SIST EN ISO 21872-1:2017

Microbiology of the food chain - Horizontal method for the determination of Vibrio spp. - Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus (ISO 21872-1:2017)

This standard describes the detection of pathogenic Vibrio parahaemolyticus and Vibrio cholerae (Reference document is ISO/TS 21872 -1)

Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum Nachweis von potentiell enteropathogenen Vibrio spp. - Teil 1: Nachweis von vibrio parahaemolyticus und vibrio cholerae (ISO 21872-1:2017)

Dieses Dokument legt ein horizontales Verfahren zum Nachweis von enteropathogenen Vibrio spp. fest, die Erkrankungen des Menschen am oder über den Darmtrakt verursachen. Die Spezies, deren Nachweis die hier aufgeführten Verfahren einbeziehen, sind Vibrio parahaemolyticus, Vibrio cholerae und Vibrio vulnificus.
Das Dokument gilt für
—   Produkte, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind;
—   Umgebungsproben im Bereich der Herstellung von Lebensmitteln und beim Umgang mit Lebensmitteln.
ANMERKUNG 1   Dieses Verfahren ist für bestimmte Produkte nicht unbedingt bis ins Detail geeignet (siehe Einleitung).
ANMERKUNG 2   Die Weltgesundheitsorganisation (WHO; en: World Health Organization) hat V. parahaemolyticus, V. cholerae und V. vulnificus als die wichtigsten lebensmittelassoziierten Vibrio spp. identifiziert. Das Verfahren in diesem Dokument kann jedoch ebenso für die Identifizierung anderer Vibrio spp., die Erkrankungen des Menschen verursachen, geeignet sein [1].

Microbiologie de la chaîne alimentaire - Méthode horizontale pour la détermination des Vibrio spp. - Partie 1: Recherche des espèces de Vibrio parahaemolyticus, Vibrio cholerae et Vibrio vulnificus potentiellement entéropathogènes (ISO 21872-1:2017)

ISO 21872-1:2017 spécifie une méthode horizontale pour la recherche des espèces entéropathogènes de Vibrio provoquant des maladies dans ou via le tractus intestinal chez l'homme. Les espèces détectables par les méthodes spécifiées incluent Vibrio parahaemolyticus, Vibrio cholerae et Vibrio vulnificus.
ISO 21872-1:2017 s'applique:
-      aux produits destinés à la consommation humaine et à l'alimentation animale;
-      aux échantillons environnementaux dans le domaine de la production et de la manutention de denrées alimentaires.

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje Vibrio spp. - 1. del: Ugotavljanje potencialno enteropatogene Vibrio parahaemolyticus, Vibrio cholerae in Vibrio vulnificus (ISO 21872-1:2017)

Ta standard opisuje ugotavljanje patogenih vrst Vibrio parahaemolyticus in Vibrio cholerae (referenčni dokument je standard ISO/TS 21872 -1).

General Information

• Status: Published
• Public Enquiry End Date: 29-May-2016
• Publication Date: 20-Aug-2017
• ICS: 07.100.30 - Food microbiology
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 01-Aug-2017
• Due Date: 06-Oct-2017
• Completion Date: 21-Aug-2017
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Mandate: M/381 - Mandate for standardisation addressed to CEN in the field of Methods of analysis of foodstuffs concerning Food Hygiene
• Ref Project: EN ISO 21872-1:2017 - Microbiology of the food chain - Horizontal method for the determination of Vibrio spp. - Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus (ISO 21872-1:2017)

Relations

• Amended By: SIST EN ISO 21872-1:2017/A1:2023 - Microbiology of the food chain - Horizontal method for the determination of Vibrio spp. - Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus - Amendment 1: Inclusion of performance testing of culture media and reagents (ISO 21872 1:2017/Amd 1:2023)
• Effective Date: 01-Oct-2021

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 21872-1:2017
01-september-2017
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje Vibrio
spp. - 1. del: Ugotavljanje potencialno enteropatogene Vibrio parahaemolyticus,
Vibrio cholerae in Vibrio vulnificus (ISO 21872-1:2017)
Microbiology of the food chain - Horizontal method for the determination of Vibrio spp. -
Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio
cholerae and Vibrio vulnificus (ISO 21872-1:2017)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum
Nachweis von potentiell enteropathogenen Vibrio spp. - Teil 1: Nachweis von vibrio
parahaemolyticus und vibrio cholerae (ISO 21872-1:2017)
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la détermination des
Vibrio spp. - Partie 1: Recherche des espèces de Vibrio parahaemolyticus, Vibrio
cholerae et Vibrio vulnificus potentiellement entéropathogènes (ISO 21872-1:2017)
Ta slovenski standard je istoveten z: EN ISO 21872-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 21872-1:2017 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------

