抗坏血酸克吕沃菌脉冲场凝胶电泳分型方法的建立

目的建立适于抗坏血酸克吕沃菌的PFGE分型方法。方法参考PulseNet中大肠杆菌O157∶H7的PFGE分型方法,选择不同的限制性内切酶和不同的电泳参数,进行反复试验,来确定用于抗坏血酸克吕沃菌PFGE分型的具有高分辩能力的限制性核酸内切酶和电泳参数。结果5株抗坏血酸克吕沃菌经限制性内切酶XbaI、SpeI、BlnI酶切后,再经我们选定的参数电泳,可呈现理想的PFGE带型,具有良好的分型能力。而NotI、SmaI酶切后产生的片段绝大部分集中在200kb以下,且图谱上条带过于密集而难以明确区分。结论利用限制性内切酶XbaI、BlnI和SpeI的PFGE分型方法对抗坏血酸克吕沃菌都具有较好的分辨能力,都可以用于该菌的分子分型,但应优选限制性内切酶BlnI、XbaI。

Establishment of the pulsed-field gel electrophoresis protocols for Kluyvera ascorbata

To optimize endonucleases and electrophoretie parameters of PFGE for Kluyvera ascorbata, we analyzed 5 isolates of Kluyvera ascorbata by PFGE. and adopted 5 endonucleases including Xba I, Spe I, Bln I, Not I and Srna I as well as used different electrophoretic parameters. The PFGE patterns were analyzed with BioNumerics software. All of the 5 strains showed different patterns with endonucleases Xba I, Spe I and Bln I, respectively. But the fragments produced by endonucleases Not I and Sma I were so small and thick that could not be easily analyzed. The PFGE patterns with endonueleases Xba I, Spe I and Bln I hed higher discriminatory power for the molecular subtyping of Kluyvera ascorbata. All of the three methods using the endonucleases mentioned above, preferably, endonucleases Xba I or Bln I, can be used for the molecular subtyping of K lu yvera ascorbata.