ISO/TS 21872-2:2020

TECHNICAL ISO/TS
SPECIFICATION 21872-2
First edition
2020-10
Microbiology of the food chain —
Horizontal method for the
determination of Vibrio spp. —
Part 2:
Enumeration of total and
potentially enteropathogenic Vibrio
parahaemolyticus in seafood using
nucleic acid hybridization
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la détermination des Vibrio spp. —
Partie 2: Dénombrement de Vibrio parahaemolyticus total et
potentiellement entéropathogène dans les fruits de mer, par
hybridation des acides nucléiques
Reference number
©
ISO 2020
© ISO 2020
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ii © ISO 2020 – All rights reserved

Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principle . 2
4.1 General . 2
4.2 Plating . 2
4.3 Preparation of membranes . 2
4.4 Hybridization. 2
4.5 Detection . 2
5 Culture media and reagents . 3
5.1 General . 3
5.2 Hybridization reagents . 3
5.2.1 Hybridization buffer . 3
5.2.2 Digoxigenin-labelled oligoprobes . 3
5.2.3 Control materials . 3
6 Equipment and consumables . 3
7 Sampling . 4
8 Procedure. 4
8.1 General . 4
8.2 Preparation of the sample . 4
8.3 Plating . 4
8.4 Preparation of process control . 4
8.5 Preparation of membranes . 4
8.5.1 Colony lift . 4
8.5.2 Cross-linking of DNA to the membrane . 5
8.5.3 Proteinase K treatment . 5
8.6 Hybridization. 5
8.6.1 General. 5
8.6.2 Hybridization of tdh and trh probes . 6
8.6.3 Colorimetric detection . 6
8.6.4 Enumeration of tdh and/or trh positive colonies . 7
8.6.5 Isolation of colonies (optional) . 7
8.6.6 Hybridization of toxR probe . 7
8.6.7 Colorimetric detection . 8
8.6.8 Enumeration of toxR positive colonies . 8
9 Interpretation of the results . 8
10 Expression of results . 9
11 Test report . 9
Annex A (normative) Flow diagram for the enumeration of total and potentially
enteropathogenic Vibrio parahaemolyticus in seafood using nucleic acid hybridization .10
Annex B (normative) Culture media and reagents .13
Annex C (informative) Hybridization of tdh, trh and toxR genes of V. parahaemolyticus .19
Bibliography .27
Foreword
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This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
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iv © ISO 2020 – All rights reserved

Introduction
Potentially enteropathogenic strains of Vibrio parahaemolyticus possess thermostable direct haemolysin
(TDH) and/or thermostable direct hemolysin-related hemolysin (TRH). TDH positive strains manifest
[1]
Kanagawa phenomenon (KP) . This characteristic is traditionally utilized in the identification of
enterotoxigenic strains of V. parahaemolyticus. Strains possessing TRH do not share the haemolytic
characteristics of TDH positive isolates and no conventional identification assay has been reported for
[2]
TRH identification. Pathogenic strains in the environment are a minority and differentiation between
enteropathogenic and total V. parahaemolyticus presence is therefore useful.
This document enables the enumeration of potentially enteropathogenic V. parahaemolyticus and/or of
total V. parahaemolyticus.
TECHNICAL SPECIFICATION ISO/TS 21872-2:2020(E)
Microbiology of the food chain — Horizontal method for
the determination of Vibrio spp. —
Part 2:
Enumeration of total and potentially enteropathogenic
Vibrio parahaemolyticus in seafood using nucleic acid
hybridization
1 Scope
This document specifies a method for the direct enumeration of potentially enteropathogenic V.
parahaemolyticus (tdh and/or trh positive) and/or the enumeration of total V. parahaemolyticus in
seafood.
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 6887-1, 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 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media
3 Terms and definitions
For the purposes of this document, th
...