Microbiology of food and animal feed - Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens - Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups (ISO/TS 13136:2012)

ISO/TR 13136:2012 describes the identification of Shiga toxin-producing Escherichia coli (STEC) by means of the detection of the following genes: a) the major virulence genes of STEC, stx and eae; b) the genes associated with the serogroups O157, O111, O26, O103, and O145.
In any case, when one or both of the stx genes is/are detected, the isolation of the strain is attempted.
The isolation of STEC from samples positive for the presence of the genes specifying the serogroups in the scope of this method can be facilitated by using serogroup-specific enrichment techniques (e.g. immunomagnetic separation, IMS).
The protocol uses real-time PCR as the reference technology for detection of the virulence and serogroup-associated genes.
ISO/TR 13136:2012 is applicable to: 1) products intended for human consumption and the feeding of animals; 2) environmental samples in the area of food production and food handling; 3) environmental samples in the area of primary production.

Mikrobiologie von Lebensmitteln und Futtermitteln - Real-time-Polymerase-Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln - Horizontales Verfahren für den Nachweis von Shiga-Toxin bildenden Escherichia coli (STEC) und Bestimmung der Serogruppen O157, O111, O26, O103 und O145 (ISO/TS 13136:2012)

Diese Technische Spezifikation beschreibt die Identifizierung von Shiga Toxin bildenden Escherichia coli (STEC) mit Hilfe des Nachweises folgender Gene:
a)   der wichtigsten Virulenzgene von STEC, stx und eae (Literaturhinweise [2], [3]);
b)   der mit den Serogruppen O157, O111, O26, O103 und O145 assoziierten Gene (Literaturhinweise [3], [4]).
In jedem Fall, wenn eines oder beide der stx Gene nachgewiesen wird/werden, wird versucht, den Stamm zu isolieren.
Die Isolierung von STEC aus Proben, die positiv hinsichtlich der Anwesenheit von Genen solcher Serogruppen sind, die im Anwendungsbereich dieses Verfahrens liegen, kann durch die Anwendung serogruppenspezifischer Anreicherungsverfahren (z. B. immunomagnetische Separation, IMS) erleichtert werden.
In dem beschriebenen Verfahren wird die Real-time-PCR als das Referenzverfahren zum Nachweis der Virulenzgene und der mit bestimmten Serogruppen-assoziierten Gene angewendet.
Die vorliegende Technische Spezifikation ist anwendbar auf:
1)   Erzeugnisse, die für den menschlichen Verzehr und als Futtermittel vorgesehen sind;
2)   Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln;
3)   Umgebungsproben im Bereich der Primärproduktion.

Microbiologie des aliments - Méthode basée sur la réaction de polymérisation en chaîne (PCR) en temps réel pour la détection des micro-organismes pathogènes dans les aliments - Méthode horizontale pour la détection des Escherichia coli producteurs de Shigatoxines (STEC) et la détermination des sérogroupes O157, O111, O26, O103 et O145 (ISO/TS 13136:2012)

L'ISO/TS 13136:2012 décrit une méthode horizontale pour la détection: a)des principaux gènes de virulence des STEC; b) des gènes associés aux sérogroupes O157, O111, O26, O103 et O145.
Dans le cas de la détection de ces gènes, une procédure d'isolement de souches est mise en place afin de confirmer la présence simultanée des gènes dans une seule et même cellule bactérienne vivante.
L'ISO/TS 13136:2012 repose sur l'utilisation de la PCR en temps réel comme technologie de référence pour la détection des gènes de virulence et des gènes associés aux sérogroupes. Un protocole de PCR en temps réel est par conséquent décrit en détail.
L'ISO/TS 13136:2012 s'applique: 1) aux produits destinés à être consommés par l'Homme et aux aliments pour animaux; 2) aux échantillons environnementaux dans la zone de production et de manipulation des aliments; 3) aux échantillons environnementaux dans la zone de production primaire.

