prEN ISO 13136-1

SLOVENSKI STANDARD
01-april-2024
Mikrobiologija v prehranski verigi - Odkrivanje prisotnosti, izolacija in
karakterizacija Escherichia coli (STEC), ki proizvaja Shiga toksin - 1. del:
Horizontalna metoda za odkrivanje prisotnosti in izolacijo 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 1: Horizontal method for the detection and
isolation of Shiga toxin-producing Escherichia coli (STEC) (ISO/DIS 13136-1:2024)
Mikrobiologie der Lebensmittelkette - Nachweis, Isolierung und Charakterisierung von
Shiga-Toxin bildenden Escherichia coli (STEC) - Teil 1: Horizontales Verfahren zum
Nachweis und zur Isolierung von Shiga-Toxin bildenden Escherichia coli (STEC)
(ISO/DIS 13136-1:2024)
Microbiologie de la chaîne alimentaire - Détection, isolement et caractérisation des
Escherichia coli producteurs de Shigatoxines (STEC) - Partie 1: Méthode horizontale
pour la détection et l’isolement des Escherichia coli producteurs de Shigatoxines (STEC
(ISO/DIS 13136-1:2024)
Ta slovenski standard je istoveten z: prEN ISO 13136-1
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 13136-1
ISO/TC 34/SC 9
Microbiology of the food
Secretariat: AFNOR
chain — Detection, isolation and
Voting begins on:
characterization of Shiga toxin-
2024-02-14
producing Escherichia coli (STEC) —
Voting terminates on:
2024-05-08
Part 1:
Horizontal method for the detection
and isolation of Shiga toxin-
producing Escherichia coli (STEC)
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
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Reference number
ISO/DIS 13136-1:2024(en)
DRAFT
ISO/DIS 13136-1:2024(en)
International
Standard
ISO/DIS 13136-1
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 1:
Horizontal method for the detection
and isolation of Shiga toxin-
producing Escherichia coli (STEC)
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
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Published in Switzerland Reference number
ISO/DIS 13136-1:2023(en)
ii
ISO/DIS 13136-1:2023(en)
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 .2
4.4 Target genes .3
4.5 Detection and isolation .3
5 Culture media and reagents . 3
6 Equipment and consumables . 3
7 Sampling . 4
8 Preparation of test sample . 5
9 Procedure . 5
9.1 Test portion and initial suspension .5
9.2 Enrichment .5
9.3 Nucleic acid extraction .6
9.4 PCR amplification (for real-time PCR) .6
9.4.1 General .6
9.4.2 Interpretation of real-time PCR results .6
9.4.3 Internal Amplification control for real-time PCR .6
9.5 STEC strain isolation .7
10 Expression of the results . 8
11 Validation of the method . 9
12 Test report . 9
13 Quality assurance . 10
Annex A (normative) Flow diagram of the procedure .11
Annex B (normative) Culture media and reagents .12
Annex C (normative) Flow diagram of the isolation of STEC strains .16
Annex D (normative) Primers and probes for the real-time PCR assays . 17
Annex E (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 .18
Annex F (informative) Acid treatment of enrichment cultures .22
Annex G (informative) Detection of Escherichia coli producing the Stx2f subtype by real-time
[5]
PCR (extract of the EURL-VTEC_Method_10 ) .23
Bibliography .25

iii
ISO/DIS 13136-1:2023(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.
This document was prepared by 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 first edition of ISO 13136-1, together with ISO 13136-2, cancels and replaces the first edition Technical
Specification (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)
— Change in the enrichment conditions (including medium and temperature)
— Removal of the determination of the presence of the gene eae and the genes associated to serogroups
O157, O111, O26, O103 and O145 from the screening of the enrichment cultures and in the STEC isolates
(included in Part 2 as characterization of the STEC isolates)
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 13136-1:2023(en)
Introduction
Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens causing human disease ranging from
uncomplicated diarrhoea to severe illness, such as haemorrhagic colitis and haemolytic uremic syndrome
(HUS). STEC are distinguished from other E. coli by the production of Shiga toxin (Stx), synonymous with
Verocytotoxin (VT). The Stx family is divided antigenically into two major types, Stx1 and Stx2. STEC strains
may possess Stx1- and/or Stx2-encoding genes. The serotypes of STEC causing human disease are highly
[1]
diverse and continuously evolving, and so all STEC isolates are considered potential human pathogens.
