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)

ISO 15216-1:2017 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.

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)

Dieses Dokument legt ein Verfahren für die quantitative Bestimmung des Gehalts an RNA von HAV und Noroviren der Genogruppen I (GI) und II (GII) in Untersuchungsproben von Lebensmitteln (weiches Obst, Blatt , Stängel und Zwiebelgemüse, in Flaschen abgefülltes Trinkwasser, zweischalige Weichtiere (Muscheln)) oder Lebensmitteloberflächen fest. Nach der Freisetzung der Viren aus der Untersuchungsprobe wird die virale RNA durch Aufschluss mit Guanidinthiocyanat und Adsorption an Silica( Partikeln) extrahiert. Die Zielsequenzen in der viralen RNA werden amplifiziert und mittels Real time RT PCR detektiert.
Dieses Verfahren ist weder für den Nachweis der Zielviren in anderen Lebensmitteln (einschließlich zusammengesetzter Lebensmittel) oder anderen Matrices noch für den Nachweis anderer Viren in Lebens-mitteln, auf Lebensmitteloberflächen oder anderen Matrices validiert.

Microbiologie dans la chaine alimentaire - Méthode horizontale pour la recherche des virus de l'hépatite A et norovirus par la technique RT-PCR en temps réel - Partie 1: Méthode de quantification (ISO 15216-1:2017)

ISO 15216-1:2017 décrit une méthode de quantification des niveaux d'ARN de VHA et norovirus des génogroupes I (GI) et II (GII) présents dans des échantillons pour essai d'aliments (fruits tendres, légumes feuilles, tiges et bulbes, eau embouteillée, MBV) ou sur des surfaces alimentaires. Après libération des virus contenus dans l'échantillon pour essai, l'ARN viral est extrait par lyse à l'aide de thiocyanate de guanidine et par adsorption sur silice. Les séquences cibles de l'ARN viral sont amplifiées et détectées par la technique RT-PCR en temps réel.
Cette méthode n'est pas validée pour la détection des virus ciblés dans d'autres aliments (y compris les aliments à plusieurs composants) ou d'autres matrices, ni pour la détection d'autres virus dans les aliments, sur les surfaces alimentaires ou dans d'autres matrices.

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje virusa hepatitisa A in norovirusov z RT-PCR v realnem času - 1. del: Metoda za kvantifikacijo (ISO 15216-1:2017)

Ta dokument določa kvantitativno metodo za določevanje ravni genske skupine virusa hepatitisa A in norovirusa I (GI) in II (GII) RNK v vzorcih ali živilih (mehkem sadju, listnati in stebelni zelenjavi, čebulnicah, ustekleničeni vodi, BMS) oz. na površinah živil. Po sprostitvi virusov iz vzorca se nato virusni RNK odstrani z lizo z gvanidinijevim tiocianatom in adsorpcijo na silicijev oksid. Ciljna zaporedja v virusni RNK poudari in zazna RT-PCR v realnem času.
Ta metoda ni potrjena za odkrivanje ciljnih virusov v drugih živilih (vključno z večkomponentnimi živili) ali drugih matricah ali odkrivanje drugih virusov v živilih, na površinah živil ali drugih matricah.

General Information

Status
Published
Publication Date
28-Mar-2017
Current Stage
6060 - Definitive text made available (DAV) - Publishing

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SLOVENSKI STANDARD
SIST EN ISO 15216-1:2017
01-junij-2017
1DGRPHãþD
SIST-TS CEN ISO/TS 15216-1:2013
0LNURELRORJLMDYSUHKUDQVNLYHULJL+RUL]RQWDOQDPHWRGD]DXJRWDYOMDQMHYLUXVD
KHSDWLWLVD$LQQRURYLUXVRY]573&5YUHDOQHPþDVXGHO0HWRGD]D
NYDQWLILNDFLMR ,62

Microbiology of the food chain - Horizontal method for determination of hepatitis A virus

and norovirus using real-time RT-PCR - Part 1: Method for quantification (ISO 15216-

1:2017)
Mikrobiologie der Lebensmittelkette - Horizontales Verfahren zur Bestimmung von
Hepatitis A-Virus und Norovirus mittels Real-time-RT-PCR - Teil 1: Verfahren zur
Quantifizierung (ISO 15216-1:2017)

Microbiologie dans la chaine alimentaire - Méthode horizontale pour la recherche des

virus de l'hépatite A et norovirus par la technique RT-PCR en temps réel - Partie 1:

Méthode de quantification (ISO 15216-1:2017)
Ta slovenski standard je istoveten z: EN ISO 15216-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 15216-1:2017 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 15216-1:2017
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SIST EN ISO 15216-1:2017
EN ISO 15216-1
EUROPEAN STANDARD
NORME EUROPÉENNE
March 2017
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes CEN ISO/TS 15216-1:2013
English Version
Microbiology of the food chain - Horizontal method for
determination of hepatitis A virus and norovirus using
real-time RT-PCR - Part 1: Method for quantification (ISO
15216-1:2017)

