柠檬酸杆菌属基质辅助激光解析电离飞行时间质谱图谱库的扩展及效果评价

Expansion and effect evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry database of Citrobacter spp.

目的扩展基质辅助激光解析电离飞行时间质谱(matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS)中柠檬酸杆菌属数据库。方法采用PCR方法扩增并测序管家基因leuS进行系统发育分析鉴定菌种;采用平行对照实验优化菌株培养和样品制备条件;按照自建库方法手动采集优化培养后的菌株谱图,构建实验室内部数据库,并进行灵敏度与特异度评价。结果经系统发育分析,有7个种共66株柠檬酸杆菌。最佳培养条件为哥伦比亚血平板做培养基、转种1次、培养24 h,甲酸溶液体积25μL。新增沃克曼氏柠檬酸杆菌和巴氏柠檬酸杆菌2个菌种谱图,补充中国株弗氏、布氏、吉氏、杨氏、无丙二酸柠檬酸杆菌等5个菌种本地区谱图。结论leuS基因可以有效区分柠檬酸杆菌属菌种,MALDI-TOF图谱库的扩展提升了质谱对本地区柠檬酸杆菌属菌种鉴定准确性。

Objective To expand the database of Citrobacter in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS).Methods The housekeeping genes leuSof Citrobacter spp.was amplified by PCR and sequenced for phylogenetic analysis.Through the parallel control experiment,the conditions of strains culture and sample preparation were optimized.According to the method of self-built database,the spectrum of the optimized strains was collected manually,the internal database of the laboratory was constructed,and the sensitivity and specificity were evaluated.Results Phylogenetic analysis showed that there were 66 strains of 7 species Citrobacter.The optimal conditions were as follows:cultured with Colombian blood plate,transplanted once,cultured for 24 h and the amount of formic acid solution was 25μL.The 2 new species spectrum of C.werkmaniiand C.pasteurii were established,and 5 species of local strains spectrum including C.freundii,C.braakii,C.gillenii,C.youngae and C.amalonaticus were supplemented.Conclusions The leuS gene can effectively distinguish Citrobacter spp.and the expansion of the MALDI-TOF database has improved the accuracy of identification of Citrobacter spp.in this region.