基质辅助激光解析飞行时间质谱在临床病原菌检测中的应用进展

近年来质谱仪技术有了快速的发展.20世纪80年代,基质辅助激光解析飞行时间质谱( matrix assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF-MS)的诞生,奠定了基于蛋白质指纹图谱鉴定微生物的硬件基础.每种微生物都有自身独特的蛋白质组成,质谱仪具有测定荷电离子质量的能力,可测得待检微生物的蛋白质质量指纹图谱,经软件对这些指纹图谱进行处理并和数据库中各种已知微生物的标准指纹图谱进行比对,即可以完成对微生物的鉴定.由于测定的图谱中主要的分子离子峰为菌体内高丰度,且表达稳定、进化保守的核糖体蛋白,因此这一方法不仅鉴定结果可靠,而且还可以通过聚类分析获得微生物间的进化和亲缘关系.     

基质辅助激光解吸电离飞行时间质谱技术在快速检测病原菌中的研究进展

摘要：传统的临床微生物学检验依赖于长时间的对病原菌的分离、培养及复杂的生化鉴定,有些病原微生物的生化反应不典型则难以准确鉴定。分子生物学技术如PCR技术要求较高,常规实验室不易开展。基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）作为一种跨学科的检测新技术,在细菌和真菌的快速鉴定、细菌的耐药性检测、流行病学分析方面呈现出较传统鉴定法较多的优势。     

基质辅助激光解吸电离飞行时间质谱在临床病原菌鉴定中的应用

目的探讨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)系统用于快速鉴定临床分离菌的可靠性和实用性。方法收集南京军区南京总医院2013年7~10月自临床标本分离的非重复细菌1 061株,分别使用MALDI-TOF MS和Vitek 2 Compact全自动细菌鉴定仪系统进行鉴定,结果不一致的菌株采用16S r DNA测序验证。结果 1 061株临床分离菌中1 058株(99.7%)经MALDI-TOF MS系统正确鉴定到属水平,1 016株(95.8%)正确鉴定到种,其余3株(0.3%)未给出鉴定结果。5株鉴定结果不一致的菌株经16S r DNA测序确认,结果与MALDI-TOF MS和Vitek 2 Compact鉴定符合率分别为40.0%(2/5)和0(0/5)。结论 MALDI-TOF MS可以作为一个快速、准确和价廉的工具应用于临床分离菌的鉴定。     

基质辅助激光解吸电离飞行时间质谱鉴定布鲁菌1例

正布鲁菌病(布病,brucellosis)又称波浪热或马耳他热等,是由布鲁菌引起的人畜共患的烈性传染病,主要以牛、羊、猪为传染源。布鲁菌可通过皮肤、呼吸道、消化道进入人体引起感染,以长期发热、多汗、关节痛及全身乏力、疼痛为主要特征。该病以往多见于牧区,近年来散发于大中城市,以青壮年为主。我院近期从1例来自山西的反复发热的患儿血培     

基质辅助激光解析电离飞行时间质谱在大肠埃希菌分群中的应用

摘要 目的 评价基质辅助激光解析/电离飞行时间质谱(MALDI-TOF MS)对大肠埃希菌菌群的鉴别能力。方法 通过VITEC 2 Compact全自动细菌鉴定仪及药敏分析系统对人和动物源性的大肠埃希菌敏感菌株进行初步筛选,然后将获得的21株人源和42株动物源性大肠埃希菌进行16S rRNA基因测序及同源性分析,同时通过MALDI-TOF MS技术对大肠埃希菌指纹图谱进行鉴定。结果 与16S rRNA基因测序相比,不同来源的菌株蛋白质特征图谱呈现明显的分群趋势,MALDI-TOF MS法对不同来源的菌株具有较强的鉴别区分能力。结论 MALDI-TOF MS法可用于菌株来源差异性的鉴别。     

基质辅助激光解吸电离飞行质谱用于血培养中病原菌的快速鉴定研究进展

临床微生物检验与其他检验项目相比,最大的缺点是报告时间长,远不能满足临床的需要。分子生物学方法如PCR法虽可提高检测灵敏度,缩短检测报告时间,但假阳性率高,对实验室环境的要求很高,在操作的过程中还需使用一些毒性化学     

基于基质辅助激光解吸电离飞行时间质谱的病原菌耐药性分析研究进展

基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）微生物识别分析系统现已发展为成熟的技术体系，其凭借简单、快速、经济、高通量以及高敏感性、高特异性的特点，在病原菌耐药性分析研究领域展现了巨大的潜力。 近年来，基于MALDI-TOF MS的病原菌耐药性研究也取得了长足进展。 本文将从定性和定量分析的角度阐述MALDI-TOF MS在病原菌耐药性分析领域的研究进展，并对其应用前景进行展望。     

