基质辅助激光解吸电离飞行时间质谱微生物鉴定系统性能验证方案的建立

目的建立基质辅助激光解吸电离飞行时间质谱系统(MALDI-TOF MS)在常规临床微生物鉴定中的性能验证方法,指导临床实验室规范微生物鉴定程序。方法选取标准菌株、质控菌株和临床菌株共115株,包含革兰阳/阴性球菌30株、革兰阳/阴性杆菌31株、真菌30株,厌氧菌、苛养菌各12株,所有菌株均经Vitek Compact鉴定和/或细菌16S r DNA、真菌ITS DNA测序分析验证。任意选择3种MALDI-TOF MS微生物鉴定系统厦门质谱、布鲁克质谱、安图质谱,采用检测系统推荐方法进行菌株鉴定,进行准确度验证试验。精密度验证:选取标准菌株和临床菌株10株,1位操作者使用3个检测系统对10株菌株分别进行质谱鉴定3次,连续鉴定3 d; 3位操作者使用3个检测系统对10株菌株每d分别进行质谱鉴定3次,连续鉴定3 d,从而验证鉴定结果的重复性。结果厦门质谱、布鲁克质谱、安图质谱对标准/质控菌株(除外厌氧菌)的鉴定符合率为100%;对临床菌株的属水平鉴定符合率为100%;对革兰阴/阳性杆菌的种水平鉴定符合率分别为100%、100%、96.77%;对革兰阳性球菌的种水平鉴定符合率分别为96.67%、96.67%、100%;对真菌的种水平鉴定符合率均为90%一致;对苛养菌的种水平鉴定符合率均为100%;对厌氧菌鉴定符合率为91.67%种水平一致。精密度验证试验结果重复性100%。结论 3种MALDI-TOF MS系统在革兰阳/阴性球菌、革兰阳/阴性杆菌、真菌、苛养菌鉴定的准确度和精密度符合要求,验证通过。本文建立的微生物鉴定质谱仪性能验证方案可满足综合性医院临床微生物实验室常规鉴定基本要求。

Establishment of a performance verification protocol for the microbial identification system based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Abstract: Objective: To establish the performance verification method of the routine clinical microbial identification system based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and guide clinical laboratories to standardize microbial identification procedures. Methods: One hundred and fifteen strains of standard strains, quality control strains and clinical isolates, including 31 strains of Gram positive/negative cocci, 30 strains of Gram positive/negative bacilli, 30 strains of yeast, 12 strains of anaerobic bacteria and 12 strains of fastidious bacteria, were collected. All strains were verified by the Vitek Compact system, and bacterial 16S rDNA or fungal ITS DNA sequencing. Three MALDI-TOF MS microbial identification systems, including Xiamen mass spectrometry (MS), Bruker MS and Antu MS, were selected randomly. The strains were identified by the recommended method of three detection systems to evaluate their accuracy. Ten strains of standard strains and clinical isolates were identified by one operator using three detection systems for 3 times each day and 3 days, and by three operators using three detection systems for 3 times each day and 3 days, respectively, to evaluate their precision. Results: The accuracy of three detection systems were all 100% for standard strains and quality control strains except anaerobic bacteria, and clinical genus-level isolates. The coincidence rates of Xiamen MS, Bruker MS and Antu MS systems were 100%, 100% and 96.77%, respectively, for species-level Gram positive/negative bacilli, and 96.67%, 96.67% and 100%, respectively, for species-level Gram positive cocci. The coincidence rates of three detection systems were 90% for species-level yeast, 100% for species-level fastidious bacteria, and 91.67% for species-level anaerobic bacteria, respectively. The precision were all 100% for three detection systems. Conclusion: The accuracy and precision of three MALDI-TOF MS systems for the identification of Gram positive/negative cocci, Gram positive/negative bacilli, yeast and fastidious bacteria meet the requirements and pass the verification. The identification of anaerobic bacteria is acceptable at the genus level. The performance verification protocol established in the study can meet the basic requirements of routine microbial identification in clinical microbiology laboratories of general hospitals.