布鲁菌脉冲场凝胶电泳图谱分型方法建立

Idetification of Brucella by pulsed-field gel electrophoresis

目的建立我国95株布鲁菌的染色体DNA脉冲场凝胶电泳图谱，进行布鲁菌的分子遗传学分类鉴定。方法选择我国不同地区、不同时限分离的布鲁菌100株（包括19株标准布鲁菌株），应用SeaKem Gold琼脂糖胶块纯化布鲁菌完整的染色体DNA，限制性内切酶XbaI消化后，进行脉冲场凝胶电泳分离（PFGE）。结果XbaI酶切布鲁菌基因组DNA后，可产生20～500kbp范围的15～25条酶切片段．并可以在PFGE凝胶上被很好的区分开。PFGE可在种的水平上区分布菌各种标准株，76株中国分离株可被分为39种PFGE类型，并聚为4大类。PFGE与传统分型方法比较，2者的一致性达63％。结论脉冲场凝胶电泳图谱可对布鲁菌进行遗传学分类鉴定，在布鲁菌的分子流行病学研究中具有重要意义。

Objective To establish a genetic typing maps for 95 stains of Brucella in China by pulsed - field gel elec- trophoresis（PFGE）. Methods SeaKem Gold agarose plug was used to purify the intact chromosome DNA from 95 isolates of Brucella selected from different areas and years, including 19 reference stains . Then the chromosome DNAs were digested with restriction endonuclease XbaI and the fragments were separated by PFGE. Results Genetic DNA of 19 reference type strains digested with low - cleavage - frequency restriction enzymes XbaI produced PFGE profiles that distinguished these strains to the level of species. For field strains isolated in China, chromosomal DNAs in agarose plugs were digested with XbaI, resulting in 15 to 25 bands. Thirty- nine distinctive PFGE patterns in the range of 20 to 500 kb were noted among 76 isolates by PFGE. Conclusion PFGE patterns, which can be used to classify and identify Brucella, can be used in molecular epidemiological research of Brucella.