CEN ISO/TS 13136:2012

SLOVENSKI STANDARD
01-maj-2013
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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.
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.

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
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

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
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

ISO/TS 13136:2012(E)
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.
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/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

ISO/TS 13136:2012(E)
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/TS 13136:2012(E)
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.
4 © ISO 2012 – All rights reserved

ISO/TS 13136:2012(E)
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.
ISO/TS 13136:2012(E)
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, possibly with a device for adjusting speed and time.
7 Sampling
Sampling is not part of the method specified in this Technical Specification. See the specific International
Standard dealing with the product concerned or specific regulations. If there is no specific International
Standard dealing with sampling of the product concerned, it is recommended that the parties concerned come
to an agreement on this subject.
It is important that the laboratory receive a truly representative sample which has not been damaged or changed
during transport or storage.
8 Preparation of test sample
Prepare the test sample in accordance with the specific International Standard dealing with the product
concerned. If there is no specific International Standard, it is recommended that the parties concerned come
to an agreement on this subject.
9 Procedure
9.1 Test portion and initial suspension
9.1.1 General
-1
Use the quantity of enrichment medium necessary to give a final dilution of 10 of the original test portion.
9.1.2 For matrix samples assumed to contain a high level of background flora
For solid matrices, aseptically transfer a test portion (x g) of sample to a peristaltic blender bag containing 9x ml
of mTSB to which novobiocin or acriflavin has been added (5.1.1.4). Bags with filters are preferred.
Homogenize in a peristaltic blender (see ISO 7218) (6.8).
For liquid matrices, transfer the test portion (x ml) of liquid sample, using a sterile pipette, into the tube or bottle
containing 9x ml of the enrichment mTSB to which novobiocin or acriflavin has been added (5.1.1.4).
9.1.3 For matrix sample assumed to contain stressed target bacteria
Allow frozen products to thaw at room temperature, and then transfer the test portion (x g or x ml) to a peristaltic
blender bag or tube containing 9x ml of BPW (5.1.2) and proceed as above.
6 © ISO 2012 – All rights reserved

ISO/TS 13136:2012(E)
9.2 Enrichment
9.2.1 Incubation
Incubate the peristaltic blender bag, tube or bottle (9.1.2) at 37 °C ± 1 °C for 18 h to 24 h.
9.2.2 Process control (for real-time PCR)
Perform process control according to ISO 22174.
Guidance on internal amplification control (IAC) and process control are given in Annex D and C.3.8.
9.3 Nucleic acid extraction
Use an appropriate nucleic acid extraction procedure for Gram-negative bacteria. A comprehensive collection
of methods can be found in Reference [10]. Alternatively, commercial kits may be used according to the
manufacturer’s instructions.
9.4 PCR amplification (for real-time PCR)
9.4.1 General
The PCR amplification approach described is based on real-time PCR.
Follow all requirements for the PCR amplification as specified in ISO 20838.
Primers and detection probes for conducting real-time PCR are described in Annex E.
9.4.2 Detection of PCR products
Light emission is captured by the apparatus once generated during the amplification.
9.4.3 Interpretation of PCR results
The PCR results obtained, including the controls specified in ISO 22174 and in Annex D, are interpreted by the
software linked to the apparatus. During amplification, the software monitors 5’ nuclease PCR amplification
by analysing fluorescence emissions of the reporter dye for each sample, R . DR is R minus the baseline
n n n
reporter dye intensity established in the first few cycles. At the end of the PCR cycles, a reaction is considered
positive if its DR curve exceeds the threshold, defined as 10 times the standard deviation of the mean baseline
n
emission calculated between the first few cycles. The cycle threshold, C , is defined as the cycle number at
t
which the DR fluorescence of a sample crosses the determined threshold value.
n
If the results are ambiguous, check the emission curves. Positive samples give a curve with a clear increase in
fluorescence, starting from a number of cycles corresponding to the C .
t
If the controls yield unexpected results, repeat the procedure.
The method is sequential (see the flowchart in Figure A.1):
— step 1: detection of the Stx-coding genes and the eae gene (PCR A in Annex E — this can also be done
in duplex PCR);
— step 2 a): samples positive for stx and the eae genes are tested for the molecular serogrouping (PCR
B in Annex E);
— step 2 b): samples positive for the Stx-coding genes are subjected to strain isolation — a serogroup-
specific enrichment method (e.g. IMS) can be used to improve the isolation of STEC from samples positive
for one of the serogroups within the scope of this Technical Specification (see 9.5 and Figure B.1).
ISO/TS 13136:2012(E)
9.5 Strain isolation
The isolation of the STEC strains is required to confirm that the positive PCR signals are generated from genes
present in the same live bacterial cell.
In case of positivity to one of the genes associated with the serogroups in the scope of this Technical
Specification, a serogroup-specific enrichment may be used to facilitate the isolation step followed by direct
plating on to suitable solid media and screening of the colonies for the presence of the virulence genes.
The standard PCR and the real-time PCR protocols in Annexes C or E or any other equivalent PCR protocol
shall be used in order to confirm the presence of the virulence genes in the isolated colonies.
A STEC isolation flowchart appears in Figure B.1 and a procedure in Annex F.
10 Expression of results
a) Samples negative for stx gene: STEC not detected in the test portion of x g or x ml (see ISO 7218).
In the absence of stx genes, the procedure is stopped without proceeding to the determination of the intimin-
coding eae gene or the genes associated with the serogroups in the scope of this method.
If isolation is not acheived from samples positive to the stx screening:
b) Samples positive for stx gene: Presumptive detection of STEC in the test portion of x g or x ml.
c) Samples positive for stx and eae genes: Presumptive detection of STEC causing the attaching and
effacing lesion in the test portion of x g or x ml.
d) Samples positive for stx and eae genes as well as to genes associated to one of the serogroups
1)
in the scope of this Technical Specification: Presumptive detection of STEC of XX serogroup in the
test portion of x g or x ml
If isolation and confirmation are achieved from samples positive to the stx screening:
e) Isolated E. coli strains positive for stx gene: Presence of STEC in the test portion of x g or x ml.
f) Isolated E. coli strains positive for stx and eae genes: Presence of STEC causing the attaching and
effacing lesion in the test portion of x g or x ml.
g) Isolated E. coli strains positive for stx and eae genes as well as for genes associated with one of
2)
the serogroups in the scope of this Technical Specification: Presence of STEC of XX serogroup in
the test portion of x g or x ml.
11 Performance data
11.1 The real-time PCR approach described in this Technical Specification has been the subject of a validation
[17]
study carried out according to ISO 16140:2003 rules. Since the only International Standard available as a
[19]
reference method at the time was ISO 16654 for the detection of E. coli O157 in foodstuffs, the method has
only been validated and certified by an NF validation (Reference [7]) for the detection of STEC belonging to the
O157 serogroup only.
Following this study, the part of the method regarding STEC O157 has been certified by AFNOR as equivalent
[17]
to ISO 16140:2003 (certificate number GEN 25/04-11/08). The complete validation dossier is available in
Reference [7].
1) XX indicates the serogroup specified by the presence of the genes assessed.
2) XX indicates the serogroup the isolated strain belongs to as assessed by the presence of the corresponding genes or
by phenotypic determination.
8 © ISO 2012 – All rights reserved

