摘要
目的建立基质辅助激光解析／电离飞行时间质谱（MALDI—TOFMS）鉴定非结核分枝杆菌（NTM）的方法，评估其快速鉴定临床和环境分离NTM的可行性、准确性和重复性。方法回顾性收集2012年1至3月上海复旦大学附属中山医院微生物实验室临床分离NTM15株及2011至2012年医院供水系统环境分离NTM68株。比较3种样本前处理和蛋白抽提方案，采用MALDI—TOFMS技术检测临床和环境分离NTM，并以16SrRNA测序结果为对照。结果3种方法均鉴定出相同细菌，但方法1的鉴定值高于其他2种方法。对生长于Middlebrook7H10琼脂平板的83株共10个种的NTM进行鉴定，与16SrRNA测序结果比较，所有菌株均能鉴定至属水平，67株（80．7％）鉴定至种，6株鉴定到复合群水平，鉴定值大于1．700的NTM占81．9％（68／83），且重复性好。培养后获得的菌落从前处理至鉴定完成仅需1．5h。结论MALDI．TOFMS可快速准确地鉴定临床和环境分离的NTM，在临床实验室常规鉴定方面有着较好的应用前景。
Objective The purpose of this study was to compare and develop the method for identification of Non-tuberculous Mycobacteria （NTM） using matrix assisted laser desorption/ionization time of flight mass spectrometry （MALDI-TOF MS）, and evaluate the feasibility, accuracy and repeatability of MALDI-TOF MS to discriminate NTM. Methods Fifteen clinical strains were collected from January to March in 2012 and 68 environmental strains were retrospectively collected from 2011 to 2012. A protocol for sample pre-treatment and protein extraction was developed and utilized it to identify clinical and environmental isolates. The results from 16 s rRNA sequencing were served as control. Results Method A was more effective in protein extraction. Although all the three methods got the same species result, a total of 83 strains belonging to 10 distinct species grown in Middle brook 7H10 media were analyzed. All members of the NTM were identified accurately at the genus level and 80. 7% （67/83） of strains could be identified at the species level. Six strains were identified at the complex level. 81.9% （68/83） of NTM got high spectral scores. The identification of cultured colony could be completed in 1.5 hours. And it had good reproducibility. Conclusion MALDI-TOF MS can be used as a rapid and accurate method for identification of Mycobacteria in clinical microbiology laboratories, implying its good application prospects.
出处
《中华检验医学杂志》
CAS
CSCD
北大核心
2013年第7期610-614,共5页
Chinese Journal of Laboratory Medicine
