SIST EN ISO 15216-2:2019

SLOVENSKI STANDARD
01-december-2019
Nadomešča:
SIST-TS CEN ISO/TS 15216-2:2013
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje virusa
hepatitisa A in norovirusov z RT-PCR v realnem času - 2. del: Metoda za
ugotavljanje (ISO 15216-2:2019)
Microbiology of the food chain - Horizontal method for determination of hepatitis A virus
and norovirus using real-time RT-PCR - Part 2: Method for detection (ISO 15216-2:2019)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zur Bestimmung von
Hepatitis A-Virus und Norovirus in Lebensmitteln mittels Real-time-RT-PCR - Teil 2:
Nachweisverfahren (ISO 15216-2:2019)
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 2:
Méthode de détection (ISO 15216-2:2019)
Ta slovenski standard je istoveten z: EN ISO 15216-2:2019
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 15216-2
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2019
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes CEN ISO/TS 15216-2:2013
English Version
Microbiology of the food chain - Horizontal method for
determination of hepatitis A virus and norovirus using
real-time RT-PCR - Part 2: Method for detection (ISO
15216-2:2019)
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 in Lebensmitteln mittels Real-time-RT-PCR -
Partie 2: Méthode de détection (ISO 15216-2:2019) Teil 2: Nachweisverfahren (ISO 15216-2:2019)
This European Standard was approved by CEN on 27 July 2019.

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, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, 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: Rue de la Science 23, B-1040 Brussels
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 15216-2:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 15216-2:2019) 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.
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 March 2020, and conflicting national standards shall
be withdrawn at the latest by March 2020.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
This document supersedes CEN ISO/TS 15216-2:2013.
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, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 15216-2:2019 has been approved by CEN as EN ISO 15216-2:2019 without any
modification.
INTERNATIONAL ISO
STANDARD 15216-2
First edition
2019-07
Microbiology of the food chain —
Horizontal method for determination
of hepatitis A virus and norovirus
using real-time RT-PCR —
Part 2:
Method for detection
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 2: Méthode de détection
Reference number
ISO 15216-2:2019(E)
©
ISO 2019
ISO 15216-2:2019(E)
© ISO 2019
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
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Published in Switzerland
ii © ISO 2019 – All rights reserved

ISO 15216-2:2019(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 . 3
4.3 Real-time RT-PCR . 3
4.4 Control materials . 4
4.4.1 Process control virus . 4
4.4.2 EC RNA control . 4
4.5 Test results. 4
5 Reagents . 4
5.1 General . 4
5.2 Reagents used as supplied . 4
5.3 Reagents requiring preparation . 6
6 Equipment and consumables . 7
7 Sampling . 8
8 Procedure. 8
8.1 General laboratory requirements . 8
8.2 Virus extraction . 8
8.2.1 General. 8
8.2.2 Process control virus material . 9
8.2.3 Negative process control . 9
8.2.4 Surfaces . 9
8.2.5 Soft fruit and leaf, stem and bulb vegetables. 9
8.2.6 Bottled water .10
8.2.7 Bivalve molluscan shellfish (BMS) .10
8.3 RNA extraction .11
8.4 Real-time RT-PCR .11
8.4.1 General requirements .11
8.4.2 Real-time RT-PCR analysis .12
9 Interpretation of results .14
9.1 General .14
9.2 Construction of process control virus RNA standard curve.14
9.3 Control for RT-PCR inhibition.14
9.4 Calculation of extraction efficiency .15
10 Expression of results .15
11 Performance characteristics of the method .16
11.1 Validation study.16
11.2 Sensitivity .16
11.3 Specificity .16
11.4 LOD .16
12 Test report .16
Annex A (normative) Diagram of procedure .17
Annex B (normative) Composition and preparation of reagents and buffers .18
Annex C (informative) Real-time RT-PCR mastermixes and cycling parameters .21
ISO 15216-2:2019(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) .22
Annex E (informative) Growth of mengo virus strain MC for use as a process control .25 ®
Annex F (informative) RNA extraction using the BioMerieux NucliSens system .26
Annex G (informative) Generation of external control RNA (EC RNA) stocks .28
Annex H (informative) Typical optical plate layout .31
Annex I (informative) Method validation studies and performance characteristics .32
Bibliography .40
iv © ISO 2019 – All rights reserved