Therefore, STEC strains regardless of the serogroup represent the target of this International Standard. The
following nomenclature has been adopted in this standard method:
stx: Shiga toxin genes (synonymous with vtx);
Stx: Shiga toxin (synonymous with VT);
STEC: Shiga toxin-producing Escherichia coli (synonymous with VTEC: Verocytotoxin-producing Escherichia
coli).
The main technical changes listed in the Foreword, introduced in this document compared to
[2]
ISO/TS 13136:2012, are considered as major (see ISO 17468 ).
These technical changes have a major impact on the performance characteristics of the method.
When this document is reviewed in the future, account will be taken of all information then available
regarding the extent to which this horizontal method has been followed and the reasons for deviations from
this method in the case of particular products.
Identification of patent holders: the following text shall be included if patent rights have been identified. [The
process is ongoing]
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 a patent.
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 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 may be obtained from the
patent database available at www.iso.org/patents.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights other than those in the patent database. ISO shall not be held responsible for identifying any or all
such patent rights.
v
DRAFT International Standard ISO/DIS 13136-1:2023(en)
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)
WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests for
detecting STEC are only undertaken in properly equipped laboratories, under the control of a skilled
microbiologist, and that great care is taken in the disposal of all incubated materials. Persons using
this document should be familiar with normal laboratory practice. This document does not purport
to address all the safety aspects associated with its use. It is the responsibility of the user to establish
appropriate safety and health practices in compliance with national and international regulations
concerning the handling and containment of biological agents.
1 Scope
This standard describes the detection and isolation of Shiga toxin-producing Escherichia coli (STEC). The
procedure includes the detection by real-time PCR of stx1 and stx2, the major virulence genes of STEC
(Reference [1]), in enrichment cultures. Isolation of STEC from the enrichment culture is attempted if one or
both stx genes are detected.
This document is applicable to
— products intended for human consumption,
— products intended for animal feeding,
— environmental samples in the area of food and feed production, handling, and
— samples from the primary production stage.
2 Normative references
The following documents are referred to in the text in such a way that some or all their content constitutes
requirements of this document. The latest edition of the referenced document (including any amendments)
applies.
ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension, and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and performance
testing of culture media
ISO 22118, Microbiology of food and animal feeding stuffs — Polymerase chain reaction (PCR) for the detection
and quantification of food-borne pathogens — Performance characteristics

ISO/DIS 13136-1:2023(en)
ISO 22174, Microbiology of the food chain — Polymerase chain reaction (PCR) for the detection of food-borne
pathogens — General requirements and definitions
ISO 22119, Microbiology of food and animal feeding stuffs — Real-time 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.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
Shiga toxin-producing Escherichia coli (STEC)
E. coli strain possessing one or multiple Shiga toxin (Stx)-encoding genes (stx)
3.2
Stx1 and Stx2
Shiga Toxin type 1 and Shiga Toxin type 2, respectively
3.3
stx1 and stx2
Genes encoding Stx1 and Stx2, respectively
4 Principle
4.1 General
The method specified comprises the following sequential steps see the flowchart in Annex A:
a) microbial enrichment;
b) nucleic acid extraction;
c) detection of stx virulence genes (described in Annex D — this can also be done as a multiplex PCR,
together with Internal Amplification Control, see 9.4.3);
d) isolation of STEC strains from enrichment culture positive for stx by determining the presence of Stx-
encoding genes in isolated colonies to confirm the presence of STEC—(see 9.5 and Annex C)
4.2 Microbial enrichment
The number of viable STEC cells available to be detected in the test portion is increased by static incubation
of the sample in buffered peptone water (BPW) (a non-selective liquid nutrient medium) at 41,5 °C for
21 h ± 3 h.