表面增强激光解析电离飞行时间质谱快速鉴定血液大肠埃希菌感染

目的探讨用表面增强激光解析电离飞行时间质谱(SELDI-TOF-MS)快速鉴定引起血流感染的大肠埃希菌。方法用SELDI-TOF-MS建立大肠埃希菌的蛋白质指纹模型。临床分离的168株菌经SELDI-TOF-MS检测,数据与大肠埃希菌模型进行相似度比较,对该模型进行评价。SELDI-TOF-MS检测88株微生物血培养分析仪阳性报警的革兰阴性杆菌,与大肠埃希菌蛋白质指纹模型进行相似度比较,对其中的大肠埃希菌进行鉴定。结果 19株建模组菌株经PCR及测序鉴定均为大肠埃希菌,并建立了该菌的蛋白质指纹模型;用168株验证组菌株对该模型进行验证,对大肠埃希菌鉴定的符合率为100%(60/60);用该模型对88株引起血流感染的革兰阴性杆菌进行鉴定,鉴定结果与传统微生物学鉴定结果具有较高的一致性(k=0.905)。结论 SELDI-TOF-MS可快速鉴定引起血流感染的大肠埃希菌,为血流感染细菌的快速诊断提供了新技术。     

基质辅助激光解吸电离飞行时间质谱技术在临床微生物鉴定中的应用及价值

摘要：20世纪90年代末,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）技术开始用于病原菌鉴定,目前已逐步用于临床微生物检验工作,对临床感染性疾病的快速诊断意义重大。本文阐述MALDI-TOF-MS技术鉴定微生物的原理和流程、在临床微生物鉴定中的应用和对微生物检验结果回报时间（TAT）改进的作用。     

Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

BACKGROUND: Hemoglobin (Hb) heterogeneity arises mainly from posttranslational modifications of the globin chains, and cation-exchange chromatography reveals falsely increased concentrations of some minor Hbs in the presence of abnormal Hbs. Here we describe a method for identification of the globin chains and their posttranslational modifications contained in the Hb fractions. METHODS: We used cation-exchange HPLC (PolyCAT A column) for separation of Hb fractions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for analysis of the separated globin chains. Globin chains were identified by their molecular masses. Posttranslational modifications of globin chains were identified by digestion of the proteins with endoproteinase V8 before MALDI-TOF MS of the resulting peptides. RESULTS: Analysis of the HbA2 fractions of patients with HbS revealed 4 different globin chains. We found, in addition to the expected alpha- and delta-chains, the carbamylated alpha- and the betaS-chains. Additionally, we analyzed HbH, Hb Barts, HbA 1b, pre-HbA 1c, HbA 1c, HbF1, HbF, HbA 1d3a, HbA 1d3b, HbA2, and HbC1 fractions from control and pathologic blood samples. We identified several posttranslational modifications of the globin chains, such as pyruvatization, glycation, acetylation, carbamylation, and acetaldehyde adduct formation. CONCLUSIONS: The native and posttranslationally modified globin chains in minor and major Hbs are unambiguously identified by MALDI-TOF MS. A minor Hb containing the carbamylated alpha- and the betaS-chain elutes at the same time as normal HbA2 (alpha2delta2) and thus leads to falsely increased HbA2 values in patients with HbS when blood is analyzed with PolyCAT A chromatography.     

Application of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in Alzheimer's disease

Alzheimer's disease is becoming an increasing problem in our aging society. According to our knowledge, so far, no effective pharmacotherapy to cure the cause of the disease has been developed. Therefore, early diagnosis is needed, which will result in implementation of a drug therapy aimed at decreasing and/or inhibiting disease development. Mass spectrometry techniques (MS) have a wide range of applications in proteomics and the search for biomarkers of neurodegenerative disorders, opening new possibilities in diagnostics. Identification of proteins in body fluids (like cerebrospinal fluid or blood) is possible due to MS spectra analysis. The detected changes in protein concentrations are connected with pathological states in an organism and, therefore, can be regarded as biomarkers. Developing procedures for proteome analysis might result in fast diagnosis, as well as creating better suited pharmaceuticals. This paper reviews the search of biomarkers in cerebrospinal fluid and blood. Later on, the use of matrix-assisted-laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in proteomics, focusing on blood-related biomarkers, is discussed. The aim of the work is also to highlight the advantages and disadvantages of MALDI-TOF-based analyses.     

对《MALDI-TOF-MS在临床常见菌快速鉴定中的应用研究》一文的商榷

编辑同志:拜读了贵刊2010年33卷第6期蒋颜等[1]的研究报道,令人兴奋.应用MALDI-TOF-MS技术鉴定细菌,近年来国外才有开展,作者能在国内迅速开展并取得研究成果,笔者深感敬佩.但也许由于文章字数的限制,使笔者对文中的一些内容不太理解,希作者指教.     

