百日咳鲍特菌脉冲场凝胶电泳分子分型方法的优化和应用

Optimization and application of pulsed field gel electrophoresis molecular typing of Bordetella pertussis

目的优化百日咳鲍特菌(Bordetella pertussis,B.P)脉冲场凝胶电泳(Pulsed field gel electrophoresis,PFGE)分型方法。方法依据已发表文献和实验室现有条件,优化B.P的PFGE电泳条件和条带标准化方法,将优化后的方法用于分析贵州省B.P分离株。结果当采用XbaI内切酶时B.P PFGE的最优脉冲时间、电泳时长分别为5-21s、20h,当采用SpeI内切酶时分别为5-45s、22h。同时使用两种内切酶时,λLadder电泳图谱比H9812菌株电泳图谱更适合作为B.P PFGE分析的标准化参考。2017-2018年贵州省16株B.P分离株采用XbaI可分为16种谱型(PX01-PX16),采用SpeI可分为12种谱型(PS01-PS12),XbaI的分辨力(DI=0.9375)高于SpeI(DI=0.9063)。结论优化后的方法更适于B.P PFGE分析;贵州省B.P分离株的PFGE谱型存在多态性。

ObjectiveTo optimize pulsed field gel electrophoresis(PFGE) for typing Bordetella pertussis(B. P).MethodsWe reviewed published literature and existing experimental conditions to optimize electrophoresis conditions and the band standard method of B. P PFGE. We used the optimized method to type B. P isolates in Guizhou.ResultsThe optimal pulse time and electrophoresis duration of B. P PFGE for using XbaI endonuclease were 5-21 seconds and 20 hours, respectively;for SpeI endonuclease,optimal times and durations were 5-45 seconds and 22 hours. When using both endonucleases, the electrophoresis profile of λ Ladder was more suitable for a standardized reference for B. P PFGE analysis than the electrophoresis profile of the H9812 strain. The 16 B. P isolates from 2017 to 2018 in Guizhou were identified as 16 subtypes(PX01-PX16) using XbaI endonuclease and 12 subtypes(PS01-PS12)using SpeI endonuclease;the resolution of XbaI(DI= 0. 937 5) was higher than SpeI(DI= 0. 906 3).ConclusionsThe optimized method was suitable for B. P PFGE;it showed that the PFGE subtypes of B. P isolates in Guizhou were polymorphic.