If injured cells are suspected to be present in the sample (e.g. frozen, UV or pressure treated food stuff,
flours, dried food) incubate the BPW culture at 37 °C for 6 h ± 1 h to increase the number of viable STEC cells
and then at 41,5 °C for further 18 h ± 3 h.
4.3 Nucleic acid extraction
Deoxyribonucleic acid (DNA) is extracted from enrichment cultures for analysis by real-time PCR assay for
the detection of the genes encoding Shiga toxins.

ISO/DIS 13136-1:2023(en)
4.4 Target genes
The primary virulence genes of STEC are the stx genes, encoding the Shiga toxins. The stx genes encode a
family of toxins, which includes two main types: Stx1 and Stx2. Large variability in stx sequences has been
described, and they can be divided in three subtypes of stx1 (stx1a, stx1c and stx1d) and at least seven of
stx2 (stx2a to stx2g) (Reference [3]). The PCR primers and probes described in this standard detect all the
previously mentioned stx subtypes, with the exception of stx2f. The gene sequence encoding Stx2f differs
significantly from that of other subtypes and is not targeted by the stx primers and probes indicated in this
standard. According to currently available data, stx2f has rarely been reported in foodstuff (References [4]
and [5]), nevertheless it has been recently associated with with human disease (Reference [6]:). If there is a
specific need for the detection of Stx2f-producing E. coli in food, such as prevalence studies, epidemiological
investigations, or regulatory requests, a protocol issued by the European Union Reference Laboratory
(EURL) for E. coli for the detection of stx2f by real-time PCR can be used. This procedure, EU-RL VTEC_
Method_10, Reference [7], is available at the following link: https:// www .iss .it/ documents/ 20126/ 0/ EURL
-VTEC _Method _10 _Rev+ 0 .pdf/ a4eb6e2b -fd13 -c52a -112a -255c005b4872 ?t = 1644309297880, and is included
in an informative Annex in this standard (Annex G). The EURL for E. coli protocol is used to determine the
presence or absence of stx2f in enrichment cultures that give negative result for the presence of stx genes in
the screening step described in the present standard.
In addition, new Stx subtypes are continuously identified, including Stx1e, Stx2h, Stx2i, Stx2j, Stx2k, Stx2l,
Stx2m, and Stx2o (References [8-13]). An ad-hoc inclusivity assay has shown that these sets of primers
described in this standard, including those described in Annex G, enable the efficient detection all the stx-
subtypes except stx1e, stx2h, stx2j, stx2n, stx2o (courtesy of Alexander Gill and Sarah Clark, Health Canada,
2023).
The detection of the target genes, stx1 and stx2, is performed either on DNA extracted from the enrichment
culture as a screening step, or from isolated colonies when attempting to identify STEC among colonies
recovered from stx-positive enrichment cultures.
4.5 Detection and isolation
Real-time PCR is applied to detect stx1 and stx2 genes in the enrichment culture, which is performed
according to the PCR platform used and to manufacturers’ instructions for the real-time PCR kit in use.
The isolation of the STEC strains from the enrichment culture is required to confirm that the positive PCR
signals are generated from genes present in live bacterial cells.
If stx genes are detected in the enrichment culture, the isolation of STEC shall be attempted as a mandatory
step. STEC isolation shall be attempted by plating the enrichment culture onto solid media, and isolated
colonies are tested for the presence of stx1 and stx2, either by the real-time PCR described in this standard
(Section 9.4), or by conventional PCR (Annex E).
