CEN ISO/TS 13136:2012

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
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 463/WG 2 - Shiga toxin producing Escherichia coli (STEC)
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 15-Nov-2012
• Due Date: 24-Jul-2012
• Completion Date: 15-Nov-2012
• Ref Project: SIST-TS CEN ISO/TS 13136:2013 - 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)

Relations

• Revised: prEN ISO 13136-1 - Microbiology of the food chain - Detection, isolation and characterization of Shiga toxin-producing Escherichia coli (STEC) - Part 1: Horizontal method for the detection and isolation of Shiga toxin-producing Escherichia coli (STEC) (ISO/DIS 13136-1:2024)
• Effective Date: 17-Aug-2022
• Revised: prEN ISO 13136-2 - 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)
• Effective Date: 17-Aug-2022

Standards Content (Sample)

SLOVENSKI STANDARD
01-maj-2013
0LNURELRORJLMDåLYLOLQNUPH2GNULYDQMHSULVRWQRVWLSDWRJHQLKPLNURRUJDQL]PRY]
PHWRGRQDRVQRYLSROLPHUD]QHYHULåQHUHDNFLMH3&5YUHDOQHPþDVX
+RUL]RQWDOQDPHWRGD]DXJRWDYOMDQMHSULVRWQRVWL(VFKHULFKLDFROL67(&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
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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.
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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.

Contents Page
Foreword .3
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.

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
ISO/TS 13136:2012(E)
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
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Published in Switzerland
ii © ISO 2012 – All rights reserved

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/TS 13136:2012(E)
Foreword
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