Mikrobiologija živil in krme - Odkrivanje prisotnosti patogenih mikroorganizmov z metodo na osnovi polimerazne verižne reakcije (PCR) v realnem času - Horizontalna metoda za ugotavljanje prisotnosti Escherichia coli (STEC) ki proizvaja Shiga toksin in določevanje serotipov O157, O111, O26, O103 ter O145 seroskupin (ISO/TS 13136:2012)

Ta tehnična specifikacija opisuje ugotavljanje prisotnosti Escherichia coli, ki proizvajajo toksine šigel (STEC), z odkrivanjem naslednjih genov:
a) glavnih genov virulence STEC, stx in eae (zveze [2][3]);
b) genov, povezanih s serotipi O157, O111, O26, O103 in O145 (zveze [3][4]). V vsakem primeru se poskuša sev izolirati, ko je zaznan eden ali oba gena stx. Izolacija STEC iz vzorcev, pozitivnih na prisotnost genov, ki določajo serotipe na področju uporabe te metode, se lahko olajša z uporabo tehnik obogatitve, specifičnih za serotip (npr. imunomagnetna separacija, IMS). Protokol kot referenčno tehnologijo za odkrivanje virulence in genov, povezanih s serotipom, uporablja polimerazno verižno reakcijo (PCR) v realnem času. Ta tehnična specifikacija se uporablja za:
1) izdelke, namenjene za prehrano ljudi in krmo živali;
2) okoljske vzorce na območju proizvodnje hrane in ravnanja s hrano;
3) okoljske vzorce na območju primarne proizvodnje.

General Information

Status
Published
Publication Date
14-Nov-2012
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
15-Nov-2012
Completion Date
15-Nov-2012

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SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 13136:2013
01-maj-2013

0LNURELRORJLMDåLYLOLQNUPH2GNULYDQMHSULVRWQRVWLSDWRJHQLKPLNURRUJDQL]PRY]

PHWRGRQDRVQRYLSROLPHUD]QHYHULåQHUHDNFLMH 3&5 YUHDOQHPþDVX
+RUL]RQWDOQDPHWRGD]DXJRWDYOMDQMHSULVRWQRVWL(VFKHULFKLDFROL 67(& LQ
GRORþHYDQMHVHURWLSRY2222LQ2 ,6276

Microbiology of food and animal feed - Real-time polymerase chain reaction (PCR)-

based method for the detection of food-borne pathogens - Horizontal method for the

detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of

O157, O111, O26, O103 and O145 serogroups (ISO/TS 13136:2012)
Mikrobiologie von Lebensmitteln und Futtermitteln - Real-time-Polymerase-

Kettenreaktion (PCR) zum Nachweis von pathogenen Mikroorganismen in Lebensmitteln

- Horizontales Verfahren für den Nachweis von Shiga-Toxin bildenden Escherichia coli

(STEC) der Serogruppen O157, O111, O26, O103 und O145 (ISO/TS 13136:2012)

Microbiologie des aliments - Méthode basée sur la réaction de polymérisation en chaîne

(PCR) en temps réel pour la détection des micro-organismes pathogènes dans les

aliments - Méthode horizontale pour la détection des Escherichia coli producteurs de

Shigatoxines (STEC) et la détermination des sérogroupes O157, O111, O26, O103 et
O145 (ISO/TS 13136:2012)
Ta slovenski standard je istoveten z: CEN ISO/TS 13136:2012
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST-TS CEN ISO/TS 13136:2013 en

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

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SIST-TS CEN ISO/TS 13136:2013
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SIST-TS CEN ISO/TS 13136:2013
TECHNICAL SPECIFICATION
CEN ISO/TS 13136
SPÉCIFICATION TECHNIQUE
TECHNISCHE SPEZIFIKATION
November 2012
ICS 07.100.30
English Version
Microbiology of food and animal feed - Real-time polymerase
chain reaction (PCR)-based method for the detection of food-
borne pathogens - Horizontal method for the detection of Shiga
toxin-producing Escherichia coli (STEC) and the determination
of O157, O111, O26, O103 and O145 serogroups (ISO/TS
13136:2012)

Microbiologie des aliments - Méthode basée sur la réaction Mikrobiologie von Lebensmitteln und Futtermitteln - Real-

de polymérisation en chaîne (PCR) en temps réel pour la time-Polymerase-Kettenreaktion (PCR) zum Nachweis von

détection des micro-organismes pathogènes dans les pathogenen Mikroorganismen in Lebensmitteln -

aliments - Méthode horizontale pour la détection des Horizontales Verfahren für den Nachweis von Shiga-Toxin

