All confirmed ESBL bacteria using the combined disk method were analyzed by Real time PCR for the presence of genes encoding TEM, SHV, CTX-M. Primers and Probes were designed for ESBL producing genes by LGC, Biosearch, USA based on primers used by Roschanski et al., 2014 [25 (link)].
Genomic DNA was extracted using Maxwell 16 cell DNA purification kit (Promega) on an automated DNA extraction machine (Maxwell 16 extraction system, USA). Real time PCR assay was performed on AriaMx system (Agilent Inc, USA) using 25 μL PCR reaction mixture containing 12.5 μL Perfecta master mix low ROX kit (Quanta Bioscience Inc, USA), 1 μL of 10 μM primers, 1 μL of probes, 7.5 μL Nuclease free water (Sigma-Aldrich, USA) and 2 μL DNA template. The thermal conditions were as follows: denaturation at 95°C for 15 min, then 30 cycles consisting of a denaturation step at 95°C for 15 seconds, annealing at 50°C for 15 seconds and extension at 70°C for 20 seconds.
After completion of the run, a cycle threshold (Ct) was calculated by determining the signal strength at which the fluorescence exceeded a threshold limit. This value was analyzed using the AriaMx system software version 3.1.
Genomic DNA was extracted using Maxwell 16 cell DNA purification kit (Promega) on an automated DNA extraction machine (Maxwell 16 extraction system, USA). Real time PCR assay was performed on AriaMx system (Agilent Inc, USA) using 25 μL PCR reaction mixture containing 12.5 μL Perfecta master mix low ROX kit (Quanta Bioscience Inc, USA), 1 μL of 10 μM primers, 1 μL of probes, 7.5 μL Nuclease free water (Sigma-Aldrich, USA) and 2 μL DNA template. The thermal conditions were as follows: denaturation at 95°C for 15 min, then 30 cycles consisting of a denaturation step at 95°C for 15 seconds, annealing at 50°C for 15 seconds and extension at 70°C for 20 seconds.
After completion of the run, a cycle threshold (Ct) was calculated by determining the signal strength at which the fluorescence exceeded a threshold limit. This value was analyzed using the AriaMx system software version 3.1.