MALDI-TOF MS自建数据库对非结核分枝杆菌鉴定的应用价值

目的 构建基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)非结核分枝杆菌(NTM)鉴定数据库并评价其应用价值.方法 收集2018年1月—2021年5月临床分离NTM菌株72株,分别采用直接同源序列比对法和MALDI-TOF MS法进行菌种鉴定,MALDI-TOF MS以"自建数据库+MBT DB5989数据...     

百日咳鲍特菌MALDI-TOF MS谱库的扩展及鉴定效果评价

目的对基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术鉴定百日咳鲍特菌谱库进行扩展。方法采用Microflex LT软件,用经全基因组测序确认的9株百日咳鲍特菌和1株副百日咳鲍特菌构建蛋白质指纹谱库,对布鲁克数据库进行扩展。对标准菌株ATCC 29213、ATCC 25922、ATCC 700603、ATCC 27853进行MALDI-TOF MS鉴定,评价扩展库的准确性。选取6株百日咳鲍特菌,每个菌株取3个菌落分别用MALDI-TOF MS鉴定,评价试验的精密度。采用36株临床分离株验证扩展谱库对百日咳鲍特菌的识别能力,并进行扩展库前后鉴定结果比较。结果成功构建9株百日咳鲍特菌和1株副百日咳鲍特菌参考谱库,并扩展现有布鲁克数据库Taxonomy(5989)数据库为Taxonomy(5989+)。扩展库准确鉴定4株标准菌株ATCC 29213、ATCC 25922、ATCC 700603、ATCC 27853,对鲍特菌鉴定无干扰;6株百日咳鲍特菌的重复鉴定符合率为100%,精密度良好。用于外部验证的36株菌经扩展后数据库鉴定,种水平鉴定率达86.1%。结论百日咳鲍特菌质谱库的扩展提升了地区性分离菌株鉴定能力。     

MALDI-TOF MS在耐甲氧西林金黄色葡萄球菌快速筛查中的价值

目的 探讨基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术在耐甲氧西林金黄色葡萄球菌(MRSA)快速筛查中的价值.方法 收集30株MRSA和29株甲氧西林敏感金黄色葡萄球菌(MSSA),采用MALDI-TOF MS技术进行菌种鉴定,比较鉴定结果的准确性;采用MALDI Biotyper软件对细菌的蛋白峰...     

MALDI-TOF MS自建库在红色毛癣菌感染中的临床快速诊断价值

目的建立并评价实验室自建的红色毛癣菌基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)数据库的可行性。方法将红色毛癣菌标准菌株ATCC 28188用沙保罗葡萄糖琼脂(SDA)平板28℃培养5d,分别用双甲酸夹心法和甲酸提取法作为蛋白提取方法,利用MALDI-TOF MS对标准菌株进行质谱数据采集,建立2种不同蛋白提取方法的菌株数据库"双甲酸夹心法自建库"和"甲酸提取法自建库";并用2种蛋白提取方法对21株红色毛癣菌临床分离株进行蛋白提取后,分别选取"双甲酸夹心法自建库+Bruker商品库"、"甲酸提取法自建库+Bruker商品库"和"Bruker商品库"做质谱鉴定,从而对各数据库的鉴定效果进行评价。结果"甲酸提取法自建库+Bruker商品库"对21株红色毛癣菌临床分离株的鉴定得分显著高于其余两组,差异有统计学意义(P<0.05),匹配率为21/21;"Bruker商品数据库"与"Bruker商品数据库+双甲酸夹心法自建库"的鉴定得分比较差异无统计学意义(P>0.05)。结论本实验室建立的"甲酸提取法自建库+Bruker库"对红色毛癣菌的鉴定能力好,适用于临床微生物实验室。与待测菌统一培养条件所建立的数据库丰富了原有的数据库信息,使待测菌的鉴定更为准确。双甲酸夹心法操作简便,但对红色毛癣菌蛋白提取的效果欠佳。     

MALDI-TOF MS鉴定马尔尼菲篮状菌的实验条件优化

目的 构建用于基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF MS)鉴定马尔尼菲篮状菌的数据库,并探索用于MALDI-TOF MS鉴定马尔尼菲篮状菌的最佳培养条件和前处理方法 .方法 利用8株马尔尼菲篮状菌构建自建数据库,比较不同培养基[沙保弱葡萄糖琼脂培养基(SDA)、沙氏葡萄糖肉汤培养基(SDB)]、培养...     