Microbiologie dans la chaine alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales

horizontale pour la recherche des virus de l'hépatite A Verfahren zur Bestimmung von Hepatitis A-Virus und

et norovirus par la technique RT-PCR en temps réel - Norovirus mittels Real-time-RT-PCR - Teil 1: Verfahren

Partie 1: Méthode de quantification (ISO 15216- zur Quantifizierung (ISO 15216-1:2017)

1:2017)
This European Standard was approved by CEN on 23 February 2017.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists in three official versions (English, French, German). A version in any other language made by

translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management

Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels

© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 15216-1:2017 E

worldwide for CEN national Members.
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SIST EN ISO 15216-1:2017
EN ISO 15216-1:2017 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 15216-1:2017
EN ISO 15216-1:2017 (E)
European foreword

This document (EN ISO 15216-1:2017) has been prepared by Technical Committee CEN/TC 275 “Food

analysis - Horizontal methods”, the secretariat of which is held by DIN, in collaboration with Technical

Committee ISO/TC 34 "Food products".

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by September 2017 and conflicting national standards

shall be withdrawn at the latest by September 2017.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent

rights.
This document supersedes CEN ISO/TS 15216-1:2013.

This document has been prepared under a mandate given to CEN by the European Commission and the

European Free Trade Association.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,

Cyprus, Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France,

Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands,

Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

the United Kingdom.
Endorsement notice

The text of ISO 15216-1:2017 has been approved by CEN as EN ISO 15216-1:2017 without any

modification.
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SIST EN ISO 15216-1:2017
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SIST EN ISO 15216-1:2017
INTERNATIONAL ISO
STANDARD 15216-1
First edition
2017-03
Microbiology of the food chain —
Horizontal method for determination
of hepatitis A virus and norovirus
using real-time RT-PCR —
Part 1:
Method for quantification
Microbiologie dans la chaîne alimentaire — Méthode horizontale
pour la recherche des virus de l’hépatite A et norovirus par la
technique RT-PCR en temps réel —
Partie 1: Méthode de quantification
Reference number
ISO 15216-1:2017(E)
ISO 2017
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior

written permission. Permission can be requested from either ISO at the address below or ISO’s member body in the country of

the requester.
ISO copyright office
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 1

4 Principle ........................................................................................................................................................................................................................ 3

4.1 Virus extraction ...................................................................................................................................................................................... 3

4.2 RNA extraction ........................................................................................................................................................................................ 4

4.3 Real-time RT-PCR .................................................................................................................................................................................. 4

4.4 Control materials .................................................................................................................................................................................. 4

4.4.1 Process control virus .................................................................................................................................................... 4

4.4.2 Double-stranded DNA (dsDNA) control ...................................................................................................... 4

4.4.3 EC RNA control .................................................................................................................................................................. 4

4.5 Test results.................................................................................................................................................................................................. 5

5 Reagents ........................................................................................................................................................................................................................ 5

5.1 General ........................................................................................................................................................................................................... 5

5.2 Reagents used as supplied ............................................................................................................................................................ 5

5.3 Prepared reagents ................................................................................................................................................................................ 6

6 Equipment and consumables .................................................................................................................................................................. 7

7 Sampling ........................................................................................................................................................................................................................ 9

8 Procedure..................................................................................................................................................................................................................... 9

8.1 General laboratory requirements ........................................................................................................................................... 9

8.2 Virus extraction ...................................................................................................................................................................................... 9

8.2.1 Process control virus material ............................................................................................................................. 9

8.2.2 Negative process control ........................................................................................................................................... 9

8.2.3 Food surfaces ...................................................................................................................................................................... 9

8.2.4 Soft fruit, leaf, stem and bulb vegetables .................................................................................................... 9

8.2.5 Bottled water ...................................................................................................................................................................10

8.2.6 Bivalve molluscan shellfish ..................................................................................................................................11

8.3 RNA extraction .....................................................................................................................................................................................11

8.4 Real-time RT-PCR ...............................................................................................................................................................................12

8.4.1 General requirements ...............................................................................................................................................12

8.4.2 Real-time RT-PCR analysis ....................................................................................................................................12

9 Interpretation of results ............................................................................................................................................................................14

9.1 General ........................................................................................................................................................................................................14

9.2 Construction of standard curves ..........................................................................................................................................14

9.3 Calculation of RT-PCR inhibition ..........................................................................................................................................15

9.4 Calculation of extraction efficiency ....................................................................................................................................15

9.5 Sample quantification ....................................................................................................................................................................16

10 Expression of results .....................................................................................................................................................................................17

