prEN ISO 13136-2

SLOVENSKI STANDARD
01-april-2024
Mikrobiologija v prehranski verigi - Odkrivanje prisotnosti, izolacija in
karakterizacija Escherichia coli (STEC), ki proizvaja Shiga toksin - 2. del:
Horizontalna metoda za karakterizacijo izolatov Escherichia coli (STEC), ki
proizvaja Shiga toksin (ISO/DIS 13136-1:2024)
Microbiology of the food chain - Detection, isolation and characterization of Shiga toxin-
producing Escherichia coli (STEC) - Part 2: Horizontal method for the characterization of
Shiga toxin-producing Escherichia coli (STEC) isolates (ISO/DIS 13136-2:2024)
Mikrobiologie der Lebensmittelkette - Nachweis, Isolierung und Charakterisierung von
Shiga-Toxin bildenden Escherichia coli (STEC) - Teil 2: Horizontales Verfahren zur
Charakterisierung von Shiga-Toxin bildenden Escherichia coli (STEC) Isolaten (ISO/DIS
13136-2:2024)
Microbiologie de la chaîne alimentaire - Détection, isolement et caractérisation des
Escherichia coli producteurs de Shigatoxines (STEC) - Partie 2: Méthode horizontale
pour la caractérisation d’isolats des Escherichia coli producteurs de Shigatoxines
(STEC) (STEC) (ISO/DIS 13136-2:2024)
Ta slovenski standard je istoveten z: prEN ISO 13136-2
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

DRAFT
International
Standard
ISO/DIS 13136-2
ISO/TC 34/SC 9
Microbiology of the food
Secretariat: AFNOR
chain — Detection, isolation and
Voting begins on:
characterization of Shiga toxin-
2024-02-28
producing Escherichia coli (STEC) —
Voting terminates on:
2024-05-22
Part 2:
Horizontal method for the
characterization of Shiga toxin-
producing Escherichia coli (STEC)
isolates
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
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IS THEREFORE SUBJECT TO CHANGE
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Reference number
ISO/DIS 13136-2:2024(en)
DRAFT
ISO/DIS 13136-2:2024(en)
International
Standard
ISO/DIS 13136-2
ISO/TC 34/SC 9
Microbiology of the food
Secretariat: AFNOR
chain — Detection, isolation and
Voting begins on:
characterization of Shiga toxin-
producing Escherichia coli (STEC) —
Voting terminates on:
Part 2:
Horizontal method for the
characterization of Shiga toxin-
producing Escherichia coli (STEC)
isolates
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENTS AND APPROVAL. IT
IS THEREFORE SUBJECT TO CHANGE
AND MAY NOT BE REFERRED TO AS AN
INTERNATIONAL STANDARD UNTIL
PUBLISHED AS SUCH.
This document is circulated as received from the committee secretariat.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
© ISO 2024
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
STANDARDS MAY ON OCCASION HAVE TO
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Published in Switzerland Reference number
ISO/DIS 13136-2:2024(en)
ii
ISO/DIS 13136-2:2024(en)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Principle . 3
5 Culture media and reagents . 3
6 Equipment and consumables . 3
7 Sampling . 4
8 Preparation of test sample . 4
9 Procedure . 5
9.1 Subtyping of stx genes .5
9.1.1 Template preparation .5
9.1.2 Setting up the PCR reaction .5
9.1.3 Agarose gel electrophoresis .5
9.2 Testing for the presence of eae and aggR genes .6
9.2.1 Template preparation .6
9.2.2 Real-time PCR amplification .6
9.2.3 Interpretation of Real-time PCR results .6
9.2.4 Internal Amplification Control for Real-time PCR . .6
9.3 Determination of the serogroups O157, O26, O103, O145, O111, O45 and O121 by Real-
time PCR amplification of O-associated genes .7
10 Expression of the results. . 7
11 Test Report . 7
12 Quality assurance . 8
Annex A (informative) Outline of the procedure . 9
Annex B (normative) Culture media and reagents . 10
Annex C (informative) Setting up the PCR reactions for stx genes subtyping .12
Annex D (informative) Primers and probes for the Real-time PCR assays for the detection of
eae and aggR genes .15
Annex E (informative) Primers and probes for the Real-time PCR assays for the detection of
O-associated genes . . 16
Bibliography .18

iii
ISO/DIS 13136-2:2024(en)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out through
ISO technical committees. Each member body interested in a subject for which a technical committee
has been established has the right to be represented on that committee. International organizations,
governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely
with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO documents should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any patent
rights identified during the development of the document will be in the Introduction and/or on the ISO list of
patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO's adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
Technical Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in collaboration with
the European Committee for Standardization (CEN) Technical Committee CEN/TC 463, Microbiology of
the food chain, in accordance with the Agreement on technical cooperation between ISO and CEN (Vienna
Agreement).
This second edition, together with the ISO 13136 Part 1, cancels and replaces the first edition
(ISO/TS 13136:2012), which has been technically revised.
The main changes compared to the previous edition are as follows:
— Structure of the standard (Full Standard rather than Technical Specification, split in Part 1 and Part 2)
— Description of the characterization of STEC isolates, in terms of determining the presence of a set of
virulence genes and serogroups, and subtyping of stx genes
A list of all parts in the ISO 13136 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iv
ISO/DIS 13136-2:2024(en)
Introduction
Shiga toxin (Stx)-producing Escherichia coli (STEC) are human pathogens causing severe disease, including
haemorrhagic colitis (HC) and the haemolytic uremic syndrome (HUS).
The Stx are the definative virulence factor of STEC, and belong to a heterogenous family of AB toxins,
including two antigenically distinct types, Stx1
...