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Published on: October 8, 2013
Optimization of Pulsed-field Gel Electrophoresis Procedure for Bacillus cereus
Hui Juan Zhang1, Zhuo Pan2, Jian Chun Wei1
1National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention/State Key Laboratory for Infectious Disease Prevention and Control, Beijing 102206, China; Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, Zhejiang, China.
Abstract:
In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth (Luria-Bertani agar plates for 12-18 h) and lysis conditions (4 h incubation with 500 µg/mL lysozyme) produced sharp bands on the gel. Restriction enzyme NotI was chosen as the most suitable. Twenty-two isolates were analyzed by NotI digestion, using three electrophoretic parameters (EPs). The EP-a was optimal for distinguishing between isolates. The optimized protocol could be completed within 40 h which is a significant improvement over the previous methods.