Escherichia coli producteurs de Shigatoxines (STEC) et la bildenden Escherichia coli (STEC) der Serogruppen O157,

détermination des sérogroupes O157, O111, O26, O103 et O111, O26, O103 und O145 (ISO/TS 13136:2012)

O145 (ISO/TS 13136:2012)

This Technical Specification (CEN/TS) was approved by CEN on 16 July 2012 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their

comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available

promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)

until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United

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

© 2012 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN ISO/TS 13136:2012: E

worldwide for CEN national Members.
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SIST-TS CEN ISO/TS 13136:2013
CEN ISO/TS 13136:2012 (E)
Contents Page

Foreword ..............................................................................................................................................................3

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SIST-TS CEN ISO/TS 13136:2013
CEN ISO/TS 13136:2012 (E)
Foreword

This document (CEN ISO/TS 13136:2012) 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".

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.

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

countries are bound to announce this Technical Specification: 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, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.

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SIST-TS CEN ISO/TS 13136:2013
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SIST-TS CEN ISO/TS 13136:2013
TECHNICAL ISO/TS
SPECIFICATION 13136
First edition
2012-11-15
Microbiology of food and animal feed —
Real-time polymerase chain reaction
(PCR)-based method for the detection
of food-borne pathogens — Horizontal
method for the detection of Shiga toxin-
producing Escherichia coli (STEC) and
the determination of O157, O111, O26,
O103 and O145 serogroups
Microbiologie des aliments — Méthode basée sur la réaction de
polymérisation en chaîne (PCR) en temps réel pour la détection
des micro-organismes pathogènes dans les aliments — Méthode
horizontale pour la détection des Escherichia coli producteurs de
Shigatoxines (STEC) et la détermination des sérogroupes O157, O111,
O26, O103 et O145
Reference number
ISO/TS 13136:2012(E)
ISO 2012
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SIST-TS CEN ISO/TS 13136:2013
ISO/TS 13136:2012(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2012

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

electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO’s

member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2012 – All rights reserved
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SIST-TS CEN ISO/TS 13136:2013
ISO/TS 13136:2012(E)
Contents Page

Foreword ............................................................................................................................................................................iv

Introduction ........................................................................................................................................................................ v

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

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

3 Terms and definitions ......................................................................................................................................... 2

4 Principle ................................................................................................................................................................. 2

4.1 General ................................................................................................................................................................... 2

4.2 Microbial enrichment .......................................................................................................................................... 2

4.3 Nucleic acid extraction ...................................................................................................................................... 3

4.4 Target genes ......................................................................................................................................................... 3

4.5 Detection ................................................................................................................................................................ 3

4.6 Isolation ................................................................................................................................................................. 3

5 Diluents, culture media and reagents ............................................................................................................ 3

5.1 Culture media ....................................................................................................................................................... 4

5.2 Reagents for nucleic acid extraction ............................................................................................................. 5

5.3 Reagents for PCR ................................................................................................................................................ 5

6 Equipment ............................................................................................................................................................. 5

7 Sampling ................................................................................................................................................................ 6

8 Preparation of test sample ................................................................................................................................ 6

9 Procedure .............................................................................................................................................................. 6

9.1 Test portion and initial suspension ................................................................................................................ 6

9.2 Enrichment ............................................................................................................................................................ 7

9.3 Nucleic acid extraction ...................................................................................................................................... 7

9.4 PCR amplification (for real-time PCR) ........................................................................................................... 7

9.5 Strain isolation ..................................................................................................................................................... 8

10 Expression of results ......................................................................................................................................... 8

11 Performance data ................................................................................................................................................ 8

Annex A (normative) Flow diagram of the screening procedure .........................................................................12

Annex B (normative) Flow diagram of the isolation and confirmation procedure .........................................13

Annex C (informative) Identification of Shiga toxin-producing Escherichia coli (STEC) by multiplex

PCR amplification of virulence genes and detection of PCR products by agarose

gel electrophoresis ...........................................................................................................................................14

Annex D (informative) Internal amplification control ..............................................................................................18

Annex E (informative) Primers and probes for the PCR assays ..........................................................................19

Annex F (normative) Isolation of STEC strains ........................................................................................................21

Bibliography .....................................................................................................................................................................22

© ISO 2012 – All rights reserved iii
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ISO/TS 13136:2012(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.

