Optimization of Pulsed-field Gel Electrophoresis Procedure for Bacillus cereus.
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.
National 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.
Hui Juan
HJ
Zhang
Microbiology Laboratory, Shijiazhuang Center for Disease Control and Prevention, Shijiazhuang 050016, Hebei, China.
Zhuo
Z
Pan
National 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.
Jian Chun
JC
Wei
National 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.
En Min
EM
Zhang
National 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.
Hong
H
Cai
National 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.
Xu Dong
XD
Liang
National 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.
Wei
W
Li
D001409:Bacillus cereus / Q000235:genetics / Q000302:isolation & purification*; D001431:Bacteriological Techniques; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*
D004269:DNA, Bacterial