SIST EN ISO 21872-1:2017

---------------------- Page: 2 ----------------------

SIST EN ISO 21872-1:2017


EN ISO 21872-1
EUROPEAN STANDARD

NORME EUROPÉENNE

July 2017
EUROPÄISCHE NORM
ICS 07.100.30
English Version

Microbiology of food and animal feeding stuffs - Horizontal
method for the detection of potentially enteropathogenic
Vibrio spp. - Part 1: Detection of Vibrio parahaemolyticus
and Vibrio cholerae (ISO 21872-1:2017)
Microbiologie des aliments - Méthode horizontale pour Mikrobiologie von Lebensmitteln und Futtermitteln -
la recherche des Vibrio spp. potentiellement Horizontales Verfahren zum Nachweis von potentiell
entéropathogènes - Partie 1: Recherche de Vibrio enteropathogenen Vibrio spp. - Teil 1: Nachweis von
parahaemolyticus et Vibrio cholerae (ISO 21872- vibrio parahaemolyticus und vibrio cholerae (ISO
1:2017) 21872-1:2017)
This European Standard was approved by CEN on 14 May 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 21872-1:2017 E
worldwide for CEN national Members.

---------------------- Page: 3 ----------------------

SIST EN ISO 21872-1:2017
EN ISO 21872-1:2017 (E)
Contents Page
European foreword . 3

2

---------------------- Page: 4 ----------------------

SIST EN ISO 21872-1:2017
EN ISO 21872-1:2017 (E)
European foreword
This document (EN ISO 21872-1:2017) 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 January 2018 and conflicting national standards shall
be withdrawn at the latest by January 2018.
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 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 21872-1:2017 has been approved by CEN as EN ISO 21872-1:2017 without any
modification.
3

---------------------- Page: 5 ----------------------

SIST EN ISO 21872-1:2017

---------------------- Page: 6 ----------------------

SIST EN ISO 21872-1:2017
INTERNATIONAL ISO
STANDARD 21872-1
First edition
2017-06
Microbiology of the food chain —
Horizontal method for the
determination of Vibrio spp. —
Part 1:
Detection of potentially
enteropathogenic Vibrio
parahaemolyticus, Vibrio cholerae and
Vibrio vulnificus
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la détermination des Vibrio spp. —
Partie 1: Recherche des espèces de Vibrio parahaemolyticus, Vibrio
cholerae et Vibrio vulnificus potentiellement entéropathogènes
Reference number
ISO 21872-1:2017(E)
©
ISO 2017

---------------------- Page: 7 ----------------------

SIST EN ISO 21872-1:2017
ISO 21872-1:2017(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. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2017 – All rights reserved

---------------------- Page: 8 ----------------------

SIST EN ISO 21872-1:2017
ISO 21872-1:2017(E)

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 2
4.1 General . 2
4.2 Primary enrichment in a liquid selective medium . 2
4.3 Secondary enrichment in a liquid selective medium . 3
4.4 Isolation and identification . 3
4.5 Confirmation . 3
5 Culture media and reagents . 3
5.1 Enrichment medium: alkaline saline peptone water (ASPW). 4
5.2 Solid selective isolation media . 4
5.2.1 First medium: thiosulphate, citrate, bile and sucrose agar medium (TCBS) . 4
5.2.2 Second medium. 4
5.3 Saline nutrient agar (SNA) . 4
5.4 Reagent for detection of oxidase . 4
5.5 Biochemical tests . 4
5.5.1 L-lysine decarboxylase saline medium (LDC) . 4
5.5.2 Arginine dihydrolase saline medium (ADH) . 4
5.5.3 Reagent for detection of β-galactosidase . 5
5.5.4 Saline medium for detection of indole . 5
5.5.5 Saline peptone waters . 5
5.5.6 Sodium chloride solution . 5
5.6 PCR . 5
5.6.1 Tris acetate EDTA buffer (TAE) (or a buffer allowing similar performance
for the purpose) . 5
5.6.2 Mastermix . 5
5.6.3 Primers and probes . 5
5.6.4 Positive control material . 5
5.6.5 Negative extraction control . 6
6 Equipment and consumables . 6
7 Sampling . 6
8 Preparation of the test sample . 6
9 Procedure (See Figure A.1) . 7
9.1 Test portion and initial suspension . 7
9.2 Primary selective enrichment . 7
9.3 Secondary selective enrichment . 7
9.4 Isolation and identification . 8
9.5 Confirmation . 8
9.5.1 General. 8
9.5.2 Selection of colonies for confirmation and preparation of pure cultures . 9
9.5.3 Tests for presumptive identification . 9
9.5.4 Biochemical confirmation .10
9.5.5 PCR confirmation .12
9.5.6 DNA extraction .12
9.5.7 Conventional PCR .12
9.5.8 Real-time PCR .13
10 Expression of results .13
© ISO 2017 – All rights reserved iii