5 Culture media and reagents
Follow current laboratory practices in accordance with ISO 7218. The composition of culture media and
reagents and their preparation are specified in Annex B. For performance testing of culture media, see
table B.1 and follow the procedures in accordance with ISO 11133.
6 Equipment and consumables
Disposable plasticware is an acceptable alternative to reusable glassware if it has suitable specifications.
Usual microbiological laboratory equipment (see ISO 7218) and the following:
6.1 Water bath or heating block capable of being maintained at temperatures up to 100 °C.
6.2 Incubator capable of operating at 41,5 °C ± 1 °C and at 37 °C ± 1 °C.

ISO/DIS 13136-1:2023(en)
6.3 Instruments needed for nucleic acid extraction . Appropriate equipment, e.g. heating plate,
according to the method adopted (if needed).
6.4 Sterile pipettes of several capacities (between 1 µl and 1 000 µl, ISO 7550, Reference [14]).
6.5 Thin-walled real-time PCR microtubes, other real-time PCR plasticware or consumables: multi-
well PCR microplates or other disposable plasticware or capillaries.
6.6 Real-time PCR apparatus. Thermal cycler with a detection tool for fluorescence emission following 5’
nuclease PCR assay. Several brands of apparatus are available and can be chosen according to the laboratory
policies.
6.7 Thermal cycler (if needed) and PCR microtubes. Several brands of apparatus are available and can be
chosen according to the policy of the laboratory.
6.8 Peristaltic blender with sterile bags, possibly with a device for adjusting speed and time.
6.9 Microcentrifuge or Plate spinner in case 96 well plate is used
6.10 Sterilization oven, for dry sterilization or autoclave for wet sterilization, used in accordance with
ISO 7218.
6.11 Drying cabinet or ventilated oven, capable of operating between 25 °C and 50 °C, or a laminar
airflow cabinet for plates drying (if needed).
6.12 Refrigerator for storage of prepared media, capable of operating at 5 °C ± 3 °C.
6.13 pH-meter, capable of being read to the nearest 0,01 pH unit at 25 °C, enabling measurements to be
made which are accurate to ±0,1 pH unit.
6.14 Sterile tubes, bottles, or flasks with caps, of appropriate capacity.
Bottles or flasks with non-toxic metallic or plastic screwcaps may be used.
6.15 Sterile Petri dishes, with a diameter of approximately 90 mm and (optional) large size (diameter
approximately 140 mm).
6.16 Sterile cell spreaders
6.17 Water bath, capable of operating at 47 °C to 50 °C.
6.18 Laminar flow hood
6.19 Sterile filter tips
6.20 PCR cabinet
7 Sampling
Sampling is not part of the method specified in this document. Follow the specific International Standard
dealing with the product concerned. If there is no specific International Standard dealing with the sampling
of the product concerned, it is recommended that the parties concerned come to an agreement on this
subject.
ISO/DIS 13136-1:2023(en)
Recommended sampling techniques are given in the following documents:
— ISO/TS 17728 for food and animal feed;
— ISO 707 for milk and milk products;
— ISO 13307 for primary production stage;
— ISO 17604 for carcasses;
— ISO 18593 for surfaces.
It is important that the laboratory receives a sample that is representative of the product at the time of
sampling. The sample should not have 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: follow the procedures specified in the ISO 6887 series. If there is no specific International
Standard available, it is recommended that the parties concerned come to an agreement on this subject.
The pre-treatment of spent irrigation water from sprout production may be carried out by centrifugation as
described in the EU-RL VTEC_Method_09 (Reference [15]).
9 Procedure
9.1 Test portion and initial suspension
Use the method specified in the relevant document of ISO 6887 (all parts) and any specific International
Standard appropriate to the product concerned.
Pre-warm BPW broth to room temperature, or preferably to incubation temperature (see 9.2.1) before use.
Allow frozen products to thaw at room temperature, and then mix the test portion (x g or x ml) with 9x ml of
the enrichment broth, BPW.