ISO 15216-2:2019(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 of 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 www .iso
.org/iso/foreword .html.
This document was prepared by the European Committee for Standardization (CEN) Technical
Committee CEN/TC 275, Food analysis — Horizontal methods, in collaboration with ISO Technical
Committee 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 15216-2:2013, which has been technically revised with
the following changes:
— a requirement to use a suitable buffer for the dilution of control materials has been added;
— the method for generating process control virus RNA for the standard curve has been changed;
— breakpoints with a defined temperature and time parameters in the extraction methods have
been added;
— the terminology has been changed from amplification efficiency to RT-PCR inhibition;
— extra real-time RT-PCR reactions for sample RNA and negative controls have been added;
— method characteristics and the results of method validation studies have been added.
A list of all parts in the ISO 15216 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
ISO 15216-2:2019(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 surfaces, viruses are removed by swabbing. For soft fruit and
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. 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 detection
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 15216-2:2013,
are considered as minor (see ISO 17468).
vi © ISO 2019 – All rights reserved

INTERNATIONAL STANDARD ISO 15216-2:2019(E)
Microbiology of the food chain — Horizontal method for
determination of hepatitis A virus and norovirus using
real-time RT-PCR —
Part 2:
Method for detection
1 Scope
This document specifies a method for detection of hepatitis A virus (HAV) and norovirus genogroups
I (GI) and II (GII), from test samples of foodstuffs [(soft fruit, leaf, stem and bulb vegetables, bottled
water, bivalve molluscan shellfish (BMS)] or surfaces using 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,
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 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 20838, ISO 22119, ISO 22174
and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:/ /www. iso. org/obp
— IEC Electropedia: available at http:/ /www.e lectropedia. org/
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.
3.2
surface
surface of food, food preparation surface or food contact surface
ISO 15216-2:2019(E)
3.3
soft fruit
small edible stoneless fruit
EXAMPLE Strawberries, raspberries, currants.
3.4
leaf, stem and bulb vegetables
leaves, stems and bulbs of plants, eaten as a vegetable
EXAMPLE Lettuce, green onions.
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 (3.1), contact with contaminated water or surfaces (3.2), 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 (3.1), through contact with contaminated water or surfaces (3.2), 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
detection of HAV
detection of HAV (3.5) RNA in a predetermined mass or volume of foodstuff (3.1), or on the area of a
surface (3.2)
3.8
detection of norovirus
detection of norovirus (3.6) RNA in a predetermined mass or volume of foodstuff (3.1), or on the area of
a surface (3.2)
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 (3.9) in order to produce standard curve data for the
estimation of extraction efficiency
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
2 © ISO 2019 – All rights reserved

ISO 15216-2:2019(E)
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 assess 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, which is the cycle at which the target is quantified in a given real-time RT-PCR
reaction
Note 1 to entry: This corresponds to the cycle at which reaction fluorescence rises above a threshold level.
4 Principle
4.1 Virus extraction
The foodstuffs and 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.
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 the 5’ and 3’ ends, respectively. 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.
Due to the low levels of virus template often present in foodstuffs or surfaces and the strain diversity in
the target viruses, the 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
ISO 15216-2:2019(E)
5.2.19 and 5.2.20. 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 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 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 evaluate RT-PCR inhibition in
individual samples, EC RNA (an RNA species carrying the target sequence of interest, 5.3.11) 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. In addition, in this method, the EC RNA control acts as a
positive control for real-time RT-PCR for the target viruses.
Alternative approaches for the assessment of RT-PCR inhibition are permitted, provided that the
alternative approach can be demonstrated to provide equivalent performance to the use of EC RNA
control as described in this document.
4.5 Test results
For surfaces, this method provides a result expressed either as “virus genome detected” or “virus
genome not detected” followed by “in x cm ”, where x is the approximate surface area swabbed. Where
it is not possible to record the surface area swabbed, results are expressed either as “virus genome
detected” or “virus genome not detected”. For other sample types, results are expressed as “virus
genome detected” or “virus genome not detected” followed by “in x ml” or “in x g”, where x is the amount
of sample tested.
5 Reagents
5.1 General
Use only reagents of recognized analytical grade, unless otherwise specified.
For current laboratory practice, see ISO 7218.
5.2 Reagents used as supplied
5.2.1 Molecular biology grade water.
4 © ISO 2019 – All rights reserved

