO/TS 15216-2:2013


TECHNICAL SPECIFICATION
CEN ISO/TS 15216-2

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 2: Method for qualitative detection
(ISO/TS 15216-2:2013, 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 2: Viren und Noroviren in Lebensmitteln mittels Real time
Méthode de détection qualitative (ISO/TS 15216-2:2013, PCR - Teil 2: Verfahren für den qualitativen Nachweis
Version Corrigée 2013-05-01) (ISO/TS 15216-2:2013, 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.





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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 15216-2:2013: E
worldwide for CEN national Members.

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SIST-TS CEN ISO/TS 15216-2:2013
CEN ISO/TS 15216-2:2013 (E)
Contents Page
Foreword .3
2

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SIST-TS CEN ISO/TS 15216-2:2013
CEN ISO/TS 15216-2:2013 (E)
Foreword
This document (CEN ISO/TS 15216-2:2013) 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 15216-2:2013, Corrected Version 2013-05-01 has been approved by CEN as CEN ISO/TS
15216-2:2013 without any modification.

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SIST-TS CEN ISO/TS 15216-2:2013

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SIST-TS CEN ISO/TS 15216-2:2013
TECHNICAL ISO/TS
SPECIFICATION 15216-2
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 2:
Method for qualitative detection
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 2: Méthode de détection qualitative
Reference number
ISO/TS 15216-2:2013(E)
©
ISO 2013

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SIST-TS CEN ISO/TS 15216-2:2013
ISO/TS 15216-2:2013(E)

COPYRIGHT PROTECTED DOCUMENT
© 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
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2013 – All rights reserved

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ISO/TS 15216-2:2013(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) . 3
4.4 Control materials . 4
4.5 Test results. 4
5 Reagents . 4
5.1 General . 4
5.2 Reagents used as supplied . 4
5.3 Prepared reagents . 6
6 Apparatus and materials. 6
7 Sampling . 8
8 Procedure. 8
8.1 General laboratory requirements . 8
8.2 Virus extraction . 8
8.3 RNA extraction .10
8.4 Real-time RT-PCR .10
9 Interpretation of results .12
9.1 General .12
9.2 Construction of process control virus RNA standard curve.12
9.3 Control for amplification efficiency .12
9.4 Calculation of extraction efficiency .13
9.5 Theoretical limit of detection .13
10 Expression of results .13
11 Test report .14
Annex A (normative) Diagram of procedure .15
Annex B (informative) Real-time RT-PCR mastermixes and cycling parameters .16
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) .17
Annex D (informative) Growth of mengo virus strain MC for use as a process control .19
0
®
Annex E (informative) RNA extraction using the BioMerieux NucliSens system .20
Annex F (normative) Composition and preparation of reagents and buffers .22
Annex G (informative) Generation of external control RNA (EC RNA) stocks .24
Annex H (informative) Typical optical plate layout .26
Bibliography .27
© ISO 2013 – All rights reserved iii

