Seakem gold gel

All isolates of S. flexneri were analyzed by PFGE according to the standard protocol for S. flexneri developed by the Centers for Disease Control and Prevention of the USA. Salmonella enterica serotype Braenderup H9812 was digested with XbaI and used as molecular weight standard. Slices of agarose plugs were digested with NotI (Takara, Dalian, China) at 37°C for 3 h. Electrophoresis was carried out in 1% agarose SeaKem Gold gel (Lonza, Rockland, ME, USE) with the CHEF Mapper system (Bio-Rad) with the following run parameters: 6 V/cm and a linear increase in switching times from 2.16 to 54.17 s over a period of 20 h. The interpretation of the PFGE patterns was performed with BioNumerics software version 6.0 (Applied Maths, Sint-Martens-Latem, Belgium). A tree indicating relative genetic similarity was constructed based on the unweighted pair group method of averages and a position tolerance of 1.2%.

Genotypes and transmission patterns were determined by performing PFGE according to the method described in a previous study [19 (link)]. S. flexneri isolates were digested with the restriction enzyme NotI (TaKaRa, Japan) at 37 °C for 3 h to generate a DNA fingerprinting profile. Salmonella enterica serotype Braenderup strain H9812 was digested with XbaI (TaKaRa, Japan) and used as a molecular size standard. Electrophoresis was performed on the CHEF Mapper XA system (Bio-Rad) with a 1% agarose SeaKem Gold gel (Lonza, USA). Electrophoretic parameters were determined by performing multiple screening runs and included switching times of 2.16 to 54.17 s, a voltage of 6 v/cm, a 120° angle and a run time of 21 h. PFGE images were obtained using a Universal Hood II (Bio-RAD, USA) and analyzed using BioNumerics software version 7.1 (Applied Maths, Sint-Martens-Latem, Belgium). A clustering tree that indicated relative genetic similarity was constructed using UPGMA (Unweighted Pair Group Method with Arithmetic Mean) and the Dice-predicted similarity value with a 1.0% pattern optimization and 1.5% band position tolerance.