🧬 STEC/VTEC — Verotoxigenic E. coli
Detection of Shiga toxin-producing Escherichia coli (STEC/VTEC) in food by real-time PCR method — detection of virulence genes (stx1, stx2, eae) and determination of serogroups O157, O111, O26, O103, O145.
Overview
STEC (Shiga toxin-producing Escherichia coli), also known as VTEC (Verocytotoxin-producing E. coli), are pathogenic E. coli strains producing Shiga toxins (Stx1 and/or Stx2) — among the most dangerous foodborne pathogens. STEC infection can lead to hemorrhagic colitis (bloody diarrhea) and hemolytic uremic syndrome (HUS) with kidney failure, thrombocytopenia and hemolytic anemia. HUS mortality is 3–5%, and permanent kidney damage occurs in 25% of children with HUS.
The most dangerous serogroups (so-called "Top 5" or "Big 6") are: O157 (most common, best known), O26, O111, O103 and O145. Infection source is most often beef (raw or undercooked), unpasteurized milk, raw sprouts (alfalfa, broccoli), lettuce and water. Infectious dose is extremely low — only 10–100 cells are sufficient.
The PN-EN ISO/TS 13136 standard describes molecular STEC detection method based on real-time PCR. This is the only standardized PCR method for STEC in food. The method includes: sample enrichment (18–24 h), DNA extraction, virulence gene detection (stx1, stx2 — Shiga toxin genes; eae — intimin gene, adhesion protein) and serogroup determination (O157, O111, O26, O103, O145) using real-time PCR.
If stx and eae genes and "Top 5" serotype are detected — strain isolation is required, often using immunomagnetic separation (IMS). Testing is mandatory in EU for sprouts and sprouting seeds (Regulation (EC) 2073/2005) and ground beef in some countries. The STEC O104:H4 epidemic crisis in Germany in 2011 (fenugreek sprouts, >4000 cases, 54 deaths) significantly strengthened testing requirements.
Method principle
Three-stage method: (1) Enrichment — sample is incubated in enrichment broth (e.g., BPW or mTSB with novobiocin) at 37°C/18–24 h. (2) DNA extraction — DNA is isolated from enriched culture (thermolysis or extraction kits). (3) Real-time PCR — real-time PCR reactions with fluorescent probes (TaqMan) are performed to detect: stx1 and stx2 genes (Shiga toxins), eae gene (intimin — adhesion to enterocytes), and serogroup genes (wzgA-O157, wzx-O111, wzx-O26, wzx-O103, wzx-O145). If sample is positive for stx + eae + "Top 5" serogroup — STEC strain is isolated, often using IMS (immunomagnetic separation) with anti-O antibody-coated beads.
Applications
- Sprouts and sprouting seeds control — Regulation (EC) 2073/2005
- Ground beef testing (minced, steaks, burgers)
- Unpasteurized milk and raw milk cheeses control
- Lettuce, leafy vegetables and fruits testing (ready-to-eat)
- Epidemiological investigation in HUS/hemorrhagic colitis outbreaks
- Drinking water and recreational water monitoring
- Imported food control (especially from countries with high endemicity)
- Testing within food safety control programs (RASFF)
Key parameters
| Parameter | Value |
|---|---|
| Enrichment | 37 ± 1°C, 18–24 h in BPW or mTSB + novobiocin |
| Detection method | Real-time PCR (qPCR) with TaqMan probes |
| Virulence genes | stx1, stx2 (Shiga toxins), eae (intimin) |
| Serogroups | O157, O111, O26, O103, O145 |
| Isolation (optional) | IMS (immunomagnetic separation) + plating on CT-SMAC / chromogenic |
| Sensitivity | 1–10 CFU/25 g (after enrichment) |
Standard
- Standard number
- PN-EN ISO/TS 13136:2013
- 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. Sample preparation and enrichment
Weigh 25 g sample, add 225 mL enrichment broth (BPW or mTSB with novobiocin). Homogenize in stomacher. Incubate 37 ± 1°C for 18–24 h.
2. DNA extraction
Take 1 mL enriched culture. Extract DNA using extraction kit (silica columns, magnetic beads or thermolysis: 100°C/10 min). Measure DNA concentration and purity (optional).
3. PCR screening — virulence genes
Prepare 3 PCR reactions (simplex or multiplex): stx1, stx2, eae. Add DNA, primers, TaqMan probes, Master Mix. Program: 95°C/10 min → 40 cycles (95°C/15 s, 60°C/60 s). Analyze fluorescence curves — Ct < 35 = positive result.
