Xbai enzyme

In order to determine the genetic relatedness of the S. enterica isolates, nine Salmonella Infantis and ten Salmonella Enteritidis isolates included in the study were analyzed by PFGE, according to PulseNet protocol [52 (link)] and previously described by Usein et al. [53 (link)].
Briefly, chromosomal DNA was digested with XbaI enzyme (Roche) and the DNA macrorestriction fragments were separated by electrophoresis, using a CHEF Mapper electrophoresis system (Bio-Rad, CA, USA), with pulse times of 2.16–63.8 s, during a 19 h run. Salmonella Braenderup H9812 was used as control strain.
The analyses of the gel images were carried out in BioNumerics v6.6.4 (Applied Maths, Kortrijk, Belgium). The comparisons were made by cluster analysis using Dice coefficient and dendrograms were generated using the unweighted-pair group method using average linkages (UPGMA) and a position tolerance of 1.5%.
Different profiles were assigned to XbaI-PFGE types according to differences in the band patterns, which were interpreted according to the criteria proposed by Tenover et al. [54 (link)].

PFGE was performed with AscI enzyme using the standardized PulseNet protocol (http://www.pulsenetinternational.org/protocols). Isolates were grown on 6% SBA at 35°C for 24 hrs. Cells were resuspended in buffer (100 mM Tris:100 mM EDTA) at an optical cell density of 0.45–0.50 and agarose plugs prepared. To lyse cells, plugs were incubated in 50 mM Tris:50 mM EDTA, 0.1mg/ml Proteinase K, 1% sarcosyl for 2 hrs at 55°C, then washed in sterile water followed by TE buffer. DNA in embedded plugs was digested for 5 hrs with 40 U of AscI enzyme (New England Biolabs, Ipswich, MA) at 37°C and PFGE performed for 17.5 hrs at 14°C. Salmonella enterica serotype Braenderup (H9812) cut with 50 U XbaI enzyme (Roche Diagnostics, Indianapolis, IN) was used as a reference standard. PFGE parameters were 6 V/cm, 120°C, linear ramping, and switch times of 1.79 s-18.66 s. Gels were stained with ethidium bromide and imaged using a Gel Doc 1000 (BioRad, Hercules, CA). PFGE patterns were analyzed using BioNumerics software Version 6.64 (Applied Maths, Inc., Sint-Martens-Latem, Belgium) and patterns normalized to the reference standard. Cluster analysis of PFGE patterns was performed using Dice similarity coefficients (1.0% optimization and 1.5% tolerance) and unweighted pair group method with averages (UPGMA).