基质辅助激光解析电离飞行时间质谱在医学真菌领域的应用进展

基质辅助激光解析电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS）是近年来新兴的微生物检测技术,通过核糖体蛋白分析实现对真菌快速、准确鉴定。本文针对MALDI-TOF MS用于致病真菌鉴定、分类、体外抗真菌药物敏感性检测以及临床微生物样本直接检测等方面作一综述。     

MALDI-TOF MS技术在临床病原真菌鉴定中的应用进展

基质辅助激光解吸电离飞行时间质谱技术（MALDI-TOF-MS）目前是一种快速而可靠的微生物鉴定方法.随着可鉴定真菌谱的完善,MALDI-TOF MS技术已逐步应用于临床常见致病酵母菌、酵母样真菌和丝状菌的鉴定中,本文将就此做一综述.     

应用基质辅助激光电离飞行时间质谱快速鉴定临床酵母菌

目的研究基质辅助激光解析电离飞行时间质谱(Matrix-Assisted Laser Desorption Ionization-Time of Flight MassSpectrometry,MALDI-TOF-MS)用于快速检测鉴定临床分离的酵母菌的可行性。方法应用Bruker MALDI-TOF-MS和VITEK 2-compact系统分别鉴定150株临床分离的酵母菌,结果不一致的菌株通过基因序列测定来鉴定。结果 MALDI-TOF-MS快速准确鉴定出了150株临床酵母菌,鉴定符合率在属水平上为100%,种水平上为94%。结论基于MALDI-TOF-MS鉴定方法具有很好的可重复性和准确性,并且其检测成本较低,实验准备时间很短,MALDI-TOF-MS可以用于临床分离的酵母菌的快速鉴定。     

基质辅助激光解析电离飞行时间质谱与细菌的快速鉴定

用传统的方法鉴别细菌往往需要较长的时间(≥48h)和复杂的程序,不利于细菌的快速鉴定。基质辅助激光解析电离飞行时间质谱是最近采用的用于细菌检测的生物质谱技术,可以对完整的细菌进行检测。这种技术以激光作为能量来源,将待测细菌的表面成分解析为离子,产生重现性很好的质谱图。将未知细菌质谱图与细菌质谱图库进行比较,可以达到对细菌进行鉴剐的目的。此种质谱技术的应用,对微生物的快速鉴别有重要意义。     

无花果内生真菌的分离及其鉴定

从无花果(Ficus carica)的根、茎、叶中分离得到87株内生真菌,经显微形态特征观察鉴定为14个属,其中根部35株涉及8个属,茎部37株涉及10个属,叶部15株涉及3个属。结果表明,无花果的不同部位内生真菌的数量、分布和种群存有差异。     

甘草内生真菌的分离及鉴定

植物体内普遍存在着内生菌,为了探寻传统药材甘草的质量与其内生菌之间可能存在的关系。对采自新疆的甘草根部进行了内生真菌的分离及鉴定。选用CYM真菌培养基,对其内生真菌进行分离及纯化,选择其中最占优势的两种菌落分别进行了菌落形态观察及菌丝形态(棉兰染色)观察,以及基因组DNA的18S rDNA和ITS rDNA序列鉴定和系统进化分析。本研究结果表明从甘草根部主要分离到两种内生真菌,分别属于Fusarium镰刀菌属和Gibberella赤霉菌属。     

真菌的分子生物学鉴定方法研究进展

综述了真菌的分子生物学鉴定方法,包括DNA碱基组成分析,核酸分析技术,真菌核型的脉冲电泳分析。     

基质辅助激光解吸电离飞行时间质谱在微生物鉴定中的研究进展

与传统的微生物鉴定技术相比,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)是一种准确、可靠和快速的鉴定和分型的技术。本文通过检索近年来国内外相关研究论文,总结最新的研究进展,发现MALDI-TOF MS在临床病原微生物、食源性微生物以及环境微生物等鉴定中有较大的优势,加快了微生物鉴定的进程,同时探索该技术在新领域的最新进展和面临的挑战,以期为我国基质辅助激光解吸电离飞行时间质谱技术的发展提供参考。     

嗜热真菌的分离鉴定与系统发育分析

目的:研究广西热带地区嗜热真菌的多样性.方法:从广西热带地区各地采集的土壤样品,将土样撒在PDA平板上,50℃高温培养,挑取真菌菌丝进一步划线分离纯化,20℃低温培养验证获得嗜热真菌,对其进行形态观察和ITS基因序列分析.结果:共分离到33株嗜热真菌,形态和分子生物学鉴定结果显示分离到的菌株中有20株属于Thermomyces lanuginosus,4株属于Thermoascus aurantiacus,1株属于Chaetomium thermophilum,l株属于Talaromyces emersonii,1株属于Myceliophthora thermophila,还有6株的ITS序列与已知真菌的同源性很低,尚无法鉴定到种属.结论:广西热带地区的嗜热真菌存在多样性,Thermomyces lanuginosus为该地区主要嗜热真菌种.     