ISO 15216-2:2019(E)
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).
5.2.16 Ethylenediaminetetraacetic acid (EDTA) disodium dihydrate.
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
[4]
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′-3′ exonuclease activity) and sufficiently sensitive for the detection of
virus RNA as expected in virus-contaminated foodstuffs and 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 for detection of HAV and
norovirus GI and GII (used in the development of this document).
ISO 15216-2:2019(E)
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. See
Annex D for illustrative details of primers and hydrolysis probes for detection of the process control
virus (used in the development of this document).
5.3 Reagents requiring preparation
Due to the large number of reagents requiring individual preparation, details of composition and
preparation are given in Annex B. The instructions in Annex B shall be followed when preparing
reagents listed under 5.3.1 to 5.3.8.
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 volume fraction). 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
(see 8.4.2.2). Split the diluted process control virus material into single use aliquots and store at −15 °C
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 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 of 1 × 10
to 1 × 10 template copies per microlitre. The concentration used shall be appropriate for the types of
samples under test and shall 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 °C (6.4) for up to 24 h, at
6 © ISO 2019 – All rights reserved

ISO 15216-2:2019(E)
−15 °C or below for up to 6 months, or at −70 °C or below for longer periods. See Annex G for illustrative
details of the preparation of EC RNA (used in the development of this document).
Alternative approaches for the assessment of RT-PCR inhibition are permitted, provided that the
alternative approach can be demonstrated to provide equivalent performance to the use of EC RNA
control as described in this document.
6 Equipment and consumables
Standard microbiological laboratory equipment (see 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
shall be used unless unobstructed tips are required, e.g. for aspiration (as in 6.8 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.
6.4 Refrigerator, capable of operating at (5 ± 3) °C
−1
6.5 Shaker, capable of operating at approximately 50 oscillations min .
−1
6.6 Shaking incubator, operating at (37 ± 2) °C and approximately 320 oscillations min or
equivalent.
6.7 Rocking platform(s) or equivalent for use at room temperature and (5 ± 3) °C at approximately
−1
60 oscillations min .
6.8 Aspirator or equivalent apparatus for removing supernatant.
6.9 Water bath, capable of operating at (60 ± 2) °C or equivalent.
6.10 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).
6.11 Centrifuge 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 cut-off.
ISO 15216-2:2019(E)
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 or equivalent tools for chopping BMS digestive glands.
6.23 Heavy duty safety glove.
6.24 RNA extraction equipment, suitable for extraction methods using silica and associated reagents
(5.2.17). See Annex F for illustrative details of RNA extraction apparatus (used in the development of this
document).
6.25 Real-time PCR machine(s), i.e. thermal cycler(s), equipped with an energy source suitable
for the excitation of fluorescent molecules, and an optical detection system for real-time detection of
fluorescence signals generated during real-time RT-PCR with hydrolysis probe chemistry.
6.26 Associated consumables for real-time RT-PCR, e.g. optical plates and caps, suitable for use with
the selected real-time RT-PCR machine.
7 Sampling
If there is no specific International Standard dealing with the sampling of the product concerned, it is
recommended that the parties concerned come to an agreement on the subject.
It is important the laboratory receive a sample that is representative and that has not been damaged or
changed during transport or storage, e.g. samples that were frozen on collection shall not be allowed
to defrost prior to receipt at the laboratory, samples that were not frozen upon collection shall not be
frozen prior to receipt at the laboratory.
8 Procedure
8.1 General laboratory requirements
The testing procedure shall be as shown in the schematic diagram in Annex A.
Sample extraction and real-time RT-PCR shall be carried out in separate working areas or rooms as
specified in ISO 22174.
8.2 Virus extraction
8.2.1 General
The selection of method is dependent upon the food matrix under test.
8 © ISO 2019 – All rights reserved

ISO 15216-2:2019(E)
8.2.2 Process control virus material
Immediately before a batch of test samples is processed, pool together sufficient aliquots of process
control virus material (5.3.9) for all i
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