---------------------- Page: 9 ----------------------

SIST EN ISO 21872-1:2017
ISO 21872-1:2017(E)

11 Performance characteristics of the method .13
11.1 Interlaboratory study .13
11.2 Sensitivity .14
11.3 Specificity .14
11.4 LOD .14
50
12 Test report .14
Annex A (normative) Diagram of procedure .15
Annex B (normative) Composition and preparation of the culture media and reagents .17
Annex C (informative) Conventional PCR for the detection of Vibrio parahaemolyticus,
thermostable direct haemolysin (tdh) and thermostable direct related haemolysin
(trh) genes, Vibrio cholerae and Vibrio vulnificus .23
Annex D (informative) Real-time PCR for the detection of Vibrio parahaemolyticus,
thermostable direct haemolysin gene (tdh) and Vibrio vulnificus .27
Annex E (informative) Results of an interlaboratory study .29
Bibliography .32
iv © ISO 2017 – All rights reserved

---------------------- Page: 10 ----------------------

SIST EN ISO 21872-1:2017
ISO 21872-1:2017(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 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: www . i so .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 21872-1:2007, which has been technically revised. It also
incorporates ISO/TS 21872-1:2007/Cor.1:2008.
The main changes are as follows:
— introduction of optional molecular identification methods for major food borne Vibrio spp.
(V. parahaemolyticus, including potentially enteropathogenic strains, V. vulnificus and V. cholerae);
— performance characteristics of the method have been added in Annex E.
A list of all parts in the ISO 21872 series can be found on the ISO website.
© ISO 2017 – All rights reserved v

---------------------- Page: 11 ----------------------

SIST EN ISO 21872-1:2017
ISO 21872-1:2017(E)

Introduction
Because of the large variety of food and feed products, the horizontal method described in this
document may not be appropriate in every detail for certain products. In this case, different methods,
which are specific to these products may be used if absolutely necessary for justified technical reasons.
Nevertheless, every attempt will be made to apply this horizontal method as far as possible.
The main changes, listed in the foreword, introduced in this document compared to ISO/TS 21872-
1:2007 are considered as major (see ISO 17468).
When this document is next reviewed, account will be taken of all information then available regarding
the extent to which this horizontal method has been followed and the reasons for deviations from this
method in the case of particular products.
The harmonization of test methods cannot be immediate and, for certain groups of products,
International Standards and/or national standards may already exist that do not comply with this
horizontal method. It is hoped that when such standards are reviewed they will be changed to comply
with this document so that eventually the only remaining departures from this horizontal method will
be those necessary for well-established technical reasons.
vi © ISO 2017 – All rights reserved

---------------------- Page: 12 ----------------------

SIST EN ISO 21872-1:2017
INTERNATIONAL STANDARD ISO 21872-1:2017(E)
Microbiology of the food chain — Horizontal method for
the determination of Vibrio spp. —
Part 1:
Detection of potentially enteropathogenic Vibrio
parahaemolyticus, Vibrio cholerae and Vibrio vulnificus
WARNING — In order to safeguard the health of laboratory personnel, it is essential that
tests for detection of Vibrio spp., and particularly toxigenic Vibrio cholerae, be conducted
only in laboratories equipped for this purpose and under the supervision of an experienced
microbiologist, and that great care is exercised in the disposal of contaminated material.
1 Scope
This document specifies a horizontal method for the detection of enteropathogenic Vibrio spp., which
causes human illness in or via the intestinal tract. The species detectable by the methods specified
include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
It is applicable to the following:
— products intended for human consumption and the feeding of animals;
— environmental samples in the area of food production and food handling.
NOTE 1 This method may not be appropriate in every detail for certain products (see Introduction).
NOTE 2 The World Health Organization (WHO) has identified that V. parahaemolyticus, V. cholerae and V.
vulnificus are the major food-borne Vibrio spp. However, the method in this document can also be appropriate for
[1]
the identification of other Vibrio spp. causing illness in humans.
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 6887-1:2017, Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initial
suspension and decimal dilutions
ISO 6887-3, Microbiology of the food chain — Preparation of test samples, initial suspension and decimal
dilutions for microbiological examination — Part 3: Specific rules for the preparation of fish and fishery
products
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
ISO 22118, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection and quantification of food-borne pathogens — Performance characteristics
© ISO 2017 – All rights reserved 1