4. PCR screening — serogroups (if stx+)
If stx1 and/or stx2 detected — prepare 5 PCR reactions for serogroups: O157, O111, O26, O103, O145. Same thermal program. Positive result = Ct < 35.
5. Screening interpretation
Variants: (A) stx+ / eae+ / Top5 serogroup+ → potentially positive sample, strain isolation required. (B) stx+ / eae− or serogroup negative → STEC present, but not Top5, report. (C) stx− → negative sample.
6. Strain isolation (IMS)
For variant (A): add 20 µL immunomagnetic beads (anti-O of appropriate serogroup) to 1 mL enriched culture. Incubate 15–30 min with mixing. Separate on magnet. Wash 3x. Plate on CT-SMAC (O157) or chromogenic agar.
7. Isolate confirmation
From CT-SMAC/chromogenic plate select typical colonies (O157: sorbitol-, colorless). Confirm by PCR — isolate must possess stx1/stx2 + eae + appropriate serogroup gene. Optionally: latex agglutination (anti-O157, etc.).
8. Reporting
Screening result: "STEC (stx+) detected/not detected in 25 g". If strain isolated: "STEC O157:H7 (stx2+, eae+) isolated from 25 g sample". Positive result in food = RASFF notification.
Required equipment and apparatus
| Equipment | Example | Indicative price |
|---|---|---|
| Real-time PCR thermal cycler | Bio-Rad CFX96 Touch, Applied Biosystems 7500 Fast, Roche LightCycler 480 | 60,000–180,000 PLN |
| IMS system (immunomagnetic separation) | Dynabeads anti-E. coli O157 + magnetic separator (Dynal MPC-S) | 5,000–15,000 PLN (separator) + 2,000–4,000 PLN/kit beads |
| Laboratory centrifuge | Eppendorf 5424 R, Hettich Mikro 200 R | 8,000–25,000 PLN |
| PCR cabinet (pre-PCR) | Biosan UVT-S-AR, ESCO PCR Cabinet | 5,000–15,000 PLN |
| DNA extraction kit | Qiagen DNeasy Food Kit, Promega Wizard, PrepMan Ultra | 1,500–3,000 PLN / 50 extractions |
| Incubator 37°C | Binder BD 56, Memmert INB 200 | 5,000–18,000 PLN |
| Class II laminar flow cabinet | Thermo Scientific MSC-Advantage | 25,000–60,000 PLN |
Reagents, media and consumables
| Reagent | CAS | Details |
|---|---|---|
| Real-time PCR kits for STEC (stx1, stx2, eae) | — | Kits with primers, TaqMan probes and controls: e.g., Bio-Rad iQ-Check STEC VirA, Romer RapidFinder STEC, Merck GeneDisc STEC; 96 reactions package |
| PCR kits for serogroups (O157, O111, O26, O103, O145) | — | Separate or multiplex kits with primers/probes for wzx/wzy genes; 96 reactions package |
| PCR Master Mix (polymerase + dNTP + buffer) | — | E.g., Bio-Rad SsoAdvanced Universal Probes Supermix, TaqMan Universal Master Mix; 200 reactions package |
| DNA extraction kit | — | Silica columns or magnetic beads; e.g., Qiagen DNeasy, Promega Wizard; 50 extractions package |
| mTSB + novobiocin (enrichment broth) | — | Modified tryptic soy broth with novobiocin (20 mg/L) — selective STEC enrichment; 500 g package + supplements |
| Immunomagnetic beads (IMS) | — | Dynabeads anti-E. coli O157, O26, O111, O103, O145; 5 mL package (50 reactions) |
| CT-SMAC Agar (Cefixime Tellurite Sorbitol MacConkey) | — | Medium for E. coli O157 isolation (sorbitol-negative); 500 g package + supplements |
| Buffered peptone water (BPW) | — | Alternative enrichment broth, 500 g package |
Health and safety (OHS)
- STEC — BSL-2 pathogen with extremely low infectious dose (10–100 cells!), use highest precautions
- Work with cultures exclusively in class II laminar flow cabinet
- Separate pre-PCR and post-PCR zones — prevent amplicon contamination
- Novobiocin — antibiotic, avoid skin contact
- Autoclave used materials and cultures (121°C/30 min) before disposal
- In case of O157 suspicion — consider notifying reference laboratory