In other circumstances, particularly when there is an urgent market requirement for such documents, a technical

committee may decide to publish other types of document:

— an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in

an ISO working group and is accepted for publication if it is approved by more than 50 % of the members

of the parent committee casting a vote;

— an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical

committee and is accepted for publication if it is approved by 2/3 of the members of the committee

casting a vote.

An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a further

three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is confirmed,

it is reviewed again after a further three years, at which time it must either be transformed into an International

Standard or be withdrawn.

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/TS 13136 was prepared by the European Committee for Standardization (CEN) 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).
iv © ISO 2012 – All rights reserved
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Introduction

Shiga toxin-producing Escherichia coli (STEC) are pathogenic E. coli, which can cause diarrhoea as well as more

severe diseases in humans such as haemorrhagic colitis and haemolytic uremic syndrome (HUS). Although

STEC may belong to a large number of serogroups, those that have been firmly associated with the most severe

forms of the disease, in particular HUS, belong to O157, O26, O111, O103, and O145 (Reference [1]).

The following nomenclature has been adopted in this Technical Specification:
— stx: Shiga toxin genes (synonymous with vtx);
— Stx: Shiga toxin (synonymous with Vtx: Verocytotoxin);

— STEC: Shiga toxin-producing Escherichia coli (synonymous with VTEC: Verocytotoxin-producing

Escherichia coli).

The International Organization for Standardization (ISO) draws attention to the fact that it is claimed that

compliance with this document may involve the use of patents.

ISO takes no position concerning the evidence, validity and scope of this patent right.

The holder of this patent right has assured ISO that he/she is willing to negotiate licences either free of charge

or under reasonable and non-discriminatory terms and conditions with applicants throughout the world. In this

respect, the statement of the holder of this patent right is registered with ISO. Information can be obtained from:

Agence Nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail/

French Agency for Food, Environment and Occupational Health and Safety (ANSES)
10 rue Pierre Curie
F-94700 MAISONS-ALFORT, Cedex
France

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

other than those identified above. ISO shall not be held responsible for identifying any or all such patent rights.

ISO (www.iso.org/patents) maintains an on-line database of patents relevant to their documents. Users are

encouraged to consult the databases for the most up to date information concerning patents.

© ISO 2012 – All rights reserved v
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SIST-TS CEN ISO/TS 13136:2013
TECHNICAL SPECIFICATION ISO/TS 13136:2012(E)
Microbiology of food and animal feed — Real-time polymerase
chain reaction (PCR)-based method for the detection of food-
borne pathogens — Horizontal method for the detection
of Shiga toxin-producing Escherichia coli (STEC) and the
determination of O157, O111, O26, O103 and O145 serogroups

IMPORTANT — It is necessary to consider any STEC as pathogenic to humans and potentially to cause

severe disease depending on both the risk profile of the food commodity (ready-to-eat foods vs. foods

intended to be consumed after technological treatment such as pasteurization, cooking etc. used to

reduce any bacteria present in the food) and the health status of the subject ingesting the food.

Moreover, given the high genomic plasticity of this bacterial species, it is possible that novel

arrangements of virulence features can give rise to novel sero-pathogroups such as the Shiga toxin-

producing enteroaggregative E. coli O104 that caused the HUS outbreaks in Germany and France in

2011-05/06. Novel atypical E. coli sero-pathogroups can arise from the acquisition of an stx-converting

bacteriophage by an E. coli strain belonging to pathogroups different from STEC.

Such atypical strains fall in the scope of this method and can be efficiently detected as they are positive for the

presence of the stx genes.
1 Scope

This Technical Specification describes the identification of Shiga toxin-producing Escherichia coli (STEC) by

means of the detection of the following genes:
a) the major virulence genes of STEC, stx and eae (References [2][3]);

b) the genes associated with the serogroups O157, O111, O26, O103, and O145 (References [3][4]).

In any case, when one or both of the stx genes is/are detected, the isolation of the strain is attempted.

The isolation of STEC from samples positive for the presence of the genes specifying the serogroups in the scope

of this method can be facilitated by using serogroup-specific enrichment techniques (e.g. immunomagnetic

separation, IMS).