---------------------- Page: 13 ----------------------

SIST EN ISO 21872-1:2017
ISO 21872-1:2017(E)

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 following terms and definitions 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
potentially enteropathogenic Vibrio spp.
microorganism which forms typical colonies on solid selective media and which possesses the described
biochemical or molecular characteristics when the test is performed in accordance with this document
Note 1 to entry: This document describes specific procedures for V. parahaemolyticus, V. cholerae and V. vulnificus.
3.2
detection of potentially enteropathogenic Vibrio spp.
determination of the presence or absence of potentially enteropathogenic Vibrio spp. (3.1)
(V. parahaemolyticus, V. cholerae and V. vulnificus) in a determined quantity of product, when the test is
performed in accordance with this document
4 Principle
4.1 General
The detection of potentially enteropathogenic Vibrio spp. (V. parahaemolyticus, V. cholerae and
V. vulnificus) requires four successive phases, as shown in the procedure diagram in Annex A.
Recovery of certain Vibrio spp. from foodstuffs may be improved by the use of different incubation
temperatures depending upon the target species or state of the food matrix. For example, recovery of
V. parahaemolyticus and V. cholerae in fresh products is enhanced by enrichment at 41,5 °C whereas
[2]
for V. vulnificus, and for V. parahaemolyticus and V. cholerae in deep frozen (<−18 °C), dried or salted
products, recovery is enhanced by enrichment at 37 °C. If detection of V. parahaemolyticus, V. cholerae
and V. vulnificus is required, all specified incubation temperatures should be used. If detection of
V. parahaemolyticus, V. cholerae and V. vulnificus together is not required, the specific procedure(s) may
be selected according to the species being sought. Such a selection should be clearly specified in the
test report.
NOTE V. parahaemolyticus, V. cholerae and V. vulnificus may be present in small numbers and are often
accompanied by a much larger number of other microorganisms belonging to the Vibrionaceae family or to other
families.
4.2 Primary enrichment in a liquid selective medium
Inoculation of the test portion in the primary enrichment medium alkaline saline peptone water
(ASPW) (5.1) at ambient temperature, followed by incubation at 41,5 °C for 6 h and/or 37 °C for 6 h.
The incubation conditions are determined by the target species and food product state.
For detection of all target species in deep frozen, dried or salted products, primary enrichment should
be at 37 °C.
2 © ISO 2017 – All rights reserved

---------------------- Page: 14 ----------------------

SIST EN ISO 21872-1:2017
ISO 21872-1:2017(E)

For detection of V. vulnificus in all products, primary enrichment should be at 37 °C.
For detection of V. parahaemolyticus and/or V. cholerae only, in fresh products, primary enrichment
should be at 41,5 °C.
4.3 Secondary enrichment in a liquid selective medium
Inoculation of the second enrichment medium (ASPW) with the cultures obtained in 4.2.
Incubation of inoculated enrichment medium at 41,5 °C for 18 h and/or 37 °C for 18 h.
For detection of V. vulnificus in all products, secondary enrichment should be at 37 °C.
For detection of V. parahaemolyticus and/or V. cholerae only, in all produc
...