For solid matrices, aseptically add the test portion (x g) into a sterile sample bag suitable for peristaltic
blending. Add 9x ml of the enrichment broth, BPW (Annex B) into the container with test portion, to obtain a
ten-fold dilution. The use of bags with filters is preferred. Homogenize in a peristaltic blender (see ISO 7218)
(6.8).
For liquid matrices, transfer aseptically the test portion (x ml) of liquid sample, using a sterile pipette, into a
suitable sterile container (e.g. tube, bottle) containing 9x ml of the enrichment broth BPW.
NOTE Faecal samples from primary production stage can be tested either as swab samples (aseptically added
to 10 ml of the enrichment broth) or by aseptically adding to the test portion (up to 25 g) of sample 9x ml of the
enrichment broth. For further guidance, see Reference [16].
9.2 Enrichment
Incubate the test sample enrichment at 41,5 °C ± 1 °C for 21 h ± 3 h. In case stressed cells are suspected to
be present in the sample (e.g. frozen, dried, UV or pressure treated foodstuff), incubate at 37 °C ± 1 °C for
6 h ± 1 h and then continue at 41,5 °C ± 1 °C for 18 h ± 3 h.
Keep the enrichment cultures at 4 °C ± 1 °C after incubation until the real-time PCR has been completed.
Isolation of STEC from stx positive enrichment cultures shall be attempted as soon as possible, and within a
maximum of 72 h. The enrichment may be stored for a longer period if verification/validation of the sample
type shows no negative effects on the probability of STEC isolation.

ISO/DIS 13136-1:2023(en)
9.3 Nucleic acid extraction
Nucleic acid extraction shall be carried out according to ISO 20837. Use an appropriate nucleic acid
extraction procedure for bacterial DNA purification from 1 ml of the enrichment culture. A comprehensive
list of methods can be found in Reference [19]. Alternatively, commercial kits may be used according to the
manufacturer's instructions.
9.4 PCR amplification (for real-time PCR)
The PCR screening for the presence of stx is meant to identify the samples that may contain STEC and
therefore shall be subjected to isolation. Isolation of viable cells is important to confirm the presence of
potentially infectious STEC cells, as a PCR positive signal may be generated from the stx sequences or
partially homologous sequences from other sources (i.e. free stx-phages or dead and injured cells) that do
not represent a hazard.
9.4.1 General
The PCR amplification protocol described is based on real-time PCR.
Follow all requirements for the PCR amplification as specified in ISO 22174.
Primers and detection probes for conducting real-time PCR for the detection of stx1 and stx2 are described
in Annex D.
9.4.2 Interpretation of real-time PCR results
The PCR results obtained, including the controls specified in ISO 22174 and hereafter, are interpreted by
the software provided with the real-time PCR apparatus. During amplification, the software monitors 5’
nuclease PCR amplification by detecting the fluorescence emitted by the reporter dye in each sample, R .
n
ΔR (change in the reported dye emission) is R minus the baseline reporter dye intensity established in the
n n
first few cycles, the point at which the reaction reaches a fluorescence intensity above the background. At
the end of the PCR cycles, a reaction is considered positive if its ΔR curve exceeds the threshold, defined
n
as 10 times the standard deviation of the mean baseline emission. The cycle threshold, C , is defined as the
t
cycle number at which the ΔR fluorescence of a sample crosses the determined threshold value.
n
As a general laboratory practice, particularly when the results are ambiguous, the laboratory personnel
should check the amplification curves to assess the results. Positive samples give a curve with a clear
increase in fluorescence, starting from the number of cycles corresponding to the C .
t
Each PCR assay must include positive and negative controls. The positive control is DNA extracted from a
STEC strain harbouring both stx1 and stx2 (e.g. E. coli O157 EDL933 reference strain WDCM 00188). The
negative control is added as described in ISO 22174. Further information about these controls can be found
in Annex E.3.8.
