传代培养对基于肽质量指纹谱的细菌识别稳定性的影响分析  

Effect of passage on the stability of bacterial identification based on peptide mass fingerprinting

作　　者：张慧芳[1] 陶晓霞[1] 何利华[1] 闫笑梅[1] 王忱诚[2] 张建中[1] 肖迪[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所,北京102206 [2]浙江大学医学院附属第一医院传染病诊治国家重点实验室,感染性疾病诊治协同创新中心,浙江杭州310003

出　　处：《疾病监测》2016年第9期734-739,共6页Disease Surveillance

基　　金：传染病预防控制国家重点实验室重点项目(No.2014SKLID102);中国疾病预防控制中心传染病预防控制所科研课题~~

摘　　要：目的应用肽质量指纹谱技术对多次传代细菌进行识别,分析细菌传代对细菌识别能力的影响。方法将幽门螺杆菌、大肠埃希菌、金黄色葡萄球菌分别连续传57、50、100代,金黄色葡萄球菌留取抗原。每一代新鲜培养菌和冻存抗原通过乙醇/甲酸法提取蛋白,采用肽质量指纹谱技术进行谱图采集和菌株鉴定。进一步采用Nano LC-MS/MS对幽门螺杆菌基于肽质量指纹谱识别的蛋白进行批量鉴定。结果幽门螺杆菌、大肠埃希菌分别连续传57代和50代,采用肽质量指纹谱每代均能正确识别到种水平;金黄色葡萄球菌连续传100代,采用肽质量指纹谱每代新鲜菌株及冻存抗原均能正确识别到种水平,各代冻存抗原和新鲜菌株的肽质量指纹谱具有很好的一致性。幽门螺杆菌蛋白扫描分析,鉴定出206个蛋白,包括各种酶类(29.6%)、核糖体蛋白(15.5%)、外膜蛋白(10.7%)、假想蛋白(19.0%)、转运相关蛋白(2.0%)、其他蛋白(22.0%)。结论细菌连续传代和抗原冻存不影响肽质量指纹谱对菌株的正确识别,细菌的肽质量指纹谱具有传代稳定性。Objective To identify the bacteria after multiple passages by the application of peptide mass fingerprinting technology,and analyze the influence of bacterial culture on the ability of bacterial identification. Methods The strains of Helicobacter pylori,Escherichia coli and Staphylococcus aureus w ere continuously cultured for 57 generations,50 generations and 100 generations respectively,and antigen of S. aureus w as collected. Each generation of fresh cultured bacteria and frozen antigen protein w ere extractedby ethanol / formic acid method. The peptide mass fingerprinting technique w as used for the spectrum acquisition and identification of the strains. Nano LC-M S / M S w ere used to reveal the protein components of H. pylori. Results H. pylori and E. coli w ere continuously cultured for 57 generations and 50 generations respectively,every generation w as identified at the species level by the peptide mass fingerprint spectrum. S. aureus w as continuously cultured for100 generations,fresh strain and frozen antigen w ere identified at the species level by the peptide mass fingerprint spectrum.The peptide mass fingerprint spectra of each generation of frozen and fresh strains had good consistency. A total of 206 proteins of H. pylori w ere identified through protein scanning analysis,including 29. 6% of enzymes,15. 5% of ribosomal protein,10. 7% of out membrane protein,19. 0% of putative protein,2. 0% of transport relative protein and 22. 0% of the others.Conclusion Continuous passage of bacteria and frozen antigen have no affects on the correct identification of peptide mass fingerprint spectrum,and the quality of the peptide mass fingerprint is stable.

关 键 词：肽质量指纹谱 细菌鉴定 传代 稳定性 

分 类 号：R446[医药卫生—诊断学]