🧬 STEC/VTEC — verotoxigenic E. coli

Microbiology ISO/TS 13136

Overview

STATUS (as of 02.11.2026). ISO/TS 13136:2012 — technical specification, first edition, published on 07.11.2012, current; it was prepared by CEN in collaboration with ISO/TC 34/SC 9 (Vienna Agreement), in Europe as CEN ISO/TS 13136:2012. The PKN catalogue has no adoption of it (search on 02.11.2026: no results for “TS 13136”, while the same query for “TS 22964” finds PKN-ISO/TS 22964:2008) — there is no designation “PN-EN ISO/TS 13136”, and the Polish title is a working translation. The revision is being prepared as the drafts ISO/DIS 13136-1 and ISO/DIS 13136-2 (in CEN among others prEN ISO 13136-1:2024).

SCOPE (clause 1). Identification of STEC by detection of the major virulence genes of STEC (stx and eae) and of the genes associated with serogroups O157, O111, O26, O103 and O145; real-time PCR is the reference technology. When one or both stx genes are detected, the isolation of the strain is always attempted; isolation of strains of the serogroups in the scope is facilitated by serogroup-specific enrichment, e.g. immunomagnetic separation (IMS). The specification applies to products intended for human consumption and for the feeding of animals, environmental samples in the area of food production and food handling and in the area of primary production.

SIGNIFICANCE (introduction, “Important” note). STEC are pathogenic E. coli which can cause diarrhoea and more severe diseases — haemorrhagic colitis and haemolytic uraemic syndrome (HUS); the serogroups O157, O26, O111, O103 and O145 are associated with the most severe forms, in particular HUS. Any STEC is considered pathogenic to humans — the risk depends on the type of food (ready-to-eat or treated, e.g. pasteurized or cooked) and on the health status of the person. Novel arrangements of virulence features can give rise to novel pathogroups, such as the Shiga toxin-producing enteroaggregative E. coli O104 which caused HUS outbreaks in Germany and France in May and June 2011; such strains fall within the scope of the method, as they carry stx genes. Nomenclature: stx = vtx, Stx = Vtx (verocytotoxin), STEC = VTEC. According to chapter 4a of the BAM manual (FDA), the infectious dose of O157:H7 is estimated at 10–100 cells (no information is available for other EHEC serotypes), and infections are mostly food- or waterborne — implicated foods include undercooked ground beef, raw milk, cold sandwiches, water, unpasteurised apple juice, sprouts and vegetables.

DEFINITIONS (3.1–3.3). STEC — E. coli strains possessing the Stx-coding genes; STEC causing the attaching and effacing lesion — with the Stx-coding genes and the intimin-coding gene eae (a combination often associated with the most severe forms of disease); STEC belonging to highly pathogenic serogroups — with the Stx-coding genes and eae, belonging to serogroups O157, O111, O26, O103 or O145.

ENRICHMENT AND TARGET GENES (4.2–4.6, 5.1). The test portion is enriched in a non-selective liquid medium (1 in 10 dilution): in mTSB with 16 mg/l of novobiocin (mTSB+N) — samples with high background flora, in buffered peptone water (BPW) — samples with stressed cells (e.g. frozen products) and lower background flora, and in mTSB with 12 mg/l of acriflavin (mTSB+A) — milk and dairy products. Novobiocin at the usual concentration of 20 mg/l (as in ISO 16654) inhibits about one third of non-O157 strains and increases the risk of false-negative results. Target genes: stx1 and stx2 (variants stx2a, stx2b and stx2c), eae, and rfbE (O157), wbdI (O111), wzx (O26), ihp1 (O145) and wzx (O103). The strain is isolated after serogroup-specific enrichment (e.g. IMS) on tryptone-bile-glucuronic (TBX) agar or on a specific medium, if available.