If the controls yield unexpected results, repeat the procedure.
9.4.3 Internal Amplification control for real-time PCR
When performing real-time PCR, an Internal Amplification Control (iac), is used in every real-time PCR
reaction, to check the absence of PCR inhibitors, hindering the correct amplification of the target. If
inhibition of the PCR is observed, ie. Lack of amplification of iac, the related DNA extracts prepared as in
section 9.3 may be diluted in sterile DNase free, RNase free, water or buffer. The rate of dilution needed
varies depending on the matrix, and a range of 1:10 to 1:100 is recommended. As an alternative to dilution,
commercial kits for removing PCR inhibitors may be used following the manufacturers’ instructions.
Different internal amplification controls (iac) are available for real-time PCR, for instance:
— Commercially available iac, including those contained in some real-time PCR master mix kits. They shall
be used following the manufacturers’ instructions.

ISO/DIS 13136-1:2023(en)
— pUC19. For the open formula pUC 19 based internal amplification control iac, see Reference [20].
Approximately 50 copies of target DNA (pUC 19) should be used per PCR reaction. The size of the iac is
119 bp.
9.5 STEC strain isolation
The isolation of the STEC strains is required to confirm that the positive PCR signals are generated from
genes present in a live bacterial cell.
The enrichment cultures are kept refrigerated (at 4 °C ± 1 °C) until the real-time PCR results are available.
Isolation from the positive samples should be attempted as soon as possible as the probability of isolation
of the STEC may decrease during the storage time. If needed, the enrichment culture may be stored at
4 °C ± 1 °C for a maximum of 72 h. Longer storing times may be possible, if verification/validation per sample
type shows no negative effects for the isolation of STEC.
Isolation is achieved by plating the positive enrichment culture onto at least two different selective and/
or differential solid media. It is mandatory to use the E. coli selective medium tryptone bile X-glucuronide
agar (TBX) as primary isolation medium. The laboratory chooses the other solid media to be used, which
should incorporate differential and selective principles other than the detection of beta-glucuronidase with
X-glucuronide.
The discrimination between STEC and commensal E. coli requires determining the presence of stx genes
in single colonies. The primary isolation medium, TBX, is a medium suitable for E. coli growth, for which
the selectivity is based on the presence of bile salts and the detection of beta-glucuronidase activity.
STEC strains may differ significantly in their resistance to selective agents other than bile salts, including
variability between strains belonging to the same O-group (see References [21], [22], [23] and [24]).
Most E. coli strains are beta-glucuronidase positive and form blue colonies in TBX. However some E. coli
strains, notably those belonging to serotype O157:H7, are beta-glucuronidase negative, therefore colonies
with diverse characteristics on TBX, including colorless ones, should be assayed for the presence of stx genes.
The proportion of the white colonies tested should be 10 % of the total colonies picked from TBX.
The use of different selective, chromogenic and differential media is highly recommended. The use of plates
containing containing cefixime and tellurite (CT) may be of help for the isolation of certain STEC strains, such
as CT-Sorbitol MacConkey agar for E. coli O157 and other STEC. Nevertheless, not all STEC are able to grow
in the presence of cefixime and tellurite. For STEC O26 isolation, a differential solid medium (MacConkey)
containing rhamnose instead of lactose may be used (RMAC), as typical STEC O26 strains do not utilize
rhamnose, while other E. coli usually do. Enterohaemolysin agar plates are not selective but can indicate the
production of beta-haemolysin, a characteristic shared by some STEC strains.
To obtain well-isolated colonies on the plates, spreading of more than one dilution of the enrichment culture
is required, and this is referred to as the dilution procedure. The range of dilutions to be spread depends on
the background microflora of the test sample and on whether high selective media (e.g. CT-SMAC) are used.