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。     

基于基质辅助激光解吸电离飞行时间质谱的克柔念珠菌遗传分化研究

目的在蛋白质水平明确克柔念珠菌的遗传分化特征，建立基于基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）的不同谱系克柔念珠菌的鉴定方法。方法选取20株克柔念珠菌，采用MALDI-TOF MS结合分析软件ClinProTools，确定克柔念珠菌不同谱系的质谱标志峰，构建基于遗传算法（GA）的分类模型，用于区分不同谱系的克柔念珠菌菌株，选择25株克柔念珠菌对该模型进行验证。结果利用20株克柔念珠菌构建的GA分类模型，反映模型正确鉴别能力的识别能力值（RC）为100%，反映模型处理测试谱图变异能力的交叉验证值（CV）为97.89%。 25株克柔念珠菌、150张谱图外部验证模型显示其分类能力为95.30%。 确定13个克柔念珠菌谱系相关的特征峰（m/z 3 971.40、3 136.95、3 427.33、2 405.28、2 996.73、2 913.95、3 376.97、6 736.13、5 819.03、4 045.16、5 869.00、3 618.10及3 946.14），可用于正确区分来自不同谱系的克柔念珠菌。结论本研究论证并确认了克柔念珠菌的群体遗传分化事件，同时也建立了一种简便而有效的快速谱系分型方法。     

New serum biomarkers for detection of endometriosis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This study used proteomic fingerprint technology, combining nano-sized magnetic beads with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), to screen for potential protein biomarkers for the diagnosis of endometriosis. Serum proteins from 126 patients with endometriosis and 120 healthy controls were profiled and compared. Biomarker pattern software identified 46 discriminating mass-to-charge m/z ratio peaks that were related to endometriosis. The model constructed by the software, based on three of these peaks (m/z 5988.7, 7185.3 and 8929.8), generated excellent separation between the endometriosis and control groups. The sensitivity was 91.4% and the specificity 95.0%. Blind testing on a second series of serum samples from patients with endometriosis and healthy controls indicated a sensitivity of 89.3% and a specificity of 90.0%. Biomarkers for endometriosis can be discovered in serum by MALDI-TOF-MS in combination with nano-sized magnetic beads. The pattern of combined markers provides a powerful and reliable diagnostic method for endometriosis, with high sensitivity and specificity.     

Comparison of the ASTA MicroIDSys and VITEK MS matrix-assisted laser desorption/ionization time-of-flight mass spectrometry systems for identification of clinical bacteria and yeasts

The ASTA MicroIDSys system (ASTA, Suwon, Korea) is a newly developed Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) system for identification of microorganisms. We compared the performance of the ASTA MicroIDSys system with that of the VITEK MS system (bioMérieux, Marcy l’Etoile, France) for identifying clinical microorganisms. A total 2055 isolates including 1910 bacteria and 145 yeasts were tested. Among them, the VITEK MS correctly identified 1999 (97.3%) isolates to species level and 26 (1.3%) to the genus level. The ASTA MicroIDSys correctly identified 1988 (96.7%) isolates to species level and 28 (1.4%) to the genus level. The VITEK MS and ASTA MicroIDSys misidentified one isolate and four (0.2%) isolates, respectively, and provided no identification for 29 (1.4%) and 35 (1.7%) isolates, respectively. The performance of the ASTA MicroIDSys was comparable to that of the VITEK MS for identification of clinically relevant bacterial and yeast isolates.     

Improvement of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of difficult-to-identify bacteria and its impact in the workflow of a clinical microbiology laboratory

This study evaluates matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) capability for the identification of difficult-to-identify microorganisms. A total of 150 bacterial isolates inconclusively identified with conventional phenotypic tests were further assessed by 16S rRNA sequencing and by MALDI-TOF MS following 2 methods: a) a simplified formic acid-based, on-plate extraction and b) performing a tube-based extraction step. Using the simplified method, 29 isolates could not be identified. For the remaining 121 isolates (80.7%), we obtained a reliable identification by MALDI-TOF: in 103 isolates, the identification by 16S rRNA sequencing and MALDI TOF coincided at the species level (68.7% from the total 150 analyzed isolates and 85.1% from the samples with MALDI-TOF result), and in 18 isolates, the identification by both methods coincided at the genus level (12% from the total and 14.9% from the samples with MALDI-TOF results). No discordant results were observed. The performance of the tube-based extraction step allowed the identification at the species level of 6 of the 29 unidentified isolates by the simplified method. In summary, MALDI-TOF can be used for the rapid identification of many bacterial isolates inconclusively identified by conventional methods.     

MALDI-TOF-MS技术快速鉴定临床酵母样真菌的应用评价

目的评价基质辅助激光解析/电离飞行时间质谱(MALDI-TOF-MS)在快速鉴定临床分离的酵母样真菌中的应用价值。方法收集临床分离的酵母样真菌135株,分别用VITEK MS、API20C和CHROMargar显色培养同时进行鉴定与比较,χ2检验分析VITEK MS和API及显色培养基鉴定结果的准确率,以真菌内转录间隔区(ITS)基因测序作为鉴定结果的金标准,同时比较各种方法在临床使用中的优缺点。结果 VITEK MS、API20C和显色培养基鉴定的准确率分别为98.5%(133/135)、78.5%(106/135)、59.3%(80/135),经χ2检验分析,差异有统计学意义(χ2=51.996,P0.05)。结论 MALDI-TOF-MS可将酵母样真菌快速、准确地鉴定到种。