益母草内生真菌分离与初步鉴定

利用平板分离法从药用植物益母草(Leonurus heterophyllus Sweet)中分离到15株内生真菌,来源于根、茎、叶分别有2,6,7株。经形态学观察和染色等方法,初步鉴定了益母草内生真菌有3个属,曲霉属(Aspergillus sp.)2株、头孢霉属(Cephalosporiumsp.)3株和囊孢霉属(Capsule sp.)10株。益母草内生真菌生物多样性较为单一,但不同部位内生菌的数量、种类及分布存在明显差异。     

Diagnostic accuracy of MALDI-TOF mass spectrometry for non-small cell lung cancer: a meta-analysis

Objective: To assess the overall accuracy of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying non-small cell lung cancer (NSCLC).

Methods: A comprehensive search of PubMed, EMBASE and CNKI databases as well as the reference lists from relevant articles was performed prior to July 2017. Two authors independently screened articles based on inclusion and exclusion criteria and assessed the quality of each study using the Quality Assessment of Diagnostic Accuracy Studies 2 (QADAS-2) tool. Meta-disc 1.4 and Stata12.0 software programs were used for the statistical analysis.

Results: Eleven eligible articles comprising 16 studies and representing 935 subjects were included in this meta-analysis. The pooled sensitivity and specificity were 0.84 (95% CI: 0.80–0.87) and 0.77 (95% CI: 0.74–0.80), respectively. The overall diagnostic performance as measured by the area under the curve (AUC) for the summary receiver-operating characteristic (SROC) curve was 0.9380.

Conclusions: MALDI-TOF MS has a high diagnostic accuracy for NSCLC.     

Identification of Gram-positive anaerobic cocci by MALDI-TOF mass spectrometry

Gram-positive anaerobic cocci (GPAC) are part of the commensal microbiota of humans and are a phylogenetically heterogeneous group of organisms. To evaluate the suitability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of GPAC, a database was constructed, using reference strains of commonly encountered GPAC and clinical isolates of which the sequence of the 16S rRNA gene was determined. Subsequently, the database was validated by identifying 107 clinical isolates of GPAC. Results were compared with the identifications obtained by 16S sequencing or fluorescent in situ hybridization (FISH). Strains belonging to the same species grouped together, in most cases, by MALDI-TOF MS analyses. Strains with sequence similarities less than 98% to their closest relatives, formed clusters distinct from recognized species in the MALDI-TOF MS dendrogram and, therefore could not be identified. These strains probably represent new species. Only three clinical isolates (2 strains of Finegoldia magna and 1 strain of Anaerococcus vaginalis) could not be identified. For all the other GPAC strains (96/107), reliable identifications were obtained. Therefore, we concluded that MALDI-TOF MS is an excellent tool for the identification of phylogenetically heterogeneous groups of micro-organisms such as GPAC.     

Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting

Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species.     

Novel truncated isoforms of constitutive serum amyloid A detected by MALDI mass spectrometry

The main focus of the serum amyloid A (SAA) family has been on the acute phase isoforms. However, the constitutive isoform (SAA4) may have a strong effect on the metabolism of human serum lipoproteins. In this study, the SAA4 protein was examined in the high-density lipoprotein fraction of both healthy and diseased individuals. Novel isoforms of SAA4 were detected using ultracentrifugation combined with solid-phase extraction and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Three truncated isoforms were identified as well as two glycosylated isoforms. Patterns of isoform distribution may be significant for assessment of cardiovascular risk as well as direction of patient treatment.     

Subunit analysis of bovine heart complex I by reversed-phase high-performance liquid chromatography, electrospray ionization-tandem mass spectrometry, and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

An effective method was developed for isolation and analysis of bovine heart complex I subunits. The method uses C18 reversed-phase high-performance liquid chromatography (HPLC) and a water/acetonitrile gradient containing 0.1% trifluoroacetic acid. Employing this system, 36 of the 45 complex I subunits elute in 28 distinct chromatographic peaks. The 9 subunits that do not elute are B14.7, MLRQ, and the 7 mitochondrial-encoded subunits. The method, with ultraviolet (UV) detection, is suitable for either analytical (<50 μg protein) or preparative (>250 μg protein) applications. Subunits eluting in each chromatographic peak were initially determined by matrix-assisted laser desorption/ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS) with subsequent positive identification by reversed-phase HPLC-electrospray ionization (ESI)/tandem mass spectrometry (MS/MS) analysis of tryptic digests. In the latter case, subunits were identified with a 99% probability using Mascot for database searching and Scaffold for assessment of protein identification probabilities. The reversed-phase HPLC subunit analysis method represents a major improvement over previous separation methods with respect to resolution, simplicity, and ease of application.     