-6 -4 -5 - 6
Prepare dilutions to 10 and spread 100 µl of the appropriate dilutions (e.g. 10 , 10 and 10 ) onto TBX
agar and at least one additional solid medium chosen by the laboratory. Dilutions can be prepared in BPW,
physiologic solution (NaCl 9 g/l) or maximum recovery diluent (Peptone 1 g/l, NaCl 8,5 g/l). The inoculated
agar plates shall be incubated at 37 °C ± 1 °C for 21 h ± 3 h. Should insufficient isolated colonies be obtained,
repeat plating of the enrichment broth at different dilutions to achieve discreet colonies. In case stressed or
poorly growing cells are suspected to be present in the sample (e.g. frozen, dried, UV, or pressure treated
-2 -3
foodstuff), plating dilutions of 10 and 10 may be needed for successful STEC isolation.
NOTE C values can provide an indication of the correct dilution plate to pick the single colonies from. When high
t
C values are obtained, this indicates relatively low numbers of STEC in the enrichment and this might hamper the
t
-5 -6
strain isolation when only colonies are selected from the 10 and 10 dilution plates. In such circumstances, it is
-4
advisable to also select some typical E. coli colonies from the 10 dilution plate(s) even though they might not be well-
isolated.
Test up to 50 typical/suspicious colonies (E. coli morphology, growth on E. coli suitable medium) per sample
for the presence of stx genes by conventional or real-time PCR, with 50 % of the colonies from TBX agar and
the remaining selected from the alternative medium or any other additional media used. Pick individual

ISO/DIS 13136-1:2023(en)
colonies based on different colony characteristics of typical and suspect colonies from the plating media
used, so that the different colony characteristics are represented in the total of 50 colonies picked.
Colonies may be tested in pools made up of no more than 10 colonies each in water (see paragraphs below). If
different pools show different stx patterns, single colonies from all stx positive pools should be investigated
for colony confirmation. When a colony or the colonies with a stx profile similar to the pool profile(s)
are detected, the procedure can be stopped, even if less than 50 colonies are tested. The colonies under
investigation shall be streaked onto a nutrient agar plate and incubated at 37 °C ± 1 °C for minimum 18 h and
up to 24 h, to allow further characterization.
There are several types of nutrient agar media commercially available either ready-to-use plates or prepared
in house from dehydrated powders. Every type of non-selective nutrient agar media, e.g. tryptone soya agar
(TSA) is suitable for the purpose of maintaining the colonies for further investigations.
Colony selection and pooling for the identification of stx-positive colonies can be conducted as follows. Select
colonies bearing in mind the highly variable colony characteristics of STEC (e.g. not all STEC show beta-
glucuronidase activity, such as STEC O157). Pick a portion of a single colony with a sterile 1 µl loop or needle;
touch the loop or needle onto a non-selective agar, e.g. Nutrient agar plate (labelled with a grid to keep track
of individual colonies). Suspend the residual colony material in 300 µl to 500 µl of 0,22 µm molecular-grade
sterile water in a microcentrifuge tube. Repeat the procedure and suspend the remaining up to nine colonies
that constitute a pool into the same microcentrifuge tube. If real-time PCR is applied, proceed with the DNA
extraction method in use in the laboratory. In case conventional PCR (as the protocol described in Annex E)
is carried out, the tube is boiled for 10 min to provide a DNA template.
DNA template from individual colonies composing the positive pools is prepared as described above.
The standard PCR protocols in Annexes D or E or other equivalent PCR protocols are used to confirm the
presence of the virulence genes in the isolated colonies.
Optionally, when testing vegetable samples including sprouts or other matrices with an acid sensitive
background flora, isolation of STEC may be aided by the application of an acid treatment (pH 2,0 ± 0,2), to
decrease the background microflora. A protocol of acid treatment method to aid STEC isolation in vegetables
is provided in the informative Annex F. Following acid treatment, spread an aliquot of the enrichment media,
select up to 30 colonies for confirmation of the presence of the s
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