The protocol uses real-time PCR as the reference technology for detection of the virulence and serogroup-

associated genes.
This Technical Specification is applicable to:
1) products intended for human consumption and the feeding of animals;
2) environmental samples in the area of food production and food handling;
3) environmental samples in the area of primary production.
2 Normative references

The following referenced documents are indispensable for the application 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 2012 – All rights reserved 1
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ISO/TS 13136:2012(E)

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 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.

Definitions 3.1 to 3.3 have been compiled from the epidemiological data on disease caused by STEC managed

by organizations such as the US Centers for Disease Control and, in the EU, by the European Centre for

Disease Prevention and Control and the European Food Safety Authority.
3.1
Shiga toxin-producing Escherichia coli
STEC
E. coli strains possessing the Stx-coding genes
3.2
Shiga toxin-producing Escherichia coli causing the attaching and effacing lesion
STEC causing the attaching and effacing lesion
E. coli strains possessing the Stx-coding genes and the intimin-coding gene eae

NOTE This combination of virulence genes is often associated with the most severe forms of STEC-induced disease.

3.3
Shiga toxin-producing Escherichia coli belonging to highly pathogenic serogroups
STEC belonging to highly pathogenic serogroups

E. coli strains possessing the Stx-coding genes, the intimin-coding gene eae and belonging to one of the

serogroups O157, O111, O26, O103, and O145
4 Principle
4.1 General
The method specified comprises the following sequential steps:
a) microbial enrichment;
b) nucleic acid extraction;
c) detection of virulence genes;
d) detection of serogroup-associated genes;
e) isolation from positive samples.
Figure A.1 is a flow diagram of the screening procedure.
4.2 Microbial enrichment

The number of STEC cells to be detected is increased by incubating the test portion in a non-selective liquid

nutrient medium chosen from:

a) modified tryptone-soy broth (tryptone soy broth supplemented with 1,5 g/l bile salts No.3, mTSB)

supplemented with 16 mg/l of novobiocin (mTSB+N);
b) buffered peptone water (BPW);
2 © ISO 2012 – All rights reserved
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c) modified tryptone-soy broth (tryptone-soy broth supplemented with 1,5 g/l bile salts No.3, mTSB)

supplemented with 12 mg/l of acriflavin (mTSB+A) for analysis of milk and dairy products.

The mTSB shall be used when analysing matrices suspected to contain high levels of contaminating microflora.

Novobiocin and acriflavin inhibit the growth of Gram-positive bacteria and promote the growth of Gram-negative

cells, including STEC. The BPW shall be used to analyse samples which are assumed to contain stressed

target bacteria (such as frozen products), to resuscitate stressed STEC cells, and expected lower levels of

contaminating microflora than in fresh samples.

NOTE The addition of novobiocin is controversial and has been investigated by several authors. It has been observed

that the minimum inhibitory concentration of the antibiotic for non-O157 STEC is lower than for O157 strains (Reference

[19]

[5]). The addition of novobiocin in the enrichment mTSB at the usual concentration of 20 mg/l, as specified in ISO 16654,

seems to inhibit the growth of about one-third of non-O157 strains (Reference [6]) increasing the risk of false-negative results.

4.3 Nucleic acid extraction

The nucleic acid is extracted according to the requirements of the adopted detection system.

4.4 Target genes

The purified nucleic acid is used for the detection of the following target genes:

— the main virulence genes for STEC: stx genes, encoding the Shiga toxins and the eae gene, encoding a

90 kDa protein, the intimin, involved in the attaching and effacing mechanism of adhesion, a typical feature

of the STEC strains causing severe disease. The stx genes encode a family of toxins including two main

types: stx1 and stx2. The latter comprises seven recognized variants (from stx2a to stx2g) (Reference

[22]). Only the variants stx2a, stx2b, and stx2c have been found to be produced by the STEC strains

included in Clause 1, and therefore constitute the target Stx-coding genes of this Technical Specification.

The GenBank accession numbers corresponding to the stx2 variants-coding genes are:

— stx2a: X07865
— stx2b: L11078
— stx2c: M59432
— the intimin-coding eae gene

— the rfbE(O157), wbdl(O111), wzx(O26), ihp1(O145) and wzx(O103) genes, to identify the corresponding

serogroups.
4.5 Detection

The detection of the target genes is performed according to the adopted detection system.