SLOVENSKI STANDARD
oSIST prEN ISO 21872:2016
01-maj-2016
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje
potencialno enteropatogene Vibrio parahaemolyticus, Vibrio cholerae in Vibrio
vulnificus (ISO/DIS 21872:2016)
Microbiology of the food chain - Horizontal method for the detection of potentially
enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus (ISO/DIS
21872:2016)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum
Nachweis von potentiell enteropathogenen Vibrio spp. - Teil 1: Nachweis von vibrio
parahaemolyticus und vibrio cholerae
Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche des
Vibrio spp. potentiellement entéropathogènes - Recherche des espèces de Vibrio
parahaemolyticus, Vibrio cholerae et Vibrio vulnificu (ISO/DIS 21872:2016)
Ta slovenski standard je istoveten z: prEN ISO 21872
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 21872:2016 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

---------------------- Page: 1 ----------------------
oSIST prEN ISO 21872:2016

---------------------- Page: 2 ----------------------
oSIST prEN ISO 21872:2016
DRAFT INTERNATIONAL STANDARD
ISO/DIS 21872
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2016-03-17 2016-06-17
Microbiology of the food chain — Horizontal method
for the detection of potentially enteropathogenic Vibrio
parahaemolyticus, Vibrio cholerae and Vibrio vulnificus
Microbiologie de la chaîne alimentaire — Méthode horizontale pour la recherche des Vibrio spp.
potentiellement entéropathogènes — Recherche des espèces de Vibrio parahaemolyticus, Vibrio cholerae
et Vibrio vulnificu
ICS: 07.100.30
ISO/CEN PARALLEL PROCESSING
This draft has been developed within the European Committee for Standardization
(CEN), and processed under the CEN lead mode of collaboration as defined in the
Vienna Agreement.
This draft is hereby submitted to the ISO member bodies and to the CEN member
bodies for a parallel three month enquiry.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
To expedite distribution, this document is circulated as received from the
IN ADDITION TO THEIR EVALUATION AS
committee secretariat. ISO Central Secretariat work of editing and text
BEING ACCEPTABLE FOR INDUSTRIAL,
composition will be undertaken at publication stage.
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 21872:2016(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
©
PROVIDE SUPPORTING DOCUMENTATION. ISO 2016

---------------------- Page: 3 ----------------------
oSIST prEN ISO 21872:2016
ISO/DIS 21872:2016(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, 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. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2016 – All rights reserved

---------------------- Page: 4 ----------------------
oSIST prEN ISO 21872:2016
ISO DIS 21872:2016 (E)
Summary
Pages
Foreword. 5
Introduction . 6
1 Scope. 7
2 Normatives references . 7
3 Terms and definitions . 8
3.1 Potentially enteropathogenic Vibrio spp. 8
3.2 Detection of potentially enteropathogenic Vibrio spp. . 8
4 Principle . 8
4.1 General . 8
4.2 Primary enrichment in a liquid selective medium . 8
4.3 Secondary enrichment in a liquid selective medium . 8
4.4 Isolation and identification. 9
4.5 Confirmation . 9
5 Culture media and reagents. 9
5.1 Enrichment medium: alkaline saline peptone water (ASPW) . 9
5.2 Solid selective isolation media . 9
5.3 Saline nutrient agar (SNA) . 10
5.4 Reagent for detection of oxidase . 10
5.5 Biochemical tests. 10
5.6 PCR . 10
6 Apparatus and glassware . 11
7 Sampling . 12
8 Preparation of the test sample . 12
9 Procedure . 12
9.1 Test portion and initial suspension. 12
9.2 Primary selective enrichment. 13
9.3 Secondary selective enrichment . 13
9.4 Isolation and identification. 14
9.5 Confirmation . 14
10 Expression of results . 20
11 Performance characteristics of the method. 20
11.1 Interlaboratory study. 20
11.2 Sensitivity . 20
11.3 Specificity . 20
11.4 LOD . 20
50
12 Test report . 20
Annex A (normative) Diagram of procedure . 22
Annex B (normative) Composition and preparation of the culture media and reagents. 25
B.1 Alkaline saline peptone water (ASPW) . 25
B.2 Thiosulfate Citrate Bile and Sucrose agar (TCBS) . 25
B.3 Saline nutrient agar (SNA) . 26
B.4 Reagent for detection of oxidase . 27
© ISO 2016 – All rights reserved 3

