CEN ISO/TS 15216-1:2013

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.

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Kingdom.
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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
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E-mail copyright@iso.org
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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
ISO/TS 15216-1:2013(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.
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 15216-1 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 15216-1:2013 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 I; Annex I was formerly Annex F.
— Many cross-references to reagents or apparatus subclauses are added.
−1 −1
— Where units of shaking operations are mentioned, “oscillations min ” replaces “min ”.
— 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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ISO/TS 15216-1:2013(E)
— 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 3.6 and 3.7, “estimation of number of copies” replaces “quantification”.
— 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 9.3, Note 1, “For a dsDNA standard curve with an idealized slope of −3,32, if the C value of
q
the sample RNA + EC RNA well is <2,00 greater than the C value of the water + EC RNA well, the
q
amplification efficiency is >25 % and therefore acceptable; if the C value of the sample RNA + EC
q
RNA well is >2,00 greater than the C value of the water + EC RNA well, the amplification efficiency
q
is <25% and therefore not acceptable.” is added.
— In 9.4, Note 1 “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 “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.”
ISO/TS 15216-1:2013(E)
— In C.4, “This primer set amplifies a product of 100 bp corresponding to nucleotides 110–209 of
the deletant mengo virus strain MC0 used in the development of this part of ISO/TS 15216. This
corresponds to nucleotides 110–270 of the non-deletant mengo virus isolate M (GenBank accession
number L22089).” is added as paragraph 2.”
— In H.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
vi © ISO 2013 – All rights reserved

ISO/TS 15216-1:2013(E)
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 quantification of levels of virus RNA in the test sample. A
schematic diagram of the testing procedure is shown in Annex A.
TECHNICAL SPECIFICATION ISO/TS 15216-1:2013(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 1:
Method for quantification
1 Scope
This part of ISO/TS 15216 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.
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
ISO/TS 15216-1:2013(E)
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
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
3.7
quantification of norovirus
estimation of number of copies of norovirus RNA in a predetermined mass or volume of foodstuff, or
area of food surface
3.8
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
2 © ISO 2013 – All rights reserved

ISO/TS 15216-1:2013(E)
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 to the point at which reaction fluorescence rises above a threshold level.
3.16
theoretical limit of detection
tLOD
level that constitutes the smallest quantity of target that can in theory be detected
Note 1 to entry: This corresponds to one genome copy per volume of RNA tested in the target assay, but varies
according to the test matrix and the quantity of starting material.
3.17
practical limit of detection
pLOD
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 1 to entry: The pLOD is related to the test portion, the quality or quantity of the template RNA, and the tLOD
of the method.
3.18
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 1 to entry: The LOQ is related to the test portion and the quality or quantity of the template RNA.
4 Principle
4.1 Virus extraction
The foodstuffs and food surfaces covered by this part of ISO/TS 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 PCR inhibitors. In this part of ISO/TS 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.
ISO/TS 15216-1:2013(E)
4.3 Real-time reverse transcription polymerase chain reaction (real time RT-PCR)
This part of ISO/TS 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.
Real-time 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 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 quantity
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 5.2.17
and 5.2.18. Illustrative details of reagents, primers, and probes (used in the development of this part of
ISO/TS 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 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/TS 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 double-stranded DNA carrying the target sequence of interest (5.3.8) and quantified
using spectrophotometry 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,
5.3.9) is added to an aliquot of sample RNA and tested using the 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 can be demonstrated to provide equivalent
performance to the use of EC RNA are permitted.
4 © ISO 2013 – All rights reserved

ISO/TS 15216-1:2013(E)
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;
of detection (LOD) for the sample.
5 Reagents
5.1 General
Use only reagents of recognized analytical grade, unless otherwise specified.
[10]
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 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
ISO/TS 15216-1:2013(E)
thiocyanate (Reference [1]) 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.
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/TS 15216).
5.2.17 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 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/TS 15216).
5.2.18 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/TS 15216).
5.2.19 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 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 (−80 ± 5) °C.
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/TS 15216).
5.3.7 Real-time RT-PCR mastermixes for target and process control virus. Reagents shall be added
in quantities as specified by the manufacturers (5.2.17) to allow 20 μl mastermix per reaction in a 25 μl
total volume. Optimal primer and probe concentrations shall be used after determination 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/TS 15216).
5.3.8 Double-stranded DNA (dsDNA) control material. Separate purified plasmids carrying the
target sequence for each target virus shall be used. The preparations shall not cause RT-PCR inhibition.
6 © ISO 2013 – All rights reserved

ISO/TS 15216-1:2013(E)
The concentrations of each dsDNA stock in template copies per microlitre shall be determined then the
4 5
stock shall be diluted to a concentration of 1 × 10 to 1 × 10 template copies per microlitre. 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/TS 15216).
5.3.9 External control (EC) RNA control material. Separate purified ssRNA carrying the target
sequence for each target virus shall be used. They shall contain levels of contaminating target DNA no
higher than 0,1 % and shall not cause RT-PCR inhibition. The concentrations of each EC RNA stock in
copies per microlitre shall be determined and stock shall be diluted to a concentration of 1 × 10 to
1 × 10 template copies per microlitre. Split the diluted EC RNA preparation (EC RNA control material)
into single use aliquots and store frozen at −15 °C or below. See Annex H for illustrative details of the
preparation of EC RNA (used in the development of this part of ISO/TS 15216).
6 Apparatus and materials
[10]
Standard microbiological laboratory equipment (ISO 7218) and in particular the following.
6.1 Micropipettes and tips of a range of sizes, e.g. 1 000 μl, 200 μl, 20 μl, 10 μl. Aerosol resistant tips
should be used unless unobstructed tips are required, e.g. for aspiration.
6.2 Pipette filler and pipettes of a range of sizes, e.g. 25 ml, 10 ml, 5 ml.
6.3 Vortex mixer.
−1
6.4 Shaker capable of operating at approximately 500 oscillations min .
−1
6.5 Shaking incubator operating at (37 ± 1,0) °C and (320 ± 20) oscillations min or equivalent.
6.6 Rocking platform(s) or equivalent for use at room temperature and (4 ± 2) °C at (60 ±
−1
5) oscillations min .
6.7 Aspirator or equivalent apparatus for removing supernatant.
6.8 Heating block capable of operating at (95 ± 1,0) °C or equivalent.
6.9 Water bath capable of operating at (60 ± 2,0) °C or equivalent.
6.10 Centrifuge(s) and rotor(s) capable of the following run speeds, run temperatures, and rotor capacities:
a) 10 000 × g at (5 ± 3) °C with capacity for tubes of at least 35 ml volume;
b) 10 000 × g at (5 ± 3) °C with capacity for narrow gauge (15 mm is too large) chloroform-resistant
tubes of at least 1 ml volume;
c) 4 000 × g at room temperature with capacity for centrifugal filter concentration devices (6.17).
6.11 Microcentrifuge.
6.12 Centrifuge and microcentrifuge tubes and bottles of a range of sizes, 1,5 ml, 5 ml, 15 ml, 50 ml,
etc. Narrow gauge (15 mm is too large) chloroform-resistant tubes with 1 ml capacity are necessary.
6.13 pH meter (or pH testing strips).
ISO/TS 15216-1:2013(E)
6.14 Sterile cotton swabs.
6.15 Mesh filter bags (400 ml).
6.16
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