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。     

应用基质辅助激光解吸电离飞行时间质谱技术鉴定临床常见厌氧菌

目的：应用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）技术对临床分离的厌氧菌进行鉴定,与传统Vitek32 ANI鉴定卡及16 S rRNA基因序列法鉴定进行比较,分析MALDI-TOF MS技术用于鉴定临床常见厌氧菌的可行性。方法收集从临床标本培养分离的厌氧菌56株,运用 MALDI-TOF MS 技术对其进行鉴定,同时运用 Vitke32 ANI 鉴定卡及16 S rRNA基因序列法分别进行鉴定,将三者的结果进行比较。结果56株厌氧菌中有41株采用 MALDI-TOF MS技术鉴定至种的水平（分值大于或等于2．0）,11株鉴定至属的水平（分值为1．7～2．0）,4株无可靠结果（分值小于1．7）。MALDI-TOF MS和16S rRNA基因序列法鉴定结果一致的占94．6％（53/56）,不一致的占5．6％（3/56）。MALDI-TOF MS与Vitke32 ANI 鉴定卡的鉴定结果一致的占80．4％（45/56）,不一致的占19．6％（11/56）。结论 MALDI-TOF MS与16S rRNA基因序列法鉴定的符合率高,可应用于临床常见厌氧菌的鉴定。     

MALDI-TOF MS技术在鲍曼不动杆菌鉴定及同源性分析中的应用

目的评价基质辅助激光解吸/电离飞行时间质谱(MALDI-TOF MS)技术对我院鲍曼不动杆菌属的鉴定及同源性分析的能力。方法对2014年9月至2015年2月我院29株经PhoneixTM100全自动微生物鉴定仪鉴定为鲍曼不动杆菌属的菌株进行MALDI-TOF MS鉴定,用MALDI-Biotyper软件进行同源性分析,并用16S rRNA基因测序进行验证。结果 MALDI-TOF MS鉴定结果为鲍曼不动杆菌(Acinetobacter baumannii)26株,院内不动杆菌(Acinetobacter nosocomialis)3株。MALDI-TOF MS鉴定为院内不动杆菌的3株菌株的16S rRNA基因测序结果显示,2株为院内不动杆菌,1株为鲍曼不动杆菌。26株质谱鉴定为鲍曼不动杆菌的菌株分为2大簇(Ⅰ型,Ⅱ型),Ⅱ型又分为2小簇(Ⅱa、Ⅱb)。Ⅰ型、Ⅱ型分布在我院4个病区。结论MALDI-TOF MS技术鉴定鲍曼不动杆菌精准,可以鉴别鲍曼不动杆菌和院内不动杆菌。MALDI-Biotyper数据库软件进行同源性分析十分快捷。     

MALDI-TOF MS用于临床微生物检验中常见菌鉴定的应用探讨

目的通过比较两种微生物鉴定系统基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)和Vitek 2Compact,评价MALDI-TOF MS在临床常规分离微生物中的应用价值。方法对比MALDI-TOF MS和Vitek 2Compact系统的菌种数据库,筛选出MALDI-TOF MS菌库中新增菌种,统计该院自2015年5月MALDI-TOF MS投入使用起至2016年12月新增菌种被检出的菌株及频次,以及高置信度鉴定临床常见致病菌的检出频次。结果 MALDI-TOF MS较Vitek 2Compact菌库中新增205种菌株,在2015年5月至2016年12月临床微生物检验中共检出206次,且MALDI-TOF MS系统高置信度鉴定4种临床常见致病菌共286例。结论 MALDI-TOF MS菌种数据库更大、准确度高,更能满足临床微生物检验的需求,适于更广泛地投入到临床微生物鉴定的应用中。     

MALDI-TOF MS 在呼吸道感染丝状真菌鉴定中的应用

目的：用MALDI-TOF MS技术和形态学方法对139株丝状真菌进行鉴定,评估MALDI-TOF MS技术在丝状真菌鉴定中的应用。方法将从临床呼吸道标本培养分离出来的139株丝状真菌,经过预处理后,用法国梅里埃公司生产的质谱仪（VITEK MS）进行鉴定,并将结果与传统形态学鉴定方法结果进行对比。结果质谱仪对139株丝状真菌正确鉴定率为82．7％（115／139）;对曲霉属的鉴定正确率达到90．8％（108／119）,其中烟曲霉和黄曲霉的鉴定正确率均为100％（77／77、21／21）,黑曲霉的鉴定正确率50．0％（8／16）,其他曲霉菌的鉴定正确率为40．0％（2／5）;对青霉属的鉴定正确率为58．3％（7／12）;对毛霉菌的正确鉴定率为0％（0／3）。呼吸道感染丝状真菌以曲霉菌属为主（89．2％）,其中又以烟曲霉（55．4％）、黄曲霉（15．1％）和黑曲霉（11．5％）多见。结论 MALDI-TOF MS技术能快速准确鉴定常见丝状真菌,为丝状真菌的鉴定带来革命性突破,但对少见丝状真菌的鉴定仍有待于数据库的进一步完善。     