Analysis of chlorophylls and their derivatives by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry

The analysis of chlorophylls and their derivatives by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry is described. Four matrices—sinapinic acid, a-cyano-4-hydroxycinnnamic acid, terthiophene, and 3-aminoquinoline—were examined to determine optimal conditions for analysis of the molecular mass and structure of chlorophyll a as a representative chlorophyll. Among them, terthiophene was the most efficient without releasing metal ions, although it caused fragmentation of the phytol-ester linkage. Terthiophene was useful for the analyses of chlorophyll derivatives as well as porphyrin products such as 8-deethyl-8-vinyl-chlorophyll a, pheophorbide a, pyropheophorbide a, bacteriochlorophyll a esterified phytol, and protoporphyrin IX. The current method is suitable for rapid and accurate determination of the molecular mass and structure of chlorophylls and porphyrins.     

MALDI-TOF mass spectrometry proteomic phenotyping of clinically relevant fungi

Proteomics is particularly suitable for characterising human pathogens with high life cycle complexity, such as fungi. Protein content and expression levels may be affected by growth states and life cycle morphs and correlate to species and strain variation. Identification and typing of fungi by conventional methods are often difficult, time-consuming and frequently, for unusual species, inconclusive. Proteomic phenotypes from MALDI-TOF MS were employed as analytical and typing expression profiling of yeast, yeast-like species and strain variants in order to achieve a microbial proteomics population study. Spectra from 303 clinical isolates were generated and processed by standard pattern matching with a MALDI-TOF Biotyper (MT). Identifications (IDs) were compared to a reference biochemical-based system (Vitek-2) and, when discordant, MT IDs were verified with genotyping IDs, obtained by sequencing the 25-28S rRNA hypervariable D2 region. Spectra were converted into virtual gel-like formats, and hierarchical clustering analysis was performed for 274 Candida profiles to investigate species and strain typing correlation. MT provided 257/303 IDs consistent with Vitek-2 ones. However, amongst 26/303 discordant MT IDs, only 5 appeared "true". No MT identification was achieved for 20/303 isolates for incompleteness of database species variants. Candida spectra clustering agreed with identified species and topology of Candida albicans and Candida parapsilosis specific dendrograms. MT IDs show a high analytical performance and profiling heterogeneity which seems to complement or even outclass existing typing tools. This variability reflects the high biological complexity of yeasts and may be properly exploited to provide epidemiological tracing and infection dispersion patterns.     

Applications of whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry in systematic microbiology

In the last few years matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been increasingly studied and applied for the identification and typing of microorganisms. Very recently, MALDI-TOF MS has been introduced in clinical routine microbiological diagnostics with marked success, which is remarkable considering that not long ago the technology was generally seen as being far from practical application. The identification of microbial isolates by whole-cell mass spectrometry (WC-MS) is being recognized as one of the latest tools forging a revolution in microbial diagnostics, with the potential of bringing to an end many of the time-consuming and man-power-intensive identification procedures that have been used for decades. Apart from applications of WC-MS in clinical diagnostics, other fields of microbiology also have adopted the technology with success. In this article, an over-view of the principles of MALDI-TOF MS and WC-MS is presented, highlighting the characteristics of the technology that allow its utilization for systematic microbiology.     

Delineation of Stenotrophomonas spp. by multi-locus sequence analysis and MALDI-TOF mass spectrometry

The genus Stenotrophomonas is genetically and phenotypically heterogeneous. Of the nine species now accepted, only S. maltophilia is of clinical importance. Based on DNA-sequences of seven house keeping genes, it encompasses genogroups of DNA-similarity below 97% that predominantly comprise strains of environmental origin. Therefore, in order to unravel the uneven distribution of environmental isolates within genogroups and reveal genetic relationships within the genus, there is need for an easy and reliable approach for the identification and delineation of Stenotrophomonas spp. In this first study, a multi-locus sequence analysis (MLSA) with seven housekeeping genes (atpD, gapA, guaA, mutM, nuoD, ppsA and recA) was applied for analysis of 21 S. maltophilia of environmental origin, Stenotrophomonas spp. and related genera. The genotypic findings were compared with the results of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) analyses. Our MLSA provided reliable inter- and intra-species discrimination of all tested isolates that correlated with the MALDI-TOF mass spectrometry data. One distantly related genogroup of environmental S. maltophilia strains needs to be reclassified as S. rhizophila. However, there are still remaining delineated S. maltophilia genogroups of predominantly environmental origin. Our data provide further evidence that ‘Pseudomonas’ beteli is a heterotypic synonym of S. maltophilia. Based on MLSA and MALDI-TOF data, Stenotrophomonas sp. (DSM 2408) belongs to S. koreensis.