---------------------- Page: 5 ----------------------
oSIST prEN ISO 21872:2016
ISO DIS 21872:2016 (E)
B.5 Lysine decarboxylase saline medium (LDC) . 27
B.6 Arginine dehydroxylase saline medium (ADH) . 27
B.7 Detection of -galactosidase . 28
B.8 Saline medium for detection of indole . 29
B.9 Saline peptone waters. 29
B.10 Sodium chloride solution . 30
B.11 TAE buffer . 30
Annex C (informative) Conventional PCR for the detection of Vibrio parahaemolyticus,
thermostable direct haemolysin (tdh) and thermostable direct related haemolysin
(tdh) genes, Vibrio cholerae and Vibrio vulnificus . 31
C.1 Composition of mastermix. 31
C.2 Vibrio parahaemolyticus primers (conventional PCR) . 31
C.3 Thermostable direct haemolysin (tdh) and thermostable direct related haemolysin
(tdh) genes Vibrio parahaemolyticus primers (conventional PCR). 31
C.4 Vibrio cholerae primers (conventional PCR). 32
C.5 Vibrio vulnificus primers (conventional PCR) . 32
C.6 Cycling parameters – VptoxR and VVH. 32
C.7 Cycling parameters – prVC . 32
C.8 Cycling parameters - tdh and trh. 32
C.9 Control material - conventional PCR . 33
C.10 Interlaboratory study. 33
Annex D (informative) Real-time PCR for the detection of Vibrio parahaemolyticus,
thermostable direct haemolysin (tdh) and Vibrio vulnificus . 35
D.1 Composition of mastermix. 35
D.2 Vibrio parahaemolyticus - primers and hydrolysis probes . 35
D.3 Thermostable direct haemolysin (tdh) gene Vibrio parahaemolyticus - primers and
hydrolysis probes. 35
D.4 Vibrio vulnificus - primers and hydrolysis probes . 36
D.5 Cycling parameters . 36
D.6 Control material – real-time PCR . 36
Annex E (informative) Results of interlaboratory study. 37
Bibliography . 41

4 © ISO 2016 – All rights reserved

---------------------- Page: 6 ----------------------
oSIST prEN ISO 21872:2016
ISO DIS 21872:2016 (E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national
standards bodies (ISO member bodies). The work of preparing International Standards is normally
carried out through ISO technical committees. Each member body interested in a subject for which a
technical committee has been established has the right to be represented on that committee.
International organizations, governmental and non-governmental, in liaison with ISO, also take part in
the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all
matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International
Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies
casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 21872 was prepared by the European Committee for Standardization (CEN), Technical Committee
CEN/TC 275 Food analysis — Horizontal methods, in collaboration with 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 the technical specifications ISO/TS 21872 -1:2007 and ISO/TS
21872-2:20027which have been technically revised.
Main changes :
- same procedure for the detection of major food borne Vibrio spp.
- performance characteristics of the method have been added to Annex E.
© ISO 2016 – All rights reserved 5

---------------------- Page: 7 ----------------------
oSIST prEN ISO 21872:2016
ISO DIS 21872:2016 (E)
Introduction
Because of the large variety of food and feed products, this horizontal method may not be appropriate
in every detail for certain products. In this case, different methods, which are specific to these products
may be used if absolutely necessary for justified technical reasons. Nevertheless, every attempt will be
made to apply this horizontal method as far as possible.
When this International Standard is next reviewed, account will be taken of all information then
available regarding the extent to which this horizontal method has been followed and the reasons for
deviations from this method in the case of particular products.
The harmonisation of test methods cannot be immediate, and for certain groups of products
International Standards and/or national standards may already exist that do not comply with this
horizontal method. It is hoped that when such standards are reviewed they will be changed t o comply
with this International Standard so that eventually the only remaining departures from this horizontal
method will be those necessary for well-established technical reasons.
6 © ISO 2016 – All rights reserved

---------------------- Page: 8 ----------------------
oSIST prEN ISO 21872:2016
ISO DIS 21872:2016 (E)
Microbiology of the food chain — Horizontal method for the
detection of enteropathogenic Vibrio parahaemolyticus, Vibrio
cholerae and Vibrio vulnificus
1 Scope
This standard specifies a horizontal method for the detection of enteropathogenic Vibrio species,
causing human illness in or via the intestinal tract. The species detect able by the methods specified
include Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus.
Taking into account the remarks made in the introduction, this International Standard is applicable to:
— products intended for human consumption and the feeding of animals;
— environmental samples in the area of food production and food handling.
NOTE 1 Reasons for not applying this method are discussed in the Introduction.
NOTE 2 The World Health Organisation (WHO) have identified that V. parahaemolyticus, V. cholerae and V.
vulnificus are the major food borne Vibrio spp. however this standard can also be appropriate for the identification
[1]
of other Vibrio spp. causing illness in humans .
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
detection of Vibrio, and particularly toxigenic V. cholerae, be conducted only in laboratories
equipped for this purpose and under the supervision of an experienced microbiologist, and that
great care is exercised in the disposal of contaminated material.
2 Normatives references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO 6887, Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspension
and decimal dilutions for microbiological examination.
ISO 7218, Microbiology of food and animal feeding stuffs — General rules for microbiological
examinations.
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
ISO 22174, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection of food-borne pathogens — General requirements and definitions.
ISO 22118, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the
detection and quantification of food-borne pathogens — Performance characteristics.
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 2016 – All rights reserved 7

