利用全基因组序列优化气单胞菌脉冲场凝胶电泳分型方法  

Using whole genome sequences to optimize pulsed field gel electrophoresis typing method for Aeromonas

作　　者：陈洪友[1] 陈敏[1] 屠丽红[1] 陈涌 张曦[1] 

机构地区：[1]上海市疾病预防控制中心,上海200336

出　　处：《中国食品卫生杂志》2017年第6期641-646,共6页Chinese Journal of Food Hygiene

基　　金：上海市第四轮公共卫生三年行动计划高端海外研修团队培养计划(GWTD2015S01);"十三五"国家传染病防治科技重大专项(2017ZX10103009-003)

摘　　要：目的对气单胞菌脉冲场凝胶电泳(PFGE)分型方法从限制性内切酶的选择和电泳条件设置上进行优化,减少片段共迁移,使片段在电泳中分布均匀且利于分析。方法利用6个种的气单胞菌的全基因组数据,通过BioEdit软件对基因组序列进行677种酶的限制性片段分析,选择产生片段数量适宜的、有效片段比例高的酶作为备选酶。对片段长度取以10为底的对数作为自变量,以片段在泳道中的相对迁移距离作为应变量,计算片段长度-相对位置拟合公式,并模拟备选酶的电泳效果图。利用气单胞菌分离株对备选酶进行试验验证,确定PFGE分型方法,并用其对腹泻分离株进行分型应用。结果经Bio Edit分析,根据片段数量及有效片段比例,PmeⅠ、PacⅠ、SwaⅠ比XbaⅠ更适合于气单胞菌的分型。从PFGE模拟效果及分离株试验验证看,Pac Ⅰ、PmeⅠ的限制性片段的分布比XbaⅠ和SwaⅠ更均匀,片段重叠更少。最终确定PmeⅠ、PacⅠ分别为气单胞菌PFGE分型的首选酶和次选酶,脉冲场电流转换时间为2.16~63.8 s。16株腹泻分离株PFGE分型辛普森多样性指数为99.17%。结论优化的PFGE方法使用PmeⅠ、PacⅠ作为气单胞菌PFGE分型的限制性内切酶,该方法产生的片段数量与分布都优于XbaⅠ,在腹泻分离株的分型中能获得良好的分辨率。此研究提供的优化程序可用于其他细菌PFGE方法的建立或优化。Objective The pulsed field gel electrophoresis（ PFGE） typing method of Aeromonas was optimized from aspects of restriction enzymes and electrophoresis parameters,which reduced fragments co-migration and made them welldistributed for easily analyzing. Methods Using whole genome sequences of 6 Aeromonas,restriction fragments produced by 677 enzymes were analyzed in Bio Edit software. Enzymes producing more effective fragments in appropriate number were selected,and the fitting curve and regression equation were derived from the length of restriction fragments represented as denary logarithm and the relative position of fragments in gel represented as fragments migration distance percentage of the valid length of the reference system. Digital simulation of pulsed field gel electrophoresis map was shown when length of fragments from different Aeromonas substituted into the equation. Results of digital simulation were also verified by PFGE typing assay with selected enzymes for Aeromonas typing. Isolates from diarrhea patients were subtyped by the optimized new method. Results Bio Edit analysis result showed Pme Ⅰ,Pac Ⅰ and Swa Ⅰ were better than Xba Ⅰ as enzymes for PFGE from the aspects of total and effective number of restriction fragments. According to the result of PFGE simulation and the verification test of isolates,the distribution of fragments of Pac Ⅰ and Pme Ⅰ was better distributed and less overlapped than those of Xba Ⅰ and Swa Ⅰ. Finally,Pme Ⅰ and Pac Ⅰ were selected as the first and second choice of enzymes for PFGE typing,and the pulsed field conversion time was set as 2. 16-63. 8 s. Sixteen isolates from diarrhea patients were subtyped by new method,and the Simpson diversity index was 99. 17 % according to the typing result.Conclusion As enzymes used in PFGE typing,PmeⅠ and PacⅠ were better choices than XbaⅠ,and good resolution was obtained from subtyping of isolates from diarrhea. The procedure for optimizing PFGE typing method provided in this studyis applicable to the

关 键 词：气单胞菌 脉冲场凝胶电泳 限制性内切酶 全基因组序列 食源性致病菌 分型 

分 类 号：R155.5[医药卫生—营养与食品卫生学]