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)

ISO 21872-1:2017 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.
ISO 21872-1:2017 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.

Mikrobiologie der Lebensmittelkette — Horizontales Verfahren zur Bestimmung von Vibrio spp. — Teil 1: Nachweis von potentiell enteropathenogenen Vibrio parahaemolyticus, Vibrio cholerae und Vibrio vulnificus (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

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Published
Publication Date
11-Jul-2017
Current Stage
6060 - Definitive text made available (DAV) - Publishing

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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.

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

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

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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.
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SIST EN ISO 21872-1:2017
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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
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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. +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 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
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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

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
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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
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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
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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
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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
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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
...

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