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1. Dear Laboratories, Dear Colleagues, We hereby send you ... - Favv

1. Dear Laboratories, Dear Colleagues, We hereby send you ... - Favv

The first method is as

The first method is as follows: Enrichment of 25 g of sample + 225ml buffered peptone water (BPW) incubation for 6 h (± 0.5h) and 18 h (±1 h) at 37°C ± 1°C. Attention : this is a superficial contamination, so a maximum amount should be taken from the surface of the vegetables. After 6 h and 18 h, 50 µl (6 h) and 10µl (18 h) shall be plated onto a TBX medium containing 2 mg/l ceftazidime (Sigma : C3809 - lG) and incubated at 37°C ±1°C for 24 h ±1h. As an alternative may be used cefotaxime (Sigma C7039-IG) at a concentration of 1 mg/l. When suspicious E. coli colonies are present (blue-green), these shall be subcultured onto a new TBX plate with no antibiotics added. All suspicious colonies – with a maximum of 50 colonies per sample – shall be screened for the presence of the vtx 2 gene using PCR (see flowchart). This screening shall be done by pooling the colonies per 10 in a cellysate. When a result is positive for the vtx2 gene, the 10 colonies of the pool that is positive for vtx2 shall individually be confirmed for the presence of vtx2. Subsequently, the vtx2+ colony shall be tested for the presence of the O104 wzx gene using PCR. For the detection of the vtx 2 gene may be used real time PCR, as described in ISO TS 13136, using the primers and the probe described by Perelle et al., (Mol cell Prob. 2004Vol 18- 185-95). For the detection of the O104 gene shall be used the primers and the probe as described by Bugarel et al. (Int J Food Microbiol 2010 142:318-329). The commercial kits of GeneSystems such as Life Technologies may also be used for both vtx2 and O104. As an alternative may be used a conventional PCR method that was published in a peer reviewed article. A second method is as follows : 25 g of sample is enriched in 225ml buffered peptone water (BPW) + 2mg/l ceftazidime and incubated for 6 h (± 0.5h) and 18h (±1 h) at 37°C ± 1°C. As an alternative may be used cefotaxime (Sigma : C7039-IG) at a concentration of 1 mg/l. After 6 h and 18 h, 50 µl (6 h) and 10 µl (18 h) shall be plated onto TBX with no antibiotics added. When there is growth of suspicious E. coli colonies, these shall be subcultured onto a new TBX plate and tested for the vtx2 gene and the wzxO104 gene (procedure : see above). When laboratories do not have a positive control sample for O104 PCR at their disposal, DNA may be made available upon request at the NRL. The methods that are proposed here have been validated. All questions will be gathered by the FASFC. Good luck !!!!!!! Wetenschap ten dienste van Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu. 2. Juliette Wytsmanstraat 14 1050 Brussel | België T + 32 2 642 51 11 | F + 32 2 642 50 01. info@wiv-isp.be | www.wiv-isp.be.

Kind regards, The NRL – food microbiology team Wetenschap ten dienste van Volksgezondheid, Veiligheid van de Voedselketen en Leefmilieu. 3. Juliette Wytsmanstraat 14 1050 Brussel | België T + 32 2 642 51 11 | F + 32 2 642 50 01. info@wiv-isp.be | www.wiv-isp.be.

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