Comparison of in-house and commercial 16S rRNA sequencing with high-performance liquid chromatography and genotype AS and CM for identification of nontuberculous mycobacteria

Sequencing of the 16S gene or other targets and line probe assay are in wide use for the identification of nontuberculous mycobacteria. We compared in-house and commercial sequencing with 3 sequence databases against high-performance liquid chromatography (HPLC) and line probe assay (HAIN Genotype AS and CM) for the identification of 84 reference, clinical, and unique strains representing 41 species. Consensus of methods was used as reference standard. Sequencing identification was more specific and flexible than HPLC, but it was limited by database content and quality as well as fragment length. No one database satisfied all requirements. In-house sequencing was lower in cost than commercial sequencing or line probe assay.     

Capsular serotyping of Haemophilus influenzae by using matrix-associated laser desorption ionization-time of flight mass spectrometry

Haemophilus influenzae is a major pathogenic bacteria causing invasive disease, which is classified into six capsular serotypes (a-f) and non-typeable (NT) strains. Capsular serotyping of H. influenzae is traditionally determined by serological methods and more recently by PCR methods. However, these methods are time-consuming and expensive. In the present study, matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated as an alternative method for capsular serotyping of H. influenzae clinical strains. We created an in-house database of all six serotypes and NT H. influenzae strains using the main spectrum creation standard method set to the default parameters in MADI-TOF MS. We evaluated the performance of the in-house database using 79 clinical strains already identified by PCR and 58 prospectively collected clinical strains. Measurements were performed using the Bruker MALDI BioTyper system. The peak list was matched against the reference library using the integrated pattern algorithm of the software. The best-matched spectrum was considered the serotyping result. All 137 test strains were correctly identified as H. influenzae using MALDI-TOF MS. The sensitivity and specificity for identification for type b, type e, and type f capsular serotypes and NT H. influenzae using MALDI-TOF MS were 100%/94.3%, 94.7%/97.9%, 97.4%/97.9%, and 85.5%/99.2%, respectively. Our findings indicate that MALDI-TOF MS is a useful alternative method for capsular serotyping of H. influenzae strains. This method is faster and more cost-effective than traditional methods and will therefore be useful for routine applications in clinical laboratories.     

Detection and typing of viruses using broadly sensitive cocktail-PCR and mass spectrometric cataloging: demonstration with dengue virus

Virus detection and taxonomic identification of serotypes, strains, or genotypes provide important information relevant for diagnosis, and for the epidemiological characterization and tracking of new strains in an endemic region. In the specific case of dengue virus, rapid serotype identification can also be useful in the treatment of secondary infections that may cause the more severe dengue hemorrhagic fever and dengue shock syndrome. In this work, dengue virus was used as a model to test a new approach of combining broadly sensitive RT-PCR amplification of nearly any virus strain with subsequent serotype- and finer-level identification by mass spectrometry. PCR primers were appended with promoter sequences, such that the resulting PCR products could be transcribed into RNA. RNA fragments generated by guanosine-specific RNase T(1) digestion were analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Viral serotypes were identified by comparing the pattern of observed fragment masses to a mass database. The database was created by computationally fragmenting 2517 dengue strains after each guanosine residue using the same primers. Computationally, all 2517 strains in the mass database were correctly identified at the serotype level from the predicted PCR product. The methodology was successfully demonstrated experimentally by identifying the serotypes of eight test strains using mosquito cell cultures infected with strains of all four serotypes and with full-length cDNA clones.     

1386例小儿中段尿培养病原菌分布及耐药性分析

目的分析小儿中段尿培养病原菌的分布及耐药性,以期对儿童临床泌尿系统感染的临床诊疗和预防控制提供有力理论支持。方法对2010～2011年1 386例小儿中段尿培养的病原菌,采用法国生物梅里埃(BioMerieux)VITEK-32全自动微生物分析系统进行鉴定。采用K-B纸片法对病原菌进行常用抗生素的敏感性检测。使用WHONET 5.4软件对原始数据进行分析和统计学处理。结果 2010～2011年尿培养标本1 386例共分离到病原菌138株,阳性率为9.96%。其中革兰阴性菌96株,占69.57%,居前2位的是大肠埃希菌、肺炎克雷伯菌;革兰阳性菌34株,占24.64%,居前2位的是屎肠球菌和粪肠球菌;真菌8株,占5.8%。2年间共分离出大肠埃希菌88株,其中产超广谱β-内酰胺酶菌株56株,比例高达63.64%。结论小儿中段尿培养的病原菌仍以大肠埃希菌为主,泌尿系统感染治疗形势由于细菌多重耐药和交叉耐药日趋严峻,日常了解并监测病原菌分布特点及其耐药情况对临床合理选用抗生素具有重要意义。     