11 Precision ....................................................................................................................................................................................................................17

11.1 Interlaboratory study .....................................................................................................................................................................17

11.2 Repeatability ..........................................................................................................................................................................................17

11.3 Reproducibility limit .......................................................................................................................................................................18

12 Test report ................................................................................................................................................................................................................18

Annex A (normative) Diagram of procedure .............................................................................................................................................19

Annex B (normative) Composition and preparation of reagents and buffers ........................................................20

Annex C (informative) Real-time RT-PCR mastermixes and cycling parameters ................................................23

© ISO 2017 – All rights reserved iii
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)

Annex D (informative) Real-time RT-PCR primers and hydrolysis probes for the detection of

HAV, norovirus GI and GII and mengo virus (process control) ..........................................................................24

Annex E (informative) Growth of mengo virus strain MC for use as a process control ..............................27

Annex F (informative) RNA extraction using the NucliSENS system .............................................................................28

Annex G (informative) Generation of dsDNA control stocks .....................................................................................................30

Annex H (informative) Generation of EC RNA stocks ........................................................................................................................33

Annex I (informative) Typical optical plate layout..............................................................................................................................35

Annex J (informative) Method validation studies and performance characteristics ......................................37

Bibliography .............................................................................................................................................................................................................................48

iv © ISO 2017 – All rights reserved
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
Foreword

ISO (the International Organization for Standardization) is a worldwide federation of national standards

bodies (ISO member bodies). The work of preparing International Standards is normally carried out

through ISO technical committees. Each member body interested in a subject for which a technical

committee has been established has the right to be represented on that committee. International

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following

URL: w w w . i s o .org/ iso/ foreword .html.

This document was prepared by the European Committee for Standardization (CEN) Technical

Committee CEN/TC 275, in collaboration with ISO Technical Committee ISO/TC 34, Food products,

Subcommittee SC 9, Microbiology, in accordance with the agreement on technical cooperation between

ISO and CEN (Vienna Agreement).

This first edition cancels and replaces ISO/TS 15216-1:2013, which has been technically revised with

the following changes:
— use of linear dsDNA molecules for quantification prescribed;
— use of a suitable buffer for dilution of control materials prescribed;

— change to the method for generating process control virus RNA for the standard curve;

— addition of breakpoints with defined temperature and time parameters in the extraction methods;

— change in terminology from amplification efficiency to RT-PCR inhibition;
— addition of extra real-time RT-PCR reactions for negative controls;
— addition of precision data and results of interlaboratory study.
A list of all parts in the ISO 15216 series can be found on the ISO website.
© ISO 2017 – All rights reserved v
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
Introduction

Hepatitis A virus (HAV) and norovirus are important agents of food-borne human viral illness. No

routine methods exist for culture of norovirus, and HAV culture methods are not appropriate for routine

application to food matrices. Detection is therefore reliant on molecular methods using the reverse-

transcriptase polymerase chain reaction (RT-PCR). As many food matrices contain substances that

are inhibitory to RT-PCR, it is necessary to use an extraction method that produces highly clean RNA

preparations that are fit for purpose. For food surfaces, viruses are removed by swabbing. For soft fruit,

leaf, stem and bulb vegetables, virus extraction is by elution with agitation followed by precipitation

with PEG/NaCl. For bottled water, adsorption and elution using positively charged membranes followed

by concentration by ultrafiltration is used and for bivalve molluscan shellfish (BMS), viruses are

extracted from the tissues of the digestive glands using treatment with a proteinase K solution. For all

matrices that are not covered by this document, it is necessary to validate this method. All matrices

share a common RNA extraction method based on virus capsid disruption with chaotropic reagents

followed by adsorption of RNA to silica particles. Real-time RT-PCR monitors amplification throughout

the real-time RT-PCR cycle by measuring the excitation of fluorescently labelled molecules. In real-

time RT-PCR with hydrolysis probes, the fluorescent label is attached to a sequence-specific nucleotide

probe that also enables simultaneous confirmation of target template. These modifications increase

the sensitivity and specificity of the real-time RT-PCR method, and obviate the need for additional

amplification product confirmation steps post real-time RT-PCR. Due to the complexity of the method,

it is necessary to include a comprehensive suite of controls. The method described in this document

enables quantification of levels of virus RNA in the test sample. A schematic diagram of the testing

procedure is shown in Annex A.