REGULATIONS. Commission Regulation (EC) No 2073/2005 (consolidated text as of 01.07.2026), criterion 1.29: sprouts — STEC O157, O26, O111, O103, O145 and O104:H4 absence in 25 g, n = 5, c = 0, method CEN/ISO TS 13136 taking into account the adaptations of the European Union reference laboratory for E. coli concerning the detection of STEC O104:H4 (footnote 22); the criterion does not apply to sprouts that have received a treatment effective to eliminate Salmonella and STEC (footnote 23). Sprouts are sampled at the stage with the highest probability of detecting STEC and Salmonella, in no case earlier than 48 h after the start of sprouting (Annex I, Chapter 3).

WHAT WE DO NOT GIVE HERE. The available text of the specification ends at 9.1.3 (initial suspension). We do not give the incubation conditions of enrichment (9.2), nucleic acid extraction (9.3), the PCR programme and reading criteria (9.4), primers and probes (Annexes C and E), the amplification control (Annex D) or the course of isolation (9.5, Annex F).

Method principle

The method comprises in sequence: microbial enrichment, nucleic acid extraction, detection of virulence genes, detection of serogroup-associated genes and isolation from positive samples (4.1, Annex A). The number of STEC cells is increased by incubating the test portion in a non-selective liquid medium (mTSB+N, BPW or mTSB+A), the nucleic acid is extracted according to the adopted detection system, and the stx, eae and serogroup genes are detected in it by real-time PCR (4.2–4.5). When STEC is suspected, isolation is attempted; when a serogroup of the scope is detected — after specific enrichment, e.g. IMS, and plating on TBX agar or a specific medium (4.6).

Applications

• Products intended for human consumption and for the feeding of animals (clause 1)
• Environmental samples in the area of food production and food handling and of primary production (clause 1)
• Milk and dairy products — enrichment in mTSB with acriflavin (4.2)
• Sprouts — food safety criterion for STEC O157, O26, O111, O103, O145 and O104:H4 (Regulation (EC) No 2073/2005, item 1.29)

Key parameters

Standard

Standard number

ISO/TS 13136:2012 (CEN ISO/TS 13136:2012)

Title (PL)

Mikrobiologia żywności i pasz — Metoda reakcji łańcuchowej polimerazy (PCR) w czasie rzeczywistym do wykrywania patogenów przenoszonych przez żywność — Horyzontalna metoda wykrywania Escherichia coli wytwarzających toksynę Shiga (STEC) oraz oznaczania serogrup O157, O111, O26, O103 i O145

Title (EN)

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

Step-by-step procedure

1. Initial suspension

   Use the quantity of enrichment medium necessary to give a final dilution of 10⁻¹ of the test portion (9.1.1).

2. Samples with high background flora

   Transfer a solid test portion (x g) to a peristaltic blender bag (preferably with a filter) containing 9x ml of mTSB with novobiocin or acriflavin and homogenize; transfer a liquid test portion (x ml) with a sterile pipette into 9x ml of this medium (9.1.2).

3. Samples with stressed cells

   Allow frozen products to thaw at room temperature, transfer the test portion (x g or x ml) to 9x ml of BPW and proceed as above (9.1.3).

4. Enrichment

   Incubate in an incubator at (37 ± 1) °C (6.2); time and conditions — 9.2, beyond the available text.

   🌡 Temperature: 37 ± 1 °C

5. Nucleic acid extraction

   According to the requirements of the adopted detection system (4.3, 9.3).

6. Gene detection

   Real-time PCR: virulence genes stx and eae, then the genes of serogroups O157, O111, O26, O103 and O145 (4.1, 4.4, 9.4).

7. Isolation

   When one or both stx genes are detected, attempt the isolation of the strain; when a serogroup of the scope is detected — specific enrichment (e.g. IMS) and plating on TBX agar or a specific medium (clause 1, 4.6, Annex F).

8. Result

   Result categories according to the definitions: STEC (stx genes), STEC with the eae gene, STEC of serogroups O157, O111, O26, O103 or O145 with stx and eae genes (3.1–3.3, clause 10).

Required equipment and apparatus

Reagents, media and consumables

Health and safety (OHS)

• Any STEC is considered pathogenic to humans and potentially able to cause severe disease — depending on the type of food and the health status of the person (“Important” note of the specification)
• For laboratory practice and PCR requirements the specification refers to ISO 7218, ISO 22174 and ISO 20838 (clause 2)

Frequently asked questions