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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.
In other circumstances, particularly when there is an urgent market requirement for such documents, a
technical committee may decide to publish other types of document:
— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical
experts in an ISO working group and is accepted for publication if it is approved by more than 50 %
of the members of the parent committee casting a vote;
— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a
technical committee and is accepted for publication if it is approved by 2/3 of the members of the
committee casting a vote.
An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for
a further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or
ISO/TS is confirmed, it is reviewed again after a further three years, at which time it must either be
transformed into an International Standard or be withdrawn.
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/TS 15216-2 was prepared by the European Committee for Standardization (CEN), in collaboration
with Technical committee ISO/TC 34, Food products, Subcommittee SC 9 Microbiology.
This corrected version of ISO/TS 15216-2:2013 incorporates the following corrections.
— Throughout, textual references have been updated to take reordering of the annexes into account.
Annex B was formerly Annex E; Annex C was formerly Annex D; Annex D was formerly Annex G;
Annex E was formerly Annex C; Annex F was formerly Annex B; Annex G was formerly Annex H;
Annex H was formerly Annex F.
−1 −1
— Where units of shaking operations are mentioned, “oscillations min ” replaces “min ”.
— Many cross-references to reagents or apparatus subclauses are added.
— A phrase citing Annex A is added to the end of the introduction.
— The definitions for “food surface” (formerly 3.2 and 3.3) are combined and expanded in a redrafted
3.2; in consequence, the following terms in Clause 3 are renumbered.
— In 3.4, Note 2, “There is only one serotype” is transposed to the end of Note 1. Also, “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” replaces “UK Advisory Committee on Dangerous Pathogens (ACDP)
hazard group 2 pathogen”.
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— In 3.5, Note 2, “group 2 biological agents by the European Union and as risk group 2 human
aetiological agents by the United States National Institutes of Health” replaces “ACDP hazard group
2 pathogens”.
— In 3.13, “used in” replaces “used as template in”.
— In 5.2.11, “from Aspergillus niger or A. aculeatus” is inserted after “Pectinase”.
— In 6.1,”Aerosol resistant tips should be used unless unobstructed tips are required, e.g. for
aspiration.” is inserted.
— In 6.5, “37 ± 1,0” replaces “37 ± 10”.
— A redrafted 6.10 on centrifuge(s) and rotor(s) replaces the former 6.10 and 6.11, with consequent
renumbering of the following subclauses.
— In 6.19, the square brackets are deleted.
— In 6.27, “Real-time PCR machine(s), i.e. thermal cycler(s),” replaces “Thermal cycler(s)”.
— In 6.28, “selected real-time PCR” replaces “selected PCR”.
— In 8.1, “Samples arriving already frozen should be defrosted prior to testing.” is inserted as the
second sentence.
— 8.2.3 Is redrafted.
— In 8.2.4, paragraph 2,”buffer (5.3.5) (for soft fruit samples, add 30 units pectinase from A. niger, or
1 140 units pectinase from A. aculeatus to the buffer) and” replaces “buffer (for soft fruit samples,
add 30 units pectinase to the buffer) and”.
— In 8.2.6, paragraph 2, “and the animal is supported with a rubber block” is added.
— In 8.2.6, last paragraph, “min at room temperature, decant” replaces “min, decant”
— In 8.4.2.3, paragraph 1,”using a real-time PCR machine (6.27)” is added.
— In 9.4, Note 1 “has a process control virus recovery (equal to the extraction efficiency in matrices
other than BMS) of 100 %. For a process control virus RNA standard curve with an idealized slope
of −3,32, if the C value of an undiluted sample RNA well is <6,64 greater than the C value of the
q q
undiluted process control virus RNA, the process control virus recovery for that sample is >1% and
therefore acceptable” replaces “will have an extraction efficiency of 100 %”.
— The title of Annex B has been expanded to read, “Real-time RT-PCR mastermixes and cycling parameters”.
— In Table B.1, footnote a, “real-time PCR machines” twice replaces “real-time machines”.
— In C.1, “This primer set amplifies a product of 173 bp corresponding to nucleotides 68–240 of HAV
isolate HM174 43c (GenBank accession number M59809).” is added as paragraph 2.
— In C.2, “This primer set amplifies a product of 86 bp corresponding to nucleotides 5291–5376 of
Norwalk virus (GenBank accession number M87661).” is added as paragraph 2.
— In C.3, “This primer set amplifies a product of 89 bp corresponding to nucleotides 5012–5100 of
Lordsdale virus (GenBank accession number X86557).” is added as paragraph 2.
— In C.4, “This primer set amplifies a product of 100 bp corresponding to nucleotides 110–209 of
the deletant mengo virus strain MC used in the development of this part of ISO/TS 15216. This
0
corresponds to nucleotides 110–270 of the non-deletant mengo virus isolate M (GenBank accession
number L22089).” is added as paragraph 2.
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— In G.5, “mastermix (if the C difference between EC RNA stock tested with heat-treated and
q
untreated mastermix is <10 for a dsDNA standard curve with an idealized slope of −3,32), the”
replaces “mastermix, the”.
ISO/TS 15216 consists of the following parts, under the general title 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
— Part 2: Method for qualitative detection
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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 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 PCR method, and
obviate the need for additional amplification product confirmation steps post 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/TS 15216 enables qualitative detection of virus RNA in the test sample. A schematic diagram
of the testing procedure is shown in Annex A.
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SIST-TS CEN ISO/TS 15216-2:2013
TECHNICAL SPECIFICATION ISO/TS 15216-2:2013(E)
Microbiology of food and animal feed — Horizontal
method for determination of hepatitis A virus and
norovirus in food using real-time RT-PCR —
Part 2:
Method for qualitative detection
1 Scope
This part of ISO/TS 15216 describes a method for qualitative detection of HAV and NoV genogroups I
(GI) and II (GII), 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.
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 1 to entry: For the purposes of this part of ISO/TS 15216, this definition includes bottled water.
3.2
food surface
surface of food, food preparation surface or food contact surface
3.3
fomite
inanimate object or material on which infectious agents can be conveyed
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3.4
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. 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.
3.5
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 five separate genogroups.
Note 2 to entry: 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.
3.6
detection of hepatitis A virus
detection of HAV RNA in a predetermined mass or volume of foodstuff, or area of food surface
3.7
detection of norovirus
detection of norovirus RNA in a predetermined mass or volume of foodstuff, or area of food surface
3.8
process control virus
virus added to the sample portion at the earliest opportunity prior to virus extraction to control for
extraction efficiency
3.9
process control virus RNA
RNA released from the process control virus in order to produce standard curve data for the estimation
of extraction efficiency
3.10
negative RNA extraction control
control free of target RNA carried through all steps of the RNA extraction and detection procedure to
monitor any cross-contamination events
3.11
negative process control
target pathogen-free sample of the food matrix which is run through all stages of the analytical process
3.12
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
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3.13
negative 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
3.14
external control RNA
reference RNA that can serve as target for the real-time 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
3.15
C value
q
quantification cycle; the PCR cycle at which the target is quantified in a given real-time PCR reaction
Note 1 to entry: This corresponds
...