The main changes, listed in the Foreword, introduced in this document compared to ISO/TS 15216-

1:2013 are considered as minor (see ISO 17468).
vi © ISO 2017 – All rights reserved
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SIST EN ISO 15216-1:2017
INTERNATIONAL STANDARD ISO 15216-1:2017(E)
Microbiology of the food chain — Horizontal method for
determination of hepatitis A virus and norovirus using
real-time RT-PCR —
Part 1:
Method for quantification
1 Scope

This document specifies a method for the quantification of levels of HAV and norovirus genogroup I

(GI) and II (GII) RNA, from test samples of foodstuffs (soft fruit, leaf, stem and bulb vegetables, bottled

water, BMS) or food surfaces. Following liberation of viruses from the test sample, viral RNA is then

extracted by lysis with guanidine thiocyanate and adsorption on silica. Target sequences within the

viral RNA are amplified and detected by real-time RT-PCR.

This method is not validated for detection of the target viruses in other foodstuffs (including multi-

component foodstuffs), or any other matrices, nor for the detection of other viruses in foodstuffs, food

surfaces or other matrices.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for

microbiological examinations

ISO 20838, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the

detection of food-borne pathogens — Requirements for amplification and detection for qualitative methods

ISO 22119, Microbiology of food and animal feeding stuffs — Real-time polymerase chain reaction (PCR) for

the detection of food-borne pathogens — General requirements and definitions

ISO 22174, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the

detection of food-borne pathogens — General requirements and definitions
3 Terms and definitions

For the purposes of this document, the terms and definitions given in ISO 22174, ISO 22119 and

ISO 20838 and the following apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
foodstuff
substance used or prepared for use as food

Note 1 to entry: For the purposes of this document, this definition includes bottled water.

© ISO 2017 – All rights reserved 1
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
3.2
food surface
surface of food, food preparation surface or food contact surface
3.3
soft fruit
small edible stoneless fruit
EXAMPLE Strawberries, raspberries or currants
3.4
leaf, stem and bulb vegetables
leaves, stems and bulbs of plants, eaten as a vegetable
3.5
hepatitis A virus
HAV
member of the Picornaviridae family responsible for infectious hepatitis

Note 1 to entry: Genetically, HAV can be subdivided into six genotypes on the basis of the VP1/2A region

(genotypes 1, 2, and 3 have been found in humans, while genotypes 4, 5, and 6 are of simian origin). There is only

one serotype.

Note 2 to entry: Transmission occurs via the faecal-oral route by person-to-person contact, through the

consumption of contaminated foodstuffs, contact with contaminated water or food surfaces, or contact with

contaminated fomites. HAV is classified as a group 2 biological agent by the European Union and as a risk group 2

human aetiological agent by the United States National Institutes of Health.
3.6
norovirus

member of the Caliciviridae family responsible for sporadic cases and outbreaks of acute gastroenteritis

Note 1 to entry: Genetically, norovirus can be subdivided into seven separate genogroups. Three of these

genogroups, GI, GII and GIV have been implicated in human gastrointestinal disease. GI and GII are responsible

for the vast majority of clinical cases.

Note 2 to entry: Transmission occurs via the faecal-oral route by person-to-person contact, through the

consumption of contaminated foodstuffs or through contact with contaminated water or food surfaces or contact

with contaminated fomites. GI and GII noroviruses are classified as group 2 biological agents by the European

Union and as risk group 2 human aetiological agents by the United States National Institutes of Health.

3.7
quantification of HAV

estimation of number of copies of HAV RNA in a predetermined mass or volume of foodstuff, or area of

food surface
3.8
quantification of norovirus

estimation of number of copies of norovirus RNA in a predetermined mass or volume of foodstuff, or

area of food surface
3.9
process control virus

virus added to the sample portion at the earliest opportunity prior to virus extraction to control for

extraction efficiency
3.10
process control virus RNA

RNA extracted from the process control virus in order to produce standard curve data for the estimation

of extraction efficiency
2 © ISO 2017 – All rights reserved
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SIST EN ISO 15216-1:2017
ISO 15216-1:2017(E)
3.11
negative RNA extraction control

control free of target RNA carried through all steps of the RNA extraction and detection procedure to

monitor any contamination events
3.12
negative process control

target pathogen-free sample of the food matrix, or target pathogen-free non-matrix sample, that is run

through all stages of the analytical process
3.13
hydrolysis probe

fluorescent probe coupled with a fluorescent reporter molecule and a quencher molecule, which are

sterically separated by the 5′-3′-exonuclease activity of the enzyme during the amplification process

3.14
negative real-time RT-PCR control

aliquot of highly pure water used in a real-time RT-PCR reaction to control for contamination in the

real-time RT-PCR reagents
3.15
external control RNA
EC RNA

reference RNA that can be used to assess inhibition of amplification for the real-time RT-PCR assay of

relevance by being added in a defined amount to an aliquot of sample RNA in a separate reaction

EXAMPLE RNA synthesized by in-vitro transcription from a plasmid carrying a copy of the target gene

3.16
C value

quantification cycle; the cycle at which the target is quantified in a given real-time RT-PCR reaction

Note 1 to entry: This corresponds to the poin
...

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