Chef driii chiller system

Genomic DNA from the selected strains was included into agarose plugs and digested with endonuclease XbaI (Thermos Scientific). According to PulseNet protocol (CDC), the macrorestriction fragments were separated by pulsed-field gel electrophoresis on a CHEF-DRIII Chiller system (Bio-Rad Laboratories, Richmond, CA), in 1% agarose gel using 0.5X TBE buffer at 6 V/cm and 14°C, with ramped pulse times of 2.2 to 54.2 s for 21 h. Salmonella enterica serovar Braenderup was included as molecular size standard. The DNA band profiles were analyzed with GelCompar software (version 3.0; Applied Maths, Sint-Martens-Latem, Belgium). The cluster analysis and generation of dendrograms was performed using UPGMA with a 1.5% band tolerance. The similarity between DNA profiles was determined using Dice's correlation coefficient. We defined arbitrarily pulsotypes and pulsogroups with similarities of >91.1% and 75%, respectively.

According to PulseNet protocol (CDC)¹, bacterial suspensions of 276 S. sonnei strains were embedded in agarose plugs, lysed, and then digested with endonuclease XbaI (Thermo Fischer Scientific, United States) at 37°C for 2 h. The macrorestriction of genomic DNA fragments was separated by pulsed-field gel electrophoresis on a CHEF-DRIII Chiller system (Bio-Rad Laboratories, Richmond, CA, United States) in 1% agarose gel using 0.5X TBE buffer at 6 V/cm and 14°C, with ramped pulse times of 2.2–54.2 s for 21 h. Salmonella enterica serovar Braenderup strain H9812 was included as molecular size standard three times on each gel to normalize the images and to compare the fingerprints among several gels. The DNA band profiles were analyzed with GelCompar software (version 3.0; Applied Maths, Sint-Martens-Latem, Belgium). A similarity dendrogram was constructed using the unweighted-pair group method with arithmetic mean (UPGMA) with the Dice similarity coefficient and a band tolerance of 1.5%. Pulsegroups were defined by sharing 73% similarity and more than 93.5% for pulsetypes.