---------------------- Page: 9 ----------------------
oSIST prEN ISO 21872:2016
ISO DIS 21872:2016 (E)
3 Terms and definitions
3.1 Potentially enteropathogenic Vibrio spp.
microorganisms which form typical colonies on solid selective media and which possess the described
biochemical or molecular characteristics when the test is performed according to this International
Standard.
Note 1 to entry: This International Standard describes specific procedures for V.
parahaemolyticus, V. cholerae and V. vulnificus
3.2 Detection of potentially enteropathogenic Vibrio spp.
determination of the presence or absence of potentially enteropathogenic Vibrio spp. (V.
parahaemolyticus, V. cholerae and V. vulnificus) (3.1), in a determined quantity of product, when the test
is performed in accordance with this International Standard
4 Principle
4.1 General
The detection of potentially enteropathogenic Vibrio spp. (V. parahaemolyticus, V. cholerae and V.
vulnificus) requires four successive phases (see also Annex A).
Recovery of certain Vibrio spp. from foodstuffs may be improved by the use of different incubation
temperatures depending upon the target species or state of the food matrix. For example, recovery of V.
parahaemolyticus and V. cholerae in fresh products is enhanced by enrichment at 41,5 °C whereas for V.
vulnificus, and for V. parahaemolyticus and V. cholerae in deep frozen, dried or salted products, recovery
is enhanced by enrichment at 37 °C. The method combines choleragenic and specific halophilic culture-
based methods for use where mixtures of Vibrio species are present. Users of this International
Standard requiring detection of V. parahaemolyticus, V. cholerae and V. vulnificus should use all specified
incubation temperatures. Users of this International Standard who do not require detection of V.
parahaemolyticus, V. cholerae and V. vulnificus together may select the specific procedure(s) according
to the species they are required to detect. Such a selection should be clearly specified in the test report.
NOTE V. parahaemolyticus, V. cholerae and V. vulnificus may be present in small numbers and are often
accompanied by a much larger number of other microorganisms belonging to the Vibrionaceae family or to other
families. Consequently, two successive selective enrichments can be necessary in order to detect the target
organisms.
4.2 Primary enrichment in a liquid selective medium
Inoculation of the test portion in the primary enrichment medium (ASPW) (5.1) at ambient
temperature, followed by incubation at 41,5 °C 1°C for 6 h 1 h and 37 °C 1°C for 6 h 1 h.
For deep frozen, dried or salted products primary enrichment at 41,5 °C shall be omitted irrespective of
the target species.
For detection of V. vulnificus only, primary enrichment at 41,5 °C shall be omitted.
For detection of V. parahaemolyticus and/or V. cholerae only, in fresh products, primary enrichment at
37 °C can be omitted.
4.3 Secondary enrichment in a liquid selective medium
Inoculation of the second enrichment medium (ASPW) with the cultures obtained in 4.2.
8 © ISO 2016 – All rights reserved

