EN ISO 15216-1:2017

SLOVENSKI STANDARD
01-junij-2017
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SIST-TS CEN ISO/TS 15216-1:2013
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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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.

Contents Page
European foreword . 3

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.
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
ISO 15216-1:2017(E)
© 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.
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Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

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 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 ®
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

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 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

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 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

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 point at which reaction fluorescence rises above a threshold level.
3.17
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
Note 1 to entry: The LOD is related to the test portion and the quality of the template RNA.
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
Note 1 to entry: 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 document 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.
ISO 15216-1:2017(E)
4.2 RNA extraction
It is necessary to extract RNA using a method that yields RNA preparations of suitable purity to reduce
the effect of RT-PCR inhibitors. In this document 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 RT-PCR
This document 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 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 hydrolysed and the fluorescent signal from the label increases
proportionately. Fluorescence can be measured at each stage throughout the cycle. The first cycle in the
real-time RT-PCR 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 or food surfaces 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 document) are provided in Annexes C and D.
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 account for these losses, samples are spiked at the earliest opportunity prior to virus extraction
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 or food surfaces under test.
An example of the preparation of process control virus (used in the development of this document) is
provided in Annex E.
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 dsDNA 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 RNA in detectable virus genome copies per microlitre.
4.4.3 EC RNA control
Many food matrices 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, EC RNA (an RNA species carrying the target sequence of interest, 5.3.12) is added
4 © ISO 2017 – All rights reserved

ISO 15216-1:2017(E)
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 the assessment of inhibition of RT-PCR that can be demonstrated to provide
equivalent performance to the use of EC RNA control are permitted.
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 LOD for the sample.
5 Reagents
5.1 General
Use only reagents of recognized analytical grade, unless otherwise specified.
Follow current laboratory practice, as specified in 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.
5.2.11 Pectinase from Aspergillus niger or A. aculeatus.
5.2.12 Chloroform.
5.2.13 n-Butanol.
5.2.14 Sodium hydroxide (NaOH) (≥10 mol/l).
5.2.15 Hydrochloric acid (HCl) (≥5 mol/l).
ISO 15216-1:2017(E)
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 sample extract, using lysis with a chaotropic buffer containing guanidine
[3]
thiocyanate 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 F for illustrative details of RNA extraction reagents (used in the development of the method
described in this document).
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’ to 3’ exonuclease activity) and sufficiently sensitive for the detection
of virus RNA as expected in virus-contaminated foodstuffs and food surfaces. See Annex C for illustrative
details of one step real-time RT-PCR reagents (used in the development of this document).
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 D for illustrative details of primers and hydrolysis probes (used in the development
of this document).
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 B.
5.3.1 5 × PEG/NaCl solution (500 g/l PEG 8 000, 1,5 mol/l NaCl); see B.1.
5.3.2 Chloroform/butanol mixture (1:1 v/v); see B.2.
5.3.3 Proteinase K solution (3 000 U/l); see B.3.
5.3.4 Phosphate-buffered saline (PBS); see B.4.
5.3.5 Tris/glycine/beef extract (TGBE) buffer; see B.5.
5.3.6 Tris solution (1 mol/l); see B.6.
5.3.7 EDTA solution (0,5 mol/l); see B.7.
5.3.8 Tris EDTA (TE) buffer (10 mmol/l Tris, 1 mmol/l EDTA); see B.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
6 © ISO 2017 – All rights reserved

ISO 15216-1:2017(E)
or below. See Annex E for illustrative details of the preparation of process control virus (used in the
development of the method described in this document).
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 following the
recommendations of the reagent manufacturers. See Annex C for illustrative details of real-time RT-PCR
mastermixes (used in the development of this document).
5.3.11 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 first 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 buffer e.g. TE buffer
4 5
(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
1 mmol/l. Split the diluted dsDNA preparation (dsDNA control material) into single use aliquots and
store at (5 ± 3) °C for up to 24 h, at −15 °C or below for up to six months, or at −70 °C or below for longer
periods. See Annex G for illustrative details of the preparation of dsDNA (used in the development of this
document).
5.3.12 EC RNA control material. 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 then the stock shall be diluted in a suitable buffer e.g. TE buffer (5.3.8), to a concentration
2 5
of 1 × 10 to 1 × 10 template copies per microlitre. The concentration used shall be appropriate for
the types of samples under test and ensure that RT-PCR inhibition calculations are not affected by the
presence of endogenous target RNA in the samples. As EDTA can act as an inhibitor of RT-PCR, buffers
used to dilute EC RNA shall not contain concentrations of EDTA greater than 1 mmol/l. Split the diluted
EC RNA preparation (EC RNA control material) into single use aliquots and store at (5 ± 3) °C for up to
24 h, at −15 °C or below for up to six months, or at −70 °C or below for longer periods. See Annex H for
illustrative details of the preparation of EC RNA (used in the development of this document).
6 Equipment and consumables
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 (as in 6.7 and F.3).
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 50 oscillations min .
−1
6.5 Shaking incubator operating at (37 ± 2) °C and approximately 320 oscillations min or
equivalent.
6.6 Rocking platform(s) or equivalent for use at room temperature and (5 ± 3) °C at approximately
−1
60 oscillations min .
6.7 Aspirator or equivalent apparatus for removing supernatant.
6.8 Water bath capable of operating at (60 ± 2) °C or equivalent.
ISO 15216-1:2017(E)
6.9 Centrifuge(s) and rotor(s) capable of the following run speeds, run temperatures, and rotor
capacities:
a) 10 000g at (5 ± 3) °C with capacity for tubes of at least 35 ml volume;
b) 10 000g at (5 ± 3) °C with capacity for chloroform-resistant tubes with 2 ml volume;
c) 4 000g at room temperature with capacity for centrifugal filter concentration devices (6.16).
6.10 Microcentrifuge.
6.11 Centrifuge and microcentrifuge tubes and bottles of a range of sizes, 1,5 ml, 5 ml, 15 ml, 50 ml,
etc. Chloroform-resistant tubes with 2 ml capacity are necessary.
6.12 pH meter (or pH testing strips with demarcations of 0,5 pH units or lower).
6.13 Sterile cotton swabs.
6.14 Mesh filter bags (400 ml).
6.15 Positively charged membrane filters with 0,45 μm pore size (47 mm diameter).
6.16 Centrifugal filter concentration devices with 15 ml capacity and 100 kDa relative molecular
mass cutoff.
6.17 Vacuum source or equivalent positive pressure apparatus for filtering and filtration tower with
aperture for 47 mm diameter membrane.
6.18 Sterile shucking knife or equivalent tools for opening BMS.
6.19 Rubber block or equivalent apparatus for holding BMS during opening.
6.20 Scissors and forceps or equivalent tools for dissecting BMS.
6.21 Sterile Petri dishes.
6.22 Razor blades o
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