摘要
目的建立并评价分枝杆菌液体培养基质辅助激光解吸电离飞行时间质谱(matrix assisted laser desorption ionization time of flight mass spectrometry,MALDI-TOF MS)直接鉴定技术。方法收集2014年6月至2016年5月首都医科大学附属北京同仁医院1 466例呼吸科住院患者痰标本,进行BactecTMMGITTM960分枝杆菌快速液体培养(简称液体培养),以其中报警并经萋-尼抗酸染色确认为抗酸杆菌的105例(无重复病例)为研究对象,对比本实验室自行设计并改良的"同仁"分枝杆菌液体培养直接鉴定法(培养液经特殊前处理后直接进行MALDI-TOF MS鉴定)及传统鉴定法(培养液转种于L-J固体培养基,得到菌落后再进行MALDI-TOF MS鉴定),并通过16S rRNA基因及16S^23S rRNA基因间隔区测序验证。结果以基因测序鉴定为金标准,液体培养直接鉴定与传统鉴定法结果一致率为100%;二者鉴定至属的准确率均为100%(105/105),鉴定至种的准确率均为99.05%(104/105)。液体培养后直接鉴定与传统鉴定法MALDI-TOF MS鉴定分值≥2.000分的比例差异无统计学意义(χ~2=2.952,P=0.086)。4例分值<1.700分的菌株谱图杂峰较多而有效峰较少。结论本实验室自行设计的"同仁"分枝杆菌液体培养MALDI-TOF MS直接鉴定法鉴定结果准确,缩短了分枝杆菌培养和鉴定的时间,且可保证生物安全,对分枝杆菌感染的快速诊断和及时治疗意义重大,适用于我国各级临床实验室。
Objective To establish and evaluate the direct matrix-assisted laser desorption ionization-time of flight mass spectrometry（ MALDI-TOF MS） for the identification of mycobacteria from positive liquid media. Methods Sputum samples from 1 466 patients hospitalized in Beijing Tongren Hospital affiliated to Capital Medical University during June 2014 and May 2016 were collected,and cultured by the BactecTMMGITTM960. A total of 105 patients with mycobacteria-positive liquid media were further confirmed with Ziehl-Neelsen acid-fast staining and selected in our study. They were identified by the improved MALDI-TOF MS method（ ＂ Tongren＂） in which the culture fluid was performed pretreatment and then identified by MALDI-TOF MS directly and the classic method in which the culture fluid was transferred to the L-J solid media and the obtained colonies were identified by MALDI-TOF MS,respectively. The identification results were further verifed by 16 S rRNA and 16 S ~ 23 S rRNA intergenic region sequencing. Results When the gene sequencing identification was used as the gold standard,the accordance rate between the direct and classic MALDI-TOF MS methods was 100%.The accuracy rates of the two methods at the genus level were 100%（ 105 /105）,and 99. 05%（ 104 /105） at the species level. There was no significant diffenrence in the proportion of identification score above 2. 000 between the two methods（ χ2= 2. 952,P = 0. 086）. The spectrograms of 4 isolates with identification score less than 1. 700 were filled of impurities and lack of valid peaks. Conclusion The improved direct MALDI-TOF MS method（ ＂ Tongren＂） established by our lab for the identification of mycobacteria from liquid media may obtain accurate results,shorten the culture and identification time of mycobacteria,and ensure biological safety,which may be helpful for the rapid diagnosis and treatment of mycobacteria infections,and be applied to different levels of clinical laboratories.
出处
《临床检验杂志》
CAS
CSCD
2016年第12期904-908,共5页
Chinese Journal of Clinical Laboratory Science
基金
基金项目:综合医院急性严重呼吸道传染病病原学诊断研究(2014ZX10004005)
关键词
结核分枝杆菌
非结核分枝杆菌
液体培养后直接鉴定
基质辅助激光解析/电离飞行时间质谱
Mycobacterium tuberculosis
nontuberculous mycobacterium
direct identification from liquid media
matrix-assisted laser desorption ionization-time of flight mass spectrometry
作者简介
杨本善，1964年生，男，主管技师，大学本科，主要从事生化免疫及分子生物学研究。
通信作者：鲁辛辛，主任医师，教授，博士研究生导师，E—mail：luxinxin2009@126．com。