4.6 Isolation

If the presence of a STEC is suspected, the isolation is attempted. If one of the serogroups specified in the

scope of this Technical Specification is detected, a serogroup-specific enrichment (e.g. IMS) can be performed

followed by plating on to tryptone–bile–glucuronic agar (TBX) or a specific selective medium if available (see

Annex F, Notes 2 and 3) in order to facilitate the isolation of the STEC from the background flora.

5 Diluents, culture media and reagents

During the analysis, unless otherwise stated, use only reagents of recognized analytical grade and sterile

distilled or demineralized water or water of equivalent purity.
© ISO 2012 – All rights reserved 3
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5.1 Culture media
5.1.1 Modified tryptone- soy broth (mTSB)
5.1.1.1 Basic medium
Composition and pH
Enzymatic digest of casein 17 g
Enzymatic digest of soy 3 g
d(+)-Glucose 2,5 g
Sodium chloride 5 g
Dipotassium hydrogenphosphate (K HPO ) 4 g
2 4
Bile salts No. 3 1,5 g
Water to 1 000 ml
pH 7,4 ± 0,2
Preparation

Dissolve the components or the dehydrated medium in water. Adjust pH with a pH-meter to pH 7,4 ± 0,2 at

25 °C and sterilize by autoclaving at 121 °C for 15 min.
5.1.1.2 Novobiocin solution
Composition
Novobiocin 0,16 g
Water 10 ml
Preparation

Dissolve the novobiocin in the water and sterilize by membrane filtration using 0,22 µm or 0,45 µm filters.

Prepare on the day of use.
5.1.1.3 Acriflavin solution
Composition
Acriflavin 0,12 g
Water 10 ml
Preparation

Dissolve the acriflavin in the water and sterilize by membrane filtration using 0,22 µm or 0,45 µm filters.

Prepare on the day of use.
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5.1.1.4 Preparation of the complete medium

Immediately before use, add 1 ml of novobiocin (5.1.1.2) or acriflavin solution (5.1.1.3) to 1 000 ml of cooled

mTSB (5.1.1.1).
The final concentration of novobiocin shall be 16 mg/l of mTSB.
The final concentration of acriflavin shall be 12 mg/l of mTSB.
5.1.2 Buffered peptone water (BPW)
Composition and pH
Peptone 10 g
Sodium chloride 5,0 g
Disodium phosphate (Na HPO ) 3,5 g
2 4
Potassium dihydrogenphosphate (KH PO ) 1,5 g
2 4
Water to 1 000 ml
pH 7,0 ± 0,2
Preparation

Dissolve the components or the dehydrated powder in the water. Adjust pH with a pH-meter to pH 7,0 ± 0,2 at

25 °C and sterilize by autoclaving at 121 °C for 15 min.
5.2 Reagents for nucleic acid extraction

The reagents to be used for nucleic acid extraction are not listed, being dependent on the method adopted (9.3).

5.3 Reagents for PCR
See ISO 20838.
5.3.1 Oligonucleotides (primers) and detection probes

Primers and probes for specific detection of the target gene sequences by standard and real-time PCR are

listed in Annexes C and E.
6 Equipment

Usual microbiological laboratory equipment (see ISO 7218) and, in particular, the following.

6.1 Water bath or heating block capable of being maintained at temperatures up to 100 °C.

6.2 Incubator according to ISO 7218, capable of being maintained at 37 °C± 1 °C.
6.3 Nucleic acid extraction apparatus.
Appropriate equipment according to the method adopted (if needed).
[16]
6.4 Pipettes of capacities between 1 µl and 100 µl, ISO 7550.
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6.5 Thin walled real-time PCR microtubes (0,2 ml/0,5 ml reaction tubes), multi-well PCR microplates or

other suitable light transparent disposable plasticware.

6.6 Thermal cycler. Several brands of apparatus are available and can be chosen according to the

laboratory policies.
6.7 PCR product detection apparatus.

Light emission following 5’ nuclease PCR assay is detected by the real-time PCR apparatus.

6.8 Peristaltic blender with sterile bags, pos
...

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