Optimization of pulsed-field gel electrophoresis for molecular typing of Brucella using Xba I

Abstract

Objective To optimize the protocol of pulsed-field gel electrophoresis(PFGE) to evaluate the applicability for molecular typing of Brucella using Xba].Methods Factors that influence PFGE including selecting restriction endonuclease from different manufacturers and pulse time were optimized.The bands were marked with their molecular weight and then analyzed with BioNumerics (Version 4.0,Applied Maths BVBA,Belgium) software.Results The Xba I from Promega Ltd was selected due to its better digestion effect.The best pulsed-field gel electrophoresis was performed in two phases for 21 h which included 16 h for pulse time of 0.5-8.0 s and 5 h for pulse time of 10.0-56.0 s; the angle of 120°,gradient of 6.0 V/cm,temperature of 14 ℃.The 6 Brucella species and 19 reference biovar strains were tested by optimized conditions of PFGE using Xba Ⅰ.The strains from four Brucella species which included Brucella abortus,Brucella melitensis,Brucella suis and Brucella ovis were clustered into four groups.But Brucella canis was classified in Brucella suis group.The Brucella neotomae was classified in Brucella abortus group.The results of clustering analysis by PFGE were similar to that of biotyping methods.Conclusions PFGE genotyping method using Xba I has good discriminatory power for molecular typing for Brucella.The optimized PFGE protocol is worthy of application. Key words: PFGE ; Molecular typing; Brucella