基质辅助激光解析电离飞行时间质谱技术对非结核分枝杆菌的鉴定与分型

Identification and classification of non-tuberculosis Mycobacterium by matrix-assisted laser desorption ionization-time of flight mass spectrometry

目的以CLSI－MM18A（病原菌DNA测序鉴定的解释指南）推荐的RPOB（利福平耐药）基因为参考标准，评价基质辅助激光解析电离飞行时间质谱（MALDI－TOF MS）技术对非结核分枝杆菌的鉴定和分型的水平的能力。方法收集2012年至2016年从临床分离的不同感染部位的55株NTM菌株，利用RPOB进行管家基因测序，并对测序结果进行系统发育进化分析；同时利用MALDI－TOF MS技术对菌株进行鉴定，并对蛋白指纹图谱进行聚类分析；评价两种方法对NTM鉴定和分型的一致性。结果RPOB基因对55株NTM显示了很好的鉴定（测序结果TM值均大于99%）和分型（可区分到复合群内的种或亚种）能力；法国BioMerieux MALDI－TOF MS技术对55株NTM的鉴定到属的水平为89.1%，鉴定到种的水平为78.2%，鉴定水平良好；利用SARAMS Premium软件对蛋白指纹图谱的聚类分析也显示出了良好的分型能力。结论MALDI－TOF MS技术可以有效的对非结核分枝杆菌进行鉴定和分型，其检测周期短，操作简便，在实验室中与RPOB基因联合应用有着很好的互补性。

ObjectiveReference standard of the RPOB （rifampin resistance） gene recommended by CLSI-MM18A （Interpretive Criteria for Identification of Bacteria and Fungi by DNA Target Sequencing） was used to evaluate the ability of MALDI-TOFMS techniques for the identification and classification of non-tuberculous Mycobacterium.MethodsFifty five clinicalstrains were collected from 2012 to 2016 with different sources. The RPOB gene was sequenced, and results were applied to phylogenetics analysis. MALDI-TOF MS technology was implemented to identify the strains, and cluster analysis was conducted based on protein fingerprint. The consistency of two methods for NTM identification and typing was evaluated.ResultsThe RPOB gene method showed a good ability of identification （similarity〉99.0%） and subtyping （to subspeciesof the complex level）. The French BioMérieux MALDI-TOF MS identified 89.1% of 55 strains to genus level and 78.2% to species level. The phylogeneticsanalysis of protein fingerprint by SARAMS Premium software also showed good typing ability.ConclusionsMALDI-TOF MS technology can identify and classify non-tuberculous Mycobacterium effectively, which is rapid and easy. It is complementary to RPOB gene method in laboratory application.