空肠弯曲菌脉冲场凝胶电泳分子检测方法的建立及应用

Pulsed field gel electrophoresis for determining the molecular homology of Campylobacter jejuni isolates

【目的】建立空肠弯曲菌脉冲场电泳(pulsed-field gel electrophoresis,PFGE)图谱分型方法。【方法】在PFGE基本程序基础上,通过调整菌液浓度、Seakem Gold琼脂糖凝胶浓度、蛋白酶K浓度、洗涤方式和限制性内切酶SmaⅠ浓度,进行程序的比较与优化。应用PFGE技术对不同来源分离株进行分析。【结果】37株空肠弯曲菌脉冲场凝胶电泳图谱显示分离株均产生了6～24条电泳带,条带数量适中,清晰易读;系统进化树显示,可分为4个遗传谱系,分离株主要分布于PFGE遗传谱系Ⅳ,不同源分离株在各组群中呈交叉分布。【结论】PFGE对空肠弯曲菌具有很强的分型能力和良好的溯源性,初步数据显示鸡、牛等动物源空肠弯曲菌与腹泻病人空肠弯曲菌病密切相关。

[ Objective] To establish a genetic typing map for Campylobacter jejuni in China by using Pulsed Field Gel Electrophoresis （PFGE）. [Methods] A PFGE protocol was developed for subtyping C. jejuni isolates from different sources. The effect of cell suspension concentration, Seakem Gold agarose gel strength, proteinase K density, washing style, and restriction enzyme concentration were also evaluated. [ Results] DNA from 37 isolated strains of C. jejuni digested with restriction enzyme Sma Ⅰ produced PFGE profiles that distinguished these strains to the level of species, and resulted in 6 to 24 bands of electrophoresis. A phyletic evolution tree showed that the isolates could be separated into four genetic lineages, and possessed dominance among lineage Ⅳ. The distribution of isolated strains from different sources overlapped each other in all groups. [ Conclusion] PFGE can be used for the investigation of molecular epidemiological patterns of C. jejuni and for tracing the source of C. jejuni in clinical patients. Preliminary data showed that human Campylobacteriosis was related to C. jejuni of animal origin, particularly from the chicken.