对in-house HIV-1基因型耐药性检测方法的评价

目的 评价in-house HIV-1基因型耐药检测方法的敏感性与准确性。方法 收集2004年4月至2008年10月全军艾滋病检测中心检测的来自河南、广西130份血浆标本。以美国FDA批准的HIV-1基因型耐药性检测系统(ViroSeqTM v2.0)为参考方法,并与建立起的基因型耐药性检测方法(in-house)平行检测待检样本,比较二者在扩增效率、耐药突变位点检测以及耐药报告等方面的一致性。结果已知的14 850个耐药突变位点中,2种方法可同时对99.3％(14 752/14 850)的耐药突变位点准确检出;在对不同突变位点的检测中,2种方法对蛋白酶抑制剂耐药突变位点、逆转录酶抑制剂耐药突变位点及两类抑制剂耐药突变位点检测一致率分别为99.7％、99.0％和99.3％（Kappa值分别为0.909 9、0.952 1和0.948 8,P值均＜0.01）;2种方法出具的两类药物的耐药结果报告一致率94.6％（Kappa=0.637 4,P＜0.01）。本研究共检测到34个ViroSeqTM数据库(ViroSeqTM software v2.7)未收录位点,其中2个突变位点对耐药的影响较大。结论in-house基因型耐药性检测方法与ViroSeqTM基因型耐药性检测系统在耐药突变位点检测以及评价上具有高度一致性,是一种快速准确、性价比高的HIV-1基因型耐药检测方法,同时在耐药数据库方面具有一定优势。     

Rapid identification of Enterobacteriaceae by sequencing DNA gyrase subunit B encoding gene

Real-time polymerase chain reaction and sequencing were used to characterize a 506-bp-long DNA fragment internal to the gyrB gene (gyrBint). The sequences obtained from 32 Enterobacteriaceae-type strains and those available in the Genbank nucleotide sequence database (n = 24) were used as a database to identify 240 clinical enterobacteria isolates. Sequence analysis of the gyrBint fragment of 240 strains showed that gyrBint constitutes a discriminative target sequence to differentiate between Enterobacteriaceae species. Comparison of these identifications with those obtained by phenotypic methods (Vitek 1 system and/or Rapid ID 32E; bioMérieux, Marcy l'Etoile, France) revealed discrepancies essentially with genera Citrobacter and Enterobacter. Most of the strains identified as Enterobacter cloacae by phenotypic methods were identified as Enterobacter hormaechei strains by gyrBint sequencing. The direct sequencing of gyrBint would be useful as a complementary tool in the identification of clinical Enterobacteriaceae isolates.     

Species identification of corynebacteria by cellular fatty acid analysis

We evaluated the usefulness of cellular fatty acid analysis for the identification of corynebacteria. Therefore, 219 well-characterized strains belonging to 21 Corynebacterium species were analyzed with the Sherlock System of MIDI (Newark, DE). Most Corynebacterium species have a qualitative different fatty acid profile. Corynebacterium coyleae (subgroup 1), Corynebacterium riegelii, Corynebacterium simulans, and Corynebacterium imitans differ only quantitatively. Corynebacterium afermentans afermentans and C. coyleae (subgroup 2) have both a similar qualitative and quantitative profile. The commercially available database (CLIN 40, MIDI) identified only one third of the 219 strains correctly at the species level. We created a new database with these 219 strains. This new database was tested with 34 clinical isolates and could identify 29 strains correctly. Strains that remained unidentified were 2 Corynebacterium aurimucosum (not included in our database), 1 C. afermentans afermentans, and 2 Corynebacterium pseudodiphtheriticum. Cellular fatty acid analysis with a self-created database can be used for the identification and differentiation of corynebacteria.     

Comparison of three commercial systems for the identification of germ-tube negative yeast species isolated from clinical specimens

Three commercial systems were evaluated for their ability to identify 171 germ-tube negative yeasts isolated from clinical specimens. The Yeast Biochemical Card and Analytical Profile Index 20 AUX identified 97% of 171 strains tested. The Biolog system had poor clinical utility: only 48% of strains were identified. For Yeast Biochemical Card and Analytical Profile Index 20 AUX, 9% and 6%, respectively, required repeat testing and both systems required supplemental tests for 28% of the strains. These observations indicate that considerable expertise and a battery of reagents in addition to the basic systems are required for accurate identification of germ-tube negative yeasts.