利用脉冲场凝胶电泳筛选质粒方法的建立和应用

Establishment and application of pulsed-field gel electrophoresis assay to screen plasmid from bacteria

目的改良及优化常规脉冲场凝胶电泳(pulsed-field gel electrophoresis,PFGE)方法,建立基于PFGE技术筛选细菌质粒方法,并用于军团菌质粒的筛选。方法使用含有质粒的杜莫氏军团菌及其质粒消除株对常规PFGE方法进行条件优化,调整细菌菌悬液浓度,蛋白酶K用量及作用时间,胶块清洗时间和次数,胶块不经过酶切直接进行PFGE,染色成像后选择能形成条带清晰脱尾较少的最优条件。结果去除常规PFGE方法中酶切步骤并优化裂解和清洗条件能使细菌DNA完整保留在胶块中,经过PFGE后,质粒DNA和染色体DNA在琼脂糖凝胶上能被很好的区分。结论该方法较常规方法更为快速可靠,可用于细菌质粒图谱的快速筛选。

Objective Based on conventional pulsed-field gel electrophoresis（PFGE） ,we established a modified assay to screen plasmids of bacteria and applied it in the screening of Legionella plasmids. Methods L. dumoffii strains with and without plasmid were used for the optimization of the treatment parameters, cell suspension density, content of proteinase K and its action time, wash times were adjusted, then the best conditions were chosen. Results Compared with conventional PFGE assay, the procedure of our assay was without restriction digestion. Intact plasmid DNA and chromosome DNA could therefore be kept in the agarose plug without being denatured and breakage. Plasmid DNA and chromosome DNA can be separated by the electrophoresis. Conclusion Modified PFGE processes is more accurate and rapid than conventional assay and could be applied in the screening of plasmids of bacteria.