ISO/TS 13136:2012(E)
11.2 Some performance characteristics of the real-time PCR screening section of the method have been
determined in published studies. In particular, the sensitivity and the limit of detection of the real-time PCR
have been determined by using dilutions of plasmids containing the cloned genes encoding the different targets
(Reference [8]). The results of this study are summarized in Table 1.
Table 1 — Sensitivity and limit of detection of the 5′ nuclease PCR assays for some of the genes
targeted in this Technical Specification (adapted from Reference [8])
Limit of detection Efficiency
Target gene No. copies/reaction %
stx1 5 94,7
stx2 5 94
rfbE 1 94,2
wbdI 5 94
wzx 5 97,7
ihp1 5 99,6
Another study (Reference [17]) considered the performances of the proposed real-time PCR approaches in
screening mixed cultures of STEC belonging to the serogroups in the field of application of this Technical
Specification, with a K-12 laboratory strain (C600). The results are summarized in Table 2.
11.3 This Technical Specification was used in the third collaborative study organized in 2009 by the European
Union Reference Laboratory (EURL) for E. coli including STEC. The study consisted of the examination of a set
of five simulated carcass swabs (moistened sponges) containing the organisms of interest, including both STEC
O157 and O26, together with background microbial flora (Table 3). The choice of using simulated carcass swabs
as the matrix to be analysed in the proficiency test was driven by the principles included in the European Food
[19]
Safety Authority guidelines for the forthcoming monitoring plans for STEC (ISO 16654 ).
There were 14 national reference laboratories (NRLs) for E. coli that participated in the study. The evaluation
of the performance of the method for non-O157 STEC (O26 STEC) returned the following values:
Sensitivity (Se): 100 % [95 % confidence interval (CI) 96,97 % to 100 %]
Specificity (Sp): 99,62 % (95 % CI 97,5 % to 100 %)
The analytical results per NRL are summarized in Table 4.
The NRLs were also asked to isolate the non-O157 STEC contaminating the samples. The outcome of the
isolation step is showed in Table 5.
The report with the complete analysis of the results of the 3rd EU-RL-STEC collaborative study is publicly
[9]
available.
ISO/TS 13136:2012(E)
Table 2 — Detection of low numbers of STEC in mixed cultures (adapted from Reference [15])
C values
t
STEC
rfbE wbd1 wzx ihp1 fliC wzx
CFU/ml
serotype
stx1/stx2 eae
(O157) (O111) (O103) (O145) (H7) (O26)
2 to 3 28 to 31 31 to 32 — — — — — 31 to 33
O26:H11
10 to 20 25 to 27 28 to 29 — — — — — 29
2 to 3 29 to 30 31 to 32 — — 32 to 33 — — —
O103:H2
10 to 20 26 to 27 29 to 30 — — 29 to 31 — – —
2 to 3 26 to 27 30 to 31 — 30 to 31 — — – —
O111:[H8]
10 to 20 24 to 25 29 to 30 — 27 to 28 — — – —
2 to 3 32 to 33 31 to 32 — — — 31 to 34 – —
O145:[H28]
10 to 20 30 to 32 29 to 31 — — — 30 – —
2 to 3 29 to 30 29 to 31 31 to 32 — — — 33 to 38 —
O157:H7
10 to 20 26 to 28 26 to 29 29 to 31 — — — 31 to 33 —
Table 3 — Composition of samples for the third EURL-STEC collaborative study
Values in colony-forming units per millilitre
Sample/contaminant Sample A Sample B Sample C Sample D Sample E
STEC O157
2 2 ×10 20 0 0
stx1, stx2, eae
STEC O26
0 40 4 ×10 40 0
stx1, eae
2 2 2 2 2
E. coli 10 10 10 10 10
2 2 2 2 2
K. pneumoniae 2 × 10 2 ×10 2 ×10 2 ×10 2 ×10
2 2 2 2 2
S. faecalis 5 ×10 5 ×10 5 ×10 5 ×10 5 ×10
The expanded uncertainty, U, associated with the level of inoculum was 0,22 log (CFU/ml) for E. coli strains
[20]
as determined according to ISO/TS 19036:2006.
Table 4 — Detection of virulence and serogroup-associated genes in the enrichment cultures by
real-time PCR (screening section of the method; third EURL-STEC collaborative study)
Participating laboratories
Target
Sample
gene
L L L L L L L L L L L L L L
1 2 4 7 8 9 12 14 15 17 21 22 25 30
A + + + + + + + + + + + + + + +
B + + + + + + + + + + + + + + +
a
stx C + + + + + + + + + + + + + + +
D + + + + + + + + + + + + + + +
E - - - - - - - - - - - - - - -
10 © ISO 2012 – All rights reserved

