Mgcl2 50 mm

DNA extracts were examined for the detection of different insertion sequences associated with ESBL genes, performing PCRs assays using the specific primers and conditions showed in Table 3 [27 (link),32 (link),33 (link)].
The PCRs were performed in a final volume of 25 µL containing 2 µL of DNA extract mixed with 2.5 µL of 10× buffer (Bioline, London, UK), 5 µL of dNTPs (Bioline, London, UK), 1.5 µL of MgCl₂ 50 mM (Bioline, London, UK), 2 µL of each primer (Sigma-Aldrich, Madrid, Spain), and 1.5 U of Inmolase™ DNA polymerase (Bioline, London, UK), in a DNA thermal cycler GeneAmp^® PCR system 2700 (Applied Biosystems Division, Foster City, CA, USA). Amplification conditions were modified in order to improve the specificity using an initial denaturation at 94 °C for 12 min, followed by 35 cycles of DNA denaturation at 94 °C for 1 min, and primer annealing temperature depending on the IS (Table 3), primer extension at 72 °C for 2 min, and a final elongation at 72 °C for 10 min. PCR products were separated by electrophoresis on 1% agarose gels and were visualized under UV light after staining with ethidium bromide.

To determinate the clonal dissemination of ESBL-producing E. coli, a sequence type analysis was performed following the scheme described by Wirth et al. [24 (link)] and seven housekeeping genes were amplified and sequenced for each isolate (adk, fumC, gyr, icd, mdh, purA and recA). DNA extraction was performed with a DNeasy^® Blood & Tissue kit (Qiagen, Barcelona, Spain) using a pretreatment protocol for Gram-Negative Bacteria. For amplification, 3 μL of DNA extract was mixed with 5 μL of buffer 10× (Bioline, London, UK), 5 μL of dNTPs (Bioline), 1.5 μL of MgCl₂ 50 mM (Bioline), 2 μL of each primer Sigma-Aldrich, Steinheim, Germany) and 1.5 U of Inmolase™ DNA polymerase (Bioline) in a final volume of 50 μL. The amplification conditions were as follows: 3 min at 94 °C, followed by 30 cycles of 1 min at 95 °C, 1 min at 60 °C (mdh, gyrB, and recA) or 1 min at 64 °C (fumC, icd, purA and adk), 2 min at 72 °C, and a final elongation step of 5 min at 72 °C. Sequence reactions were performed by EZ-Seq Service (Macrogen Europe) and sequences data were imported into the E. coli MLST database website [25 ] to determine MLST type. These data were analyzed using BioNumerics v7.5 software (Applied Maths, Sint-Martens-Latem, Belgium).