蜡样芽胞杆菌脉冲场凝胶电泳影响因素的研究

Study on Influencing Factors of Pulsed Field Gel Electrophoresis for Bacillus cereus

目的通过比较蜡样芽胞杆菌(以下简称蜡样杆菌)脉冲场凝胶电泳(PFGE)技术方法的影响因素,优化蜡样杆菌PFGE分型方法。方法在细菌收集、细胞裂解、胶块制备和电泳参数等环节设立对照,选取3株蜡样杆菌比较不同实验方案的电泳图谱结果。细菌收集设立LB固体培养基和液体培养基集菌法;细胞裂解设立灌制胶块前溶菌酶孵育组与未孵育组,设立单独溶菌酶裂解组和溶菌酶与溶葡萄球菌酶联合裂解组;细菌的胶块内裂解设立溶菌酶不同浓度和不同作用时间(2、4和16h)实验组;选取22株蜡样杆菌对3组电泳参数进行分析评价。结果 LB固体培养基集菌法与液体集菌法相比,获得了同样清晰或更清晰的条带。灌制胶块前溶菌酶与菌悬液进行一定时间的孵育可明显改善条带的清晰度。溶菌酶对胶块内细菌的裂解,4h为最佳裂解时间。溶葡萄球菌酶与溶菌酶联合使用未见对细菌裂解有改善作用。3组电泳参数(EP)的图谱差异有统计学意义(P<0.05),EP-a的相似性系数最小,为最佳电泳参数。结论试验确定了最佳蜡样杆菌PFGE实验方案,简化和优化了目前已有的蜡样杆菌PFGE实验方法,可应用于蜡样杆菌引起的暴发疫情分析及食源性病原菌的实验室分子分型研究。

Objective To optimize the operating procedures of pulsed - field gel electrophoresis （PFGE） for Bacillus cereus. Methods Factors influencing PFGE results, including preparation of vegetative cells embedded in agarose, lysis of embedded ceils, enzymatic digestion of intact genomic DNA and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation were controlled, resulting in a rapid and reliable method for B. cereus genotyping. Results Optimal cellular growth （ Luria - Bertani agar plates for 12 -18h） and lysis conditions （4h incubation with 500μg/ml lysozyme） provided sharp bands by PFGE,and lysozyme with enough time before plug preparation is critical for lysiug the ceils wall. Twenty - two isolates were analyzed by NotI digestion, using three electrophoretic parameters （ EPs~. The EP - a with high D values and minimal similarity coefficients was optimal for distinguishing between isolates. Conclusion The optimized protocol of B. cereus presented in this study is more rapid （4 days from culture to analysis） and robust compared to existing methods. We believe that adaption of this method will assist in the anaylsis of outbreaks caused by B. cereus and will be a benefit to laboratories performing subtyping for important food - borne pathogens.