ISO/TS 13136:2012(E)
Table 4 (continued)
Participating laboratories
Target
Sample
gene
L L L L L L L L L L L L L L
1 2 4 7 8 9 12 14 15 17 21 22 25 30
A + + + + + + + + + + + + + + +
B + + + + + + + + + + + + + + +
eae C + + + + + + + + + + + + + + +
D + + + + + + + + + + + + + + +
E - - - - - - - - - + - - - - -
A - - - - - - - - - - - - - - -
B + + + + + + + + + + + + + + +
O26 C + + + + + + + + + + + + + + +
D + + + + + + + + + + + + + + +
E - - - - - - - - - - - - - - -
A - - - - - - - - - - - - - - -
B - - - - - - - - - - - - - - -
O111 C - - - - - - - - - - - - - - -
-
D - - - - - - - - - - - - - -
-
E - - - - - - - - - - - - - -
-
A - - - - - - - - - - - - - -
B - - - - - - - - - - - - - - -
O103 C - - - - - - - - - - - - - - -
D - - - - - - - - - - - - - - -
E - - - - - - - - - - - - - - -
A - - - - - - - - - - - - - - -
B - - - - - - - - - - - - - - -
O145 C - - - - - - - - - - - - - - -
D - - - - - - - - - - - - - - -
E - - - - - - - - - - - - - - -
a
This includes either stx1 or stx2.
Table 5 — Isolation of STEC O26 from the RT PCR-positive enrichment cultures (Confirmation of
positive cultures by isolation of the infecting strain section; third EURL-STEC collaborative study)
Laboratories
Test Sample True value
L L L L L L L L L L L L L L
1 2 4 7 8 9 12 14 15 16 17 21 25 30
B + + + + + + + + + + + + + + +
Isolation of
C + + + + + + + + + + + + + + +
O26
D + + + + + + + + + + + + + + +
ISO/TS 13136:2012(E)
Annex A
(normative)
Flow diagram of the screening procedure
Test portion x g or x ml
9 x ml mTSB+N/A or BPW
Enrichment 18 h to 24 h
37 °C ± 1 °C
Test portion (1 ml) of the culture, DNA
purification and stx and eae genes detection
Positive result to stx and eae:
Negative result to stx:
Positive result to stx:
test for serogroup-associated genes.
Isolation (see Annex B)
Isolation (see Annex B)
Results reporting Results reporting
Results reporting
Figure A.1 — Flow diagram of the screening procedure
12 © ISO 2012 – All rights reserved

ISO/TS 13136:2012(E)
Annex B
(normative)
3)
Flow diagram of the isolation and confirmation procedure
If the sample was positive for one of the serogroup-associated genes in the scope of the method, a serogroup-
specific enrichment (SSE) may be performed in order to facilitate the isolation.
Enric
...