---------------------- Page: 10 ----------------------
oSIST prEN ISO 21872:2016
ISO DIS 21872:2016 (E)
Incubation of inoculated enrichment medium at 41,5 °C 1 °C for 18 h  1 h and/or 37 °C 1 °C for 18 h
 1 h.
For detection of V. parahaemolyticus and/or V. cholerae only, secondary enrichment at 37 °C can be
omitted.
4.4 Isolation and identification
From the cultures obtained in 4.2 and in 4.3, inoculation of two solid selective media:
— Thiosulfate Citrate Bile and Sucrose agar (TCBS) medium (5.2.1);
— another appropriate solid selective medium (left to the choice of the laboratory), such as
chromogenic agar, complementary to the TCBS medium (5.2.2).
Incubation of the TCBS medium at 37 °C 1 °C, then examination after 24 h  3 h. Incubation the second
selective medium according to the manufacturer’s recommendations.
4.5 Confirmation
Presumptive colonies of V. parahaemolyticus, V. cholerae and V. vulnificus isolated in 4.4 are subcultured
and confirmed by means of appropriate biochemical and/or polymerase chain (PCR) reaction tests.
Users of this International Standard may use biochemical and/or PCR confirmation of isolates.
Detection of enteropathogenic V. parahaemolyticus as determined by presence of the direct
thermostable haemolysin (tdh) and/or direct related haemolysin (trh) genes can only be carried out
using PCR tests.
NOTE PCR based identification can be achieved by conventional PCR for V. parahaemolyticus, V. vulnificus and
V. cholerae or real-time PCR for V. parahaemolyticus, and V. vulnificus. The PCR methods used in the development
of this Standard are given in Annexes C and D.
5 Culture media and reagents
For general laboratory practice, see ISO 7218.
NOTE 1 On account of the large number of culture media and reagents, it has been considered preferable, for
clarity of the text, to give their composition and their preparation in Annex B.
NOTE 2 Primers, probes and PCR running conditions used in the development of this Standard are given in
Annexes C and D.
5.1 Enrichment medium: alkaline saline peptone water (ASPW)
See B.1.
5.2 Solid selective isolation media
5.2.1 First medium: Thiosulphate, Citrate, Bile and Sucrose agar medium (TCBS)
See B.2.
See ISO 11133.
Table 1 — Performance testing of Thiosulphate, Citrate, Bile and Sucrose agar medium (TCBS)
© ISO 2016 – All rights reserved 9

---------------------- Page: 11 ----------------------
oSIST prEN ISO 21872:2016
ISO DIS 21872:2016 (E)
a
Function Incubation Control strains WCDM  Criteria Characteristic reactions
Vibrio parahaemolyticus
37 °C ± 1 °C
b
00185  Good growth Green (sucrose negative)
for 18 - 24 h

Productivity
Vibrio furnissii
37 °C ± 1 °C
b
00186  Good growth Yellow (sucrose positive)
for 18 - 24 h

c, d
Escherichia coli
37 °C ± 1 °C 00012, 00013
Selectivity Total inhibition -
for 18 - 24 h or 00090

a
World Data Centre for Microorganisms (WDCM) strain catalogue available on http://refs.wdcm.org
b
Strain to be used as a minimum
c
Some national restrictions and directions may require the use of a different E. coli serovar. Make reference to national
requirements relating to the choice of E. coli serovars.
d
 Strain free of choice; one of the strains has to be used as a minimum.
5.2.2 Second medium
The selection of the second medium is left to the choice of the test laboratory. Preparation of the
medium should be strictly according to the manufacturer’s instructions.
5.3 Saline nutrient agar (SNA)
See B.3.
5.4 Reagent for detection of oxidase
See B.4.
5.5 Biochemical tests
5.5.1 Lysine decarboxylase saline medium (LDC)
See B.5.
5.5.2 Arginine dihydrolase saline medium (ADH)
See B.6.
5.5.3 Reagent for detection of -galactosidase
See B.7.
5.5.4 Saline medium for detection of indole
See B.8.
5.5.5 Saline peptone waters
See B.9.
5.5.6 Sodium chloride solution
See B.10.
5.6 PCR
5.6.1 TAE buffer (or a buffer allowing similar performance for the purpose).
10 © ISO 2016 – All rights reserved

---------------------- Page: 12 ----------------------
oSIST prEN ISO 21872:2016
ISO DIS 21872:2016 (E)
See B.11.
5.6.2 Mastermix
Reagents shall be added in quantities as specified by the manufacturer’s instructions. See Annexes C and
D (informative) for example details of master mixes used in the development of this Standard.
5.6.3 Primers and probes
Primer (and hydrolysis probe) sequences if required shall be published in a peer -reviewed journal and
be verified for use against a broad range of target Vibrio and non-target strains.
For V. parahaemolyticus the target region should be toxR.
For determination of pathogen strains of V. parahaemolyticus genes encoding the thermostable direct
(TDH) and the thermostable direct related (TRH) haemolysins should be targeted.
For V. cholerae the target region for conventional PCR should be the 16S-23S rRNA intergenic spacer
region prVC.
For V. vulnificus the target region should be the cytotoxin-haemolsyin region.
Target regions other than those specified above for the identification of V. parahaemolyticus,
V. vulnificus and V. cholerae can be used if they have been shown to demonstrate equivalent
performance, are published in a peer-reviewed journal and are verified against a broad range of target
Vibrio and non-target strains.
See
...