Applied biosystem 2720 thermal cycler

All E. coli isolates were examined for detection of eae, bfpA, stx1, and stx2 virulence genes. DNA of each isolate was extracted from an overnight TSB culture using a QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Uniplex PCR was conducted for eae gene detection. The PCR mix contained 12.5 μL of EmeraldAmp MAX PCR master mix (Takara Bio, Kusatsu, Japan), 1 μL (20 pmol) of each primer, 5 μL of DNA template (~50 ng), and water up to a final volume of 25 μL. The PCR reaction was conducted in an Applied Biosystem 2720 thermal cycler (Applied Biosystem, Foster City, CA, USA) under the following cycling conditions: 94 °C for 7 min; 35 cycles of 94 °C for 1 min, 51 °C for 1 min, and 72 °C for 2 min; and a final extension at 72 °C for 10 min. bfpA, stx1, and stx2 genes were detected under the same PCR conditions as for eae. The primers and annealing temperature specific to each gene are illustrated in Supplementary Data (Table S1). E. coli O157:H7 Sakai (positive for stx1 and stx2 genes) and E. coli strain E2348/69 (positive for eae and bfpA genes) were used as positive controls in all PCR reactions. Isolates that were positive for eae gene and negative for stx1 and stx2 genes were defined as EPEC [1 (link)]. EPEC isolates that were negative for bfpA gene were considered atypical EPEC (aEPEC).

Chromosomal DNA was extracted from B. cereus cells with the Puregene DNA Purification kit (Gentra Systems, USA). Plasmid DNA was extracted from E. coli using QIAprep spin columns (QIAGEN, France). Restriction enzymes (New England Biolabs, USA) and T4 DNA ligase (New England Biolabs, USA) were used in accordance with manufacturer’s recommendations. Oligonucleotide primers were synthesized by Sigma-Proligo (Paris, France). PCR was performed in an Applied Biosystem 2720 Thermal cycler (Applied Biosystem, USA). Amplified fragments were purified with the QIAquick PCR Purification Kit (QIAGEN, France). Digested DNA fragments were extracted from gels with the QIAquick Gel Extraction Kit (QIAGEN, France). Nucleotide sequences